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Bridging Molecular Genetics and Biomarkers in Lewy Body and Related Disorders

Bridging Molecular Genetics and Biomarkers in Lewy Body and Related Disorders SAGE-Hindawi Access to Research International Journal of Alzheimer’s Disease Volume 2011, Article ID 842475, 18 pages doi:10.4061/2011/842475 Review Article Bridging Molecular Genetics and Biomarkers in Lewy Body and Related Disorders 1, 2 1, 2 3 3 Gilbert J. Ho, Willie Liang, Masaaki Waragai, Kazunari Sekiyama, 1 3 Eliezer Masliah, and Makoto Hashimoto Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093-0624, USA The Center for Memory and Aging, Poway, CA 92064, USA Laboratory for Chemistry and Metabolism, Tokyo Metropolitan Institute for Neuroscience, Tokyo 183-8526, Japan Correspondence should be addressed to Gilbert J. Ho, giho@ucsd.edu Received 30 December 2010; Accepted 20 April 2011 Academic Editor: G. B. Frisoni Copyright © 2011 Gilbert J. Ho et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Recent advances have been made in defining the genetic and molecular basis of dementia with Lewy bodies (DLBs) and related neurodegenerative disorders such as Parkinson’s disease (PD) and Parkinson’s disease dementia (PDD) which comprise the spectrum of “Lewy body disorders” (LBDs). The genetic alterations and underlying disease mechanisms in the LBD overlap substantially, suggesting common disease mechanisms. As with the other neurodegenerative dementias, early diagnosis in LBD or even identification prior to symptom onset is key to developing effective therapeutic strategies, but this is dependent upon the development of robust, specific, and sensitive biomarkers as diagnostic tools and therapeutic endpoints. Recently identified mutations in the synucleins and other relevant genes in PD and DLB as well as related biomolecular pathways suggest candidate markers from biological fluids and imaging modalities that reflect the underlying disease mechanisms. In this context, several promising biomarkers for the LBD have already been identified and examined, while other intriguing possible candidates have recently emerged. Challenges remain in defining their correlation with pathological processes and their ability to detect DLB and related disorders, and perhaps a combined array of biomarkers may be needed to distinguish various LBDs. 1. Introduction of AD tau pathology, further complicating the diagnosis [2]. Approximately 20–40% of Parkinson’s disease (PD) Over the past decade, dementia with Lewy bodies (DLBs) patients also eventually develop a progressive dementing has arguably become the second most common form illness designated as Parkinson’s disease dementia (PDD) of neurodegenerative dementia behind Alzheimer’s disease characterized by a frontal-subcortical clinical presentation (AD). In addition to progressive decline in cognition, [3]. DLB/Diffuse Lewy body disease (DLBD), Lewy body DLB is characterized by fluctuations in cognition with variant of AD (LBV), and PDD comprise an emerging variations in attention and alertness, recurrent formed visual spectrum of clinical phenotypes from relatively pure motor hallucinations, visuospatial dysfunction, and spontaneous PD to the more predominant cognitive and behavioral disturbance observed in PDD and DLB, yet the reason for parkinsonism. Often, DLB patients also exhibit neuroleptic sensitivity, transient loss of consciousness, falls, and rapid eye the variability remains unknown. Despite the heterogeneity movement (REM-) sleep behavior disorder [1]. The clinical of their clinical phenotypes, a significant neuropathological overlap is observed among these diseases, hence the term separation of DLB from other similar disorders is often difficult resulting in poor diagnostic accuracy, but the relative “Lewy body disorders” (LBDs) to collectively describe con- temporal co-occurrence of parkinsonian features with the ditions in which Lewy bodies (LBs) and Lewy neurites (LNs) typical DLB cognitive and behavioral symptoms such as predominate as the hallmark histological lesions. Variation visuospatial disturbance strongly suggests the diagnosis. in the distribution of Lewy body pathology is present among Clinical presentation of DLB is also influenced by the amount LBDs, with more neocortical and limbic system LB in both 2 International Journal of Alzheimer’s Disease DLB and PDD compared to the brains of PD patients while the α-secretase pathway precludes Aβ formation by without cognitive symptoms and greater neuronal loss in cleaving βAPP at a site within the Aβ sequence. Genetic substantia nigra in PDD than DLB [4]. Yet, DLB and overlap analysis of early-onset familial AD cases revealed numerous disorders such as LBV, more so than PDD, have β-amyloid mutations in the βAPP gene as well as presenilin 1 (PS1; pathology and basal forebrain cholinergic deficit similar to chromosome 14) and presenilin 2 (PS2; chromosome 1) AD patients [5]. genes, all of which accelerate the processing of βAPP, leading As with other forms of dementia, the pathobiological to increased Aβ generation [12]. Specifically, the βAPP changes in LBD likely occur decades prior to the onset of KM670/671NL (Swedish) mutation affects the β-secretase clinical symptoms and correspond to widespread irreversible site, A692G (Flemish) mutation alters the α-secretase site, neurodegeneration [6, 7]. It is increasingly clear from the and both V717F (Indiana) and V717I (London) mutations AD therapeutic experience that by the time widespread affect the γ-secretase processing, leading to elevated Aβ neuronal injury ensues, symptomatic cholinergic treatments levels. Also important in the Notch developmental signaling are minimally effective at best, and disease-modifying thera- pathway which is analogous to βAPP processing, the pre- peutic approaches in trials have thus far proven ineffective senilins are thought to be a component of the γ-secretase at altering disease course or in rescuing diseased brain [8, enzyme complex, which suggests that missense mutations 9]. To demonstrate efficacy, any potential disease-modifying in the presenilins mechanistically lead to accelerated βAPP therapy in neurodegenerative dementia must be initiated processing to Aβ [13]. prior to the expression of the clinical phenotype during the Therefore, the abnormal proteolytic cleavage of βAPP initial molecular pathogenetic events before irreversible neu- leads to elevated brain Aβ deposition, and as a result, ronal damage has occurred. At present, accurately predicting diminished peripheral levels of Aβ. Reflecting a shift from those individuals at risk for developing neurodegenerative soluble Aβ to insoluble brain deposits, significant decreases dementia is challenging, and this places greater urgency on in CSF Aβ42 levels have been demonstrated in AD and developing earlier methods of disease detection. Critically more recently in DLB cases [14]. Parnetti et al. found that important is not only distinguishing the LBD from AD and DLB, compared with PD, PDD, and AD patients, showed the other forms of dementia, but also separating DLBs and other lowest CSF levels of Aβ42 and, when combined with CSF tau, LBDs. Although there are many promising candidates for differentiated DLB from PD and PDD, but not from AD [15]. LBD, no biomarkers have yet been validated for clinical Also, Spies and colleagues showed a greater decrease in Aβ40 diagnostic use, and thus many opportunities exist to develop in clinical DLB and vascular dementia patients compared such tests. Here, we highlight from the perspective of how with control levels and even with AD. Differentiation of major genetic discoveries in the LBD and their corresponding non-AD dementias such as vascular dementia and DLB biomolecular processes might translate into useful disease was improved by comparing the ratio of Aβ42 and Aβ40 markers in biological fluids. Because of many common [16]. More recently, the detection of amyloid in dementia pathogenetic features among the LBDs, the emerging genetic patients has been greatly enhanced by the use of amyloid- influences found in PD have readily been translated to DLBs binding agents such as Pittsburgh compound B [17], which and other LBDs, providing clues to rational approaches also demonstrated amyloid burden in DLB. An Australian for selecting future DLB and LBD biomarker targets for study reported more variable cortical PiB binding in DLB exploration. This paper will highlight several PD and DLB patients than in AD [18], whereas a subsequent examination genes and their protein products as candidates for biological of PiB binding in LBDs including DLB, PDD, and PD, disease markers (Table 1). compared with AD and normal patients, showed higher amyloid burden inDLB and ADthaninPDD, PD, or NC patients [19]. Amyloid load was highest in LBD patients in the parietal and posterior cingulate regions, corresponding 2. Amyloid and Tau in Lewy Body Disorders to visuospatial impairments on neuropsychological testing, 2.1. Amyloid Genetics and Biomarkers in LBD. Aβ,akey suggesting that amyloid deposition could partly contribute component of neuritic plaques in AD brain, is overproduced to the clinical presentation of LBDs. leading to various degrees of amyloid aggregation and synaptic and neuronal toxicity [10]. As indicated previously, amyloid pathology in the form of neuritic and diffuse 2.2. Tau Genetics and Biomarkers in LBD. Mutations in plaques can be also found in varying degrees in the brain the tau gene on chromosome 17 may also present with tissue of patients with DLB, which may interact with LB phenotypic features of PDD or DLB, but they differ patho- or synuclein pathology or influence the clinical features of logically from these disorders in that LBs are generally absent LBDs [4, 11]. The genetic mechanisms of Aβ overproduction [20]. Tau-bearing neurofibrillary tangles remain one of the in AD are well established; the βAPP gene (chromosome pathological hallmarks of AD but are also central to a diverse 21), the first identified AD susceptibility gene, encodes a group of disorders termed “tauopathies” which include pro- transmembranous protein ranging from 695 to 770 residues, gressive supranuclear palsy, corticobasal ganglionic degener- which undergoes a process of regulated intramembranous ation, frontotemporal dementia (FTD) with parkinsonism proteolysis ultimately releasing Aβ peptides, primarily Aβ42 linked to chromosome 17, and other disorders [21]. Tau and Aβ40, as well as other fragments. Aβ is generated by is a microtubule binding protein, which acts to stabilize the concerted action of β-secretase and γ-secretase complex, tubulin polymerization in microtubules critical for axonal International Journal of Alzheimer’s Disease 3 Table 1: Genetics and biomarkers in LBD. Biochemical Source of Gene defect Relevance to LB disorders marker biomarker APP: K670M/N671L and so forth. Aβ PS1: H163R and so forth PS2: Deposited in plaques CSF, plasma AD lesions N141I and so forth Tauopathy: P301L, N279K, K317M, Found in NFT in AD brain, released after Tau CSF and so forth neuronal damage Mutation →↑ α-syn aggregation. α-synuclein A53T, A30P (PD), G209A (DLB), CSF, skin LB component, toxic oligomers and (PARK1/4) E46K, triplication (PD & DLB) cells, platelets protofibrils PD/DLB Inhibit α-syn aggregation: mutant causes β-synuclein P123H, V70M (DLB) CSF lesions degeneration Amyloidogenic: affects neuronal and Ventricular γ-synuclein SNP in DLBD axonal cytoskeleton CSF K161N, W453Stop, 202-203delAG, Ubiquitin E3 ligase, LOF mutation in PD Parkin (PARK 2) ND M192L, K211N, and so forth alters mitophagy Neuronal deubiquitinating hydrolase; UCHL-1 I93M, S18Y (SNP) impaired synaptic and cognitive function ND Proteostasis/ (PARK 5) in AD & PD oxidative A168P, A217D, E417G, E240K, and Mitochondrial serine/threonine kinase; stress PINK 1 (PARK 6) ND so forth (PD) LOF mutation in PD alters mitophagy M26I, D149A, G78G, R98Q (PD), Redox-dependent chaperone; LOF DJ-1 (PARK7) CSF, plasma L166P (PD & DLB) mutation in PD Gain of function mutant in PD?DLB: G2019S, duplication, triplication LRRK2 (PARK 8) interacts with α-syn and tau, and with ND (PD) parkin in apoptotic cell death Disrupted NF → abnormal axonal Cytoskeletal NF NEFM (PD) CSF transport; released in cell damage Lysosomal 84 dupl G, IVS 2 + 1, N370S, L444P Gaucher’s disease, abnormal lysosomal GBA CSF, plasma dysfunction (PD) function/autophagy in PD α-syn-induced microglial activation →↑ IL-1α,IL-1β, Inflammation SNP: IL-1β−511,TNF-α−308 secretion of neuroinflammatory CSF IL-6, TNFα mediators CSF: cerebrospinal fluid; GBA: glucocerebrosidase; Aβ: β-amyloid; NF: neurofilament; ND: not yet determined; PD: Parkinson’s disease; DLB: Dementia with Lewy body; UCHL1: ubiquitin carboxy terminal hydrolase L1; PINK 1: PTEN-induced putative kinase 1; LRRK2: leucine-rich repeat kinase 2; LOF: loss of function; SNP: single nucleotide polymorphism. cytoskeletal integrity and function. In disease, tau protein Consequently, both total tau and hyperphosphorylated truncation at Glu 391 or hyperphosphorylation causes forms have been widely investigated and detected in CSF, microtubule destabilization and aggregation of unbound tau but not serum, by enzyme-linked immunosorbent assay into paired helical filaments (PHFs) leading to characteristic methods. In the differentiation of dementia types, Arai et al. tangle formations [22]. Unlike the tauopathies, no direct initially reported elevated total CSF tau levels in AD but not pathogenetic tau mutations have been identified in LBDs, in PD, but subsequently, they showed that total tau was also but tau pathology appears to be a consistent feature among increased in DLB at similar levels to AD [24]. Yet, others have neurodegenerative dementias including AD and LBDs, and found differences for both total and phospho-tau (p-tau) in given the pathological overlap, they might share similar differentiating DLB from AD [25], and levels of total tau and pathogenetic pathways (reviewed in Stoothoff and Johnson) p-tau 181 were significantly increased in autopsy-confirmed [23]. The Ser/Thr kinase and glycogen synthase kinase- DLB patients [26]. In clinically diagnosed dementia cases, 3β (GSK3β), in concert with other molecules such as fyn CSF p-tau 231 discriminated AD from non-AD dementias kinase, normally regulate tau function but with aberrant as a group, where levels were significantly higher in AD activation accelerate the hyperphosphorylation of tau in patients compared with DLB, FTD, vascular dementia, other neurodegenerative disease. Similarly, the cell cycle family disorders, and control subjects [27]. Separation of DLB kinase and cyclin-dependent kinase 5 (Cdk5/p35), active from AD, however, was less robust, provided that CSF p-tau during normal brain development and involved in regulatory 231 levels were also increased in DLB. Clinically diagnosed tau phosphorylation during mitosis, may also contribute to DLB cases also showed elevated levels of CSF p-tau 181 PHF formation. compared with controls [28], and Hampel et al. reported 4 International Journal of Alzheimer’s Disease that p-tau 181 provided the best discrimination of DLB Another mutation, E46K, was discovered in a Spanish family from AD yielding a sensitivity of 94% and specificity of 64% presenting with autosomal dominant DLB [48], and in [29]. In autopsy-confirmed DLB and AD patients, however, genetic studies of a large family with the spectrum of Lewy sensitivity decreased to 75% and specificity to 61%, with a body phenotype ranging from PD to DLB, α-syn gene diagnostic accuracy reported as 73% [30]. triplication was described, causing α-syn overproduction similar to the trisomy effect observed in Down syndrome patients [49]. 3. Synucleins: Genetics to Biomarkers in Autosomal dominant point mutations are shown to affect the Lewy Body Disorders the aggregative properties of α-syn, which has mechanistic implications for the pathogenesis of LBD. Compared to wild- 3.1. Pathogenetics of Synucleins in LBD type α-syn, biophysical analyses reveal that α-syn aggregation is folding state dependent, where A53T and A30P mutated 3.1.1. Functions of α-Synuclein. LBs are filamentous inclu- proteins cause increased aggregation only from the partially sions consisting primarily of the presynaptic protein α- folded intermediate state and not the monomeric state [50]. synuclein (α-syn), which might have several roles in vivo. A53T α-syn transgenic mice have increased oligomerization Studies demonstrate that it is localized to multiple neural of the protein in brain regions devoid of inclusions as well tissues, including high expression in neocortex and hip- as those areas with more abundant lesions and neurodegen- pocampus, and that expression increases during acquisition- eration, and consistent with prior biophysical findings, α-syn related synaptic plasticity [31]. Interaction with tubulin toxicity in these mice was dependent on the conformation of suggests α-syn could be a microtubule-associated protein intermediate species [51]. In fact, the E46K mutation, as well similar to tau [32, 33], and it is highly active in various as the others not only increase the tendency toward aggre- membrane lipid bilayers such as in presynaptic vesicles gation, but also promote formation of annular protofibrillar acting as a chaperone for soluble NSF attachment protein structures, causes pore formation in various membranes and receptor (SNARE) complex formation [34], in neuronal neuronal damage [52]. Golgi apparatus influencing protein trafficking [35]and in the inner membrane of neuronal mitochondrial [36]. 3.1.3. β-Synuclein Mutations in DLB. α-Syn is a member of Thesynucleinsmight actto preservemembranestability, a larger family of synuclein proteins which also includes β- provide antioxidant function, and assist with membrane synuclein (β-syn) and γ-synuclein (γ-syn). β-syn has recently turnover, although the actual role of synucleins remains been implicated in PD and DLB pathogenesis, but its precise elusive [37, 38]. Because of its association with LB and the role in disease is still emerging. Despite having strong homol- tendency to self-aggregate into pathological oligomers and ogy with α-syn, it is not clearly amyloidogenic, but is highly ultimately fibrillar structures [39], α-syn plays a central role localized to presynaptic sites in neocortex, hippocampus, in the pathogenesis of LBD, hence the alternate designation and thalamus like α-syn [53, 54]. Normal β-syn may act “synucleinopathies.” The degree of α-syn immunoreactivity as a biological negative regulator of α-syn. In bigenic α- in cortical LBs correlates with cognitive severity and disease syn/β-syn-overexpressing mice and in doubly transfected progression in PDD and DLB [4, 40]. Also, the protein can be cultured cells, β-syn ameliorated amyloidogenicity, neurode- recovered from filaments in purified Lewy bodies from PDD generative changes, and motor deficits induced by α-syn and DLB brain [41], and recombinant α-syn tends to form overexpression alone [55]. On the other hand, mutated β- Lewy body-like fibrillar structures in vitro [42]. syn leads to neuronal damage and disease and augments neurodegeneration, perhaps through a loss of its natural 3.1.2. α-Synuclein Mutations in PD and DLB. In the past regulator function. Two novel β-syn point mutations, P123H decade, tremendous advances have been made in under- and V70M, were found in highly conserved regions of the β- standing the genetic factors influencing the pathogenesis of syn gene in respective familial (P123H) and sporadic (V70M) Lewy body disorders. Compelling evidence for a genetic basis DLB index cases [56], where abundant LB pathology and for PD and DLB followed the discovery of mutations in the α-syn aggregation was present without β-syn aggregation. α-syn gene (PARK1/4) in patients with autosomal dominant P123H β-syn overexpression in transgenic mice resulted in familial Parkinson’s disease, and subsequently, mutations axonal damage, gliosis, profound memory, and behavioral were identified in patients with both sporadic and familial deficits [57]. These phenomena may involve α-syn, since DLBs. From a susceptibility marker on chromosome 4q21- bigenic mice overexpressing α-syn with P123H β-syn show 23 that segregated with the PD phenotype in Italian and greater deficits compared with monogenic mice and com- Greek kindreds, A53T [43] and A30P [44]were the first pared with P123H β-syn expressed with α-syn knockout, two missense mutations in α-syn associated with familial implying that the P123H mutation has a synergistic effect Parkinson’s disease. Clinical analysis of the Italian A53T with other synucleinopathies to cause neurodegeneration. mutation revealed phenotypic variability over the disease P123H as well as V70M β-syn mutations might also injure course with several individuals demonstrating moderate to neurons by disrupting normal lysosomal pathways and severe dementia [45]. Subsequently, a case of clinically and corresponding cellular autophagic processes [58]. pathologically well-characterized DLBD in the United States and a Greek proband of DLB with a family history of PD were 3.1.4. Association of γ-Synuclein with LBD. Unlike the oth- both determined to have the A53T α-syn mutation [46, 47]. er synuclein family members, γ-syn or persyn is largely International Journal of Alzheimer’s Disease 5 expressed in the cell bodies and axons of primary sensory PD and DLB patients [69, 70], and also from postmortem neurons, sympathetic neurons, and motor neurons as well CSF from DLB and other neurodegenerative diseases [71]. as in brain [59]. In cancer biology, γ-syn is associated with Comparative findings regarding differences in CSF α-syn abnormally altering cellular mitotic checkpoints in various levels among various neurodegenerative diseases, however, types of malignancies, making them more aggressively are difficult to interpret because of inconsistent observations. metastatic [60], but as far as neurodegeneration, it is the In PD, a smaller early study showed that no differences in most recent synuclein member to be linked to LBD neu- full-length CSF 19 kDa α-syn have been found in relation ropathology and the least well understood. Single-nucleotide to control individuals [69], but a recent effort using a polymorphisms in all three synucleins have been associated new Luminex assay in a larger sample controlling for with sporadic DLBD, most prominently γ-syn [61], and in extraneous influences showed significantly decreased levels sporadic PD, DLB, and LBV patients, γ-syn antibodies, as in PD compared to controls with 92% disease sensitivity well as β-syn and α-syn reveal unique hippocampal axonal and 58% specificity [72]. Elevated α-syn levels, however, pathology [62]. In vivo, γ-syn overexpression in trans- were foundin DLB, AD, andvasculardementia with no genic mice shows age- and dose-dependent neuronal loss differences among them [71]. Perhaps more intriguing, throughout the neuraxis, especially in spinal motor neurons, higher-molecular weight aggregated α-syn species in CSF where γ-syn-bearing inclusions, gliosis, and alterations in might be associated with PD and DLB. Reduced levels of heat shock protein and neurofilament structure are found a24kD α-syn-immunoreactive band were found in DLB [63], perhaps suggesting relevance to motor neuron disease CSF and correlated directly with declining cognition [73]. associated with dementia. In vitro evidence further supports Moreover, using a specific enzyme-linked immunosorbent a cytoskeletal role for γ-syn in maintaining neurofilament assay (ELISA), soluble aggregated α-syn oligomers in CSF structure; γ-syn overexpression in cultured neurons causes were significantly increased in PD patients compared against disruption of the neurofilament network by destabilizing the control subjects, AD and progressive supranuclear palsy, structural integrity of neurofilament-H allowing degradation and specificity ranged from approximately 85 to 87%, while by calcium-dependent proteases, which has implications for sensitivity was about 53–75% range [74]. neurodegeneration [64]. Plasma α-syn detected by immunoblotting was decreased in PD compared with age-matched control subjects, and those PD patients with age-at-onset prior to 55 years (early- 3.2. Synucleins as Biomarkers of LBD onset) had significantly lower levels than those with onset after 55 years of age (late-onset) [75]. In addition, soluble 3.2.1. Synucleins in the Extracellular Compartment. Synu- oligomeric α-syn detected by specific ELISA was significantly cleins are known as intracellular molecules, but they also elevated in plasma from PD. This test demonstrated a appear in extracellular and peripheral fluids from active specificity of approximately 85%, a sensitivity of 53%, and passive processes. Evidence suggests that turnover and and a positive predictive value of 0.818 [76]. Although secretion of these proteins might occur during normal measurement of plasma α-syn appears interesting as a cellular processing, releasing synucleins into extracellular biomarker, it was reported that skin cells and platelets are space and hence into peripheral sites. In transfected and un- also sources for α-syn, and their levels did not correlate transfected cultured neuroblastoma cells, 15 kDa α-syn is with disease presence or severity [77]. Moreover, red blood released into surrounding media [65], and furthermore, not cells are also a major source of α-syn [78], and thus, only monomeric α-syn but also aggregated forms are secreted plasma could be contaminated by α-syn not originating from in an unconventional exocytic manner into extracellular fluid brain, which might render interpretation of results difficult. in response to proteasomal and mitochondrial dysfunction One promising consideration for the future exploration [66]. Remarkably, Desplats et al. recently showed that of α-syn as an LBD biomarker will be the development neuronally secreted α-syn can also be taken in endocytically of novel imaging compounds and techniques, similar to by other neurons or glia as a means of transmitting pathology amyloid imaging, to specifically target and visualize α-syn [67]. Secreted α-syn interacts with various molecules such distribution in the PD and LBD brain. The availability of enzymes; in cultures, matrix metalloproteinase-3 cleaves such methods will be a significant advance in biomarkers for native α-syn to smaller proteolytic fragments that enhance synucleinopathies. its aggregative properties [68]. Whether β-syn and γ-syn also undergo unconventional exocytosis and secretion remains unknown, but given structural and functional similarity to 3.2.3. β-Syn and γ-Syn as Potential Biomarkers in Lewy α-syn, the possibility exists. Certainly, synaptic and axonal Body Disorders. Due to their increasing importance in LBD damage reflecting neurodegeneration may also allow release pathogenesis, β-syn and γ-syn, as much as α-syn, might of synucleins into the extracellular millieu and access to be excellent targets as peripheral markers of disease. As peripheral fluids such as CSF and blood. such, levels of these synucleins might be altered in the CSF of patients with PD/PDD and DLB, reflecting the 3.2.2. α-Synuclein as a PD and DLB Biomarker. Multiple underlying degenerative processes in brain. No studies to forms of α-syn are released into cerebrospinal fluid (CSF) date have examined β-syn levels in peripheral fluids in and other biological fluids. Full-length α-syn has been relation to neurodegenerative disease, but a small study recovered from lumbar CSF from living normal control, reported elevated postmortem ventricular CSF γ-syn levels 6 International Journal of Alzheimer’s Disease in DLB, AD, and vascular dementia patients, with the highest aggregation of α-syn and LB formation [80]or contributeto levels seen in DLB patients [71]. More detailed examination pathogenesis by other molecular pathways. of both β-syn and γ-syn as a peripheral disease markers DJ-1 is found in brain across a wide range of neurodegen- in well-characterized populations of PD, DLB, and other erative diseases including PD, FTD, AD, DLB, and LBVAD, disorders is warranted to determine their specificity and and demonstrates striking association with neuropil threads sensitivity in the synucleinopathies. and neurofibrillary pathology in neocortex and subcortical brain regions in these disorders [90]. Interestingly, this association with tau pathology was seen in DLB and LBV brains, suggesting that as a chaperone molecule, DJ-1 may 4. DJ-1 in the LewyBodyDisorders be involved in tangle formation, and the binding of DJ- 4.1. Functional Role of DJ-1 in Lewy Body Diseases. Recently, 1 with these lesions could abolish the normally protective DJ-1 (PARK 7) has emerged as a significant molecular target effect of DJ-1, enhancing oxidative neurotoxicity. Wang et al. of interest in LBD principally because of its genetic associa- observed that DJ-1 knockout mice have markedly abnormal tion with PD and its increasing importance in cellular oxida- hippocampal long-term depression accompanied by a less tive neuroprotection. Although its exact role is unknown, severe abnormality in long-term potentiation, which was multiple functions have been assigned to the DJ-1 protein. reversed by the D2/3 agonist quinpirole, indicating that DJ-1 Described by Nagakubo et al. as a mitogen-dependent has a role in dopamine-dependent signaling in hippocampal oncogene involved in Ras-related signaling pathways [79], plasticity [91]. This implies that DJ-1 may be important in it shares structural homology with the carboxy-terminal the maintenance of memory and cognition. domain of Escherichia coli HPII catalase and is reported to possess catalase activity which reduces oxidative stress in cultured cells [80]. It also binds to and regulates the PIAS 4.3. DJ-1 as a Potential Biomarker for Lewy Body Dis- SUMO-1 ligase and is itself posttranslationally modified by eases. Given its pathogenetic significance, DJ-1 could be a sumoylation [81, 82]. Of relevance to Lewy body formation candidate biological marker for DLB and LB and might and neurotoxicity, DJ-1 displays redox-dependent chaperone serve as a means of monitoring in vivo oxidative damage activity conferring proper protein folding and thermal sta- and protein misfolding. Although intracellular and mito- bility, which in fact, also inhibits α-syn aggregation [80]. The chondrial in localization, DJ-1 is presumed to be secreted overexpression of DJ-1 in rats protects nigral dopaminergic perhaps specifically under disease conditions which induce neurons against degeneration involving 6-hydroxydopamine, oxidative damage. Using semiquantitative immunoblotting, while mutant DJ-1 in mice causes abnormal dopamine we previously identified DJ-1 in CSF of sporadic PD patients, reuptake and susceptibility to 1-methyl- 4-phenyl-1,2,3,6- where levels were significantly elevated compared with tetrahydropyridine (MPTP) toxicity [83]. Deletion of DJ- controls. Levels were higher in the earlier stage PD cohort 1 homologs in Drosophila renders them sensitive to H O , (Hoehn-Yahr stages I-II) than in the more severe patients 2 2 paraquat, and rotenone toxicity [84]. (Hoehn-Yahr stages III-IV) [92]. Similarly, plasma DJ-1 levels in PD patients were markedly increased compared to controls, but unlike CSF, levels were relatively higher in late 4.2. DJ-1 Mutations and Possible Relevance to LBD. No less stage (III-IV) rather than early stage PD (I-II) [93]. The than 13 gene mutations have been identified in DJ-1 in reason for this difference between plasma and CSF DJ-1 is atypical younger-onset PD patients, but their significance to unknown, but we surmised previously that since CSF DJ-1 idiopathic late-onset PD remains uncertain. In autosomal originates from a central source produced mainly by reactive recessive early-onset PD from consanguineous families, a glia, early increases in CSF DJ-1 levels probably represent complete DJ-1 deletion in a Dutch family and a point an early protective response to damage, whereas plasma mutation L166P in an Italian case were identified [85]. DJ-1, like other plasma disease markers, likely represents When expressed in cultured cells, L166P appears to be a peripheral oxidative stress damage. In fact, DJ-1 is secreted loss-of-function mutation which leads to DJ-1 functional into blood in breast cancer, melanoma, familial amyloid instability, degradation by the proteasome system [86, 87], neuropathy, and stroke [94–96]. In the largest study to date, abnormal translocation of DJ-1 to mitochondria, and loss Hong et al. developed a more sensitive and quantitative of chaperone activity [80]. The importance of DJ-1 gene Luminex assay for CSF DJ-1 to complement immunoblotting alterations in dementia and DLB, however, is uncertain. One mass spectrometric and chromatographic analysis methods report found no impact on dementia risk of the DJ-1 14kb and found decreasing rather than increasing levels of DJ-1 deletion [88], and analysis of an insertion/deletion variant in PD CSF compared with control patients [72]. The 90% (g.168 185del) in DJ-1 in a larger sample of patients also disease sensitivity and 70% disease specificity for PD using showed no association with either PD or DLB compared to this method approaches minimal desired parameters for a control patients [89]. Given these early negative findings, the clinically useful biomarker for PD. Importantly, the study relevance of DJ-1 genetic mutations to DLB and other LBD is highlighted the fact that DJ-1 levels are greatly influenced not known. At present, no patient harboring a DJ-1 mutation by such variables as the extent of blood contamination and has come to autopsy, so the precise pathology is not known. patient age, which could account for some of the variability Although DJ-1 mutant cases may ultimately not be LBDs, it is across studies. Of note, DJ-1 is also subject to oxidative possible that alterations in DJ-1 may somehow influence the modifications in PD and AD brain tissue, and this might be International Journal of Alzheimer’s Disease 7 measured in peripheral fluids as well, as another monitor of mutation carriers [107]. These observations suggest a much oxidative damage [97]. CSF DJ-1 remains a promising and broader link between GBA mutations and the dementia perhaps clinically useful biomarker for PD, but as far as DLB phenotype of LBD. In fact, examination of GBA gene and other LBD, it is unknown whether CSF levels of DJ- alterations in DLB patients, with and without concomitant 1 are altered. Since plasma DJ-1 is increased in DLB, it is LBV-type AD pathology, showed that the majority of GBA hypothesized that CSF DJ-1 may also be elevated. Further mutationswere found in DLBpatientsratherthan inPD, investigation will be necessary to clarify the utility of DJ-1 with a mutation rate in DLB ranging from 18 to 23% as a biomarker in DLB and LBD. overall [108, 109]. The proportion of DLB patients with GBA mutations was higher in those with pure neocortical LB pathology compared to those with mixed LB and AD pathology and to those with predominantly brainstem LB. 5. Glucocerebrosidase as a Novel Biomarker for A significant association was also found between GBA Lewy Body Disorders mutation status and the presence of LB, indicating that 5.1. Glucocerebrosidase Mutations Influence PD and DLB. altered GBA might play a role in their formation and in Many clinicopathologic parallels can be drawn between synucleinopathy [108]. the lysosomal storage disorders, such as Niemann-Pick, Sandhoff’s, Tay-Sachs disease and others, and the age-related 5.2. Glucocerebrosidase and Chaperone-Mediated Autophagy neurodegenerative disorders, when considering the aberrant in LBD. Important in neurodegeneration, disrupted cellular accumulation of pathological substances (e.g., lysosomal proteostasis represents a state in which an imbalance exists sphingomyelin in Niemann-Pick disease versus synucleins between effective functioning of the innate cytoprotective in PD and DLB) and the phenotypes of neuronal loss machinery and excessive accumulation and aggregation of and cognitive deterioration found in both. Common to abnormally misfolded proteins, leading to neurotoxicity. It these diseases are abnormalities in lysosomal and autophagic is increasingly apparent that chaperone-mediated autophagy mechanisms as part of a larger disruption of cellular (CMA) and lysosomal degradation pathways are important proteostasis leading to abnormal storage/accumulation of in maintaining cellular proteostasis as part of a larger toxic materials and neuronal damage. In the past few network of cellular actions, with particular relevance for years, an altogether unexpected pathogenetic relationship neurodegenerative diseases. Recently, as evidence for CMA emerged between Gaucher’s disease (GD), a prototypic dysfunction in synucleinopathies, a significant decrease storage disease, and the synucleinopathies. Despite its overall in autophagy markers was reported in substantia nigra rarity, GD is the most common inherited lysosomal storage from PD brain [110]. Soluble forms of α-syn, including disease, especially in the Ashkenazi Jewish population. It is monomers, oligomers, and even protofibrils, are normally caused by autosomal recessive gene mutations in the gluco- cleared through the CMA/lysosomal degradation by inter- cerebrosidase (GBA) gene (chromosome 1q21), leading to acting with the chaperone, heat shock cognate-70, and either partial or complete deficiency of GBA, and hence, toxic becoming internalized into lysosomes via the Lamp-2a lysosomal accumulation of its substrate, glucosylceramide, in membrane receptor [111, 112]. Studies have indicated that multiple cell types including neurons [98]. Recent reports α-syn shares a common pentapeptide structure with other documented an increased incidence of PD in heterozygous lysosomal substrates, designating it as a target for removal relatives of patients with GD [99, 100], but interest in by this pathway [111], and the lysosomal structure is critical this phenomenon was propelled by the finding that GBA to maintaining the internal acidic environment, allowing mutations were in fact more common in PD patients of lysosomal hydrolases to degrade α-syn into peptides released Ashkenazi background compared with AD patients and PD into the cytosol [112]. Mutant GBA could therefore disrupt patients in the general population [101–103]. Moreover, lysosomal activity leading to abnormal accumulation of more severe GBA mutations such as 84 dupl G and IVS nondegraded α-syn, which then aggregates to toxic solu- 2 + 1 were associated with a greater degree of PD risk, ble oligomers and protofibrils. Also, abnormalities in the compared with less severe GBA mutations such as N370S ubiquitin-proteasome system (UPS) are present in AD and [104]. The relationship between PD and GBA has now been PD, and GBA alterations might secondarily overwhelm the replicated in much larger international studies with the most ability of UPS to remove accumulated α-syn, promoting common mutations being L444P and N370S, and about 28 aggregation and neurotoxicity [113]. Pathologically, in GD GBA mutations are presently recognized [105]. with parkinsonism, α-syn-positive inclusions were observed Interestingly, in a study of British patients with PD and in neurons in hippocampal CA2-4 regions, while cortical GBA mutations, all 17 carrier patients demonstrated abun- synuclein pathology was identified in other GD cases [114]. dant α-syn neuropathology with Braak stage 5-6 severity and Further, parkin, an E3 ubiquitin ligase also implicated common neocortical LB pathology. Clinically, these patients in PD, has been shown to affect the stability of mutant had earlier age at onset, and hallucinations were present GBA and increase its degradation causing further lysosomal in 45% of patients, while 48% had cognitive impairment dysfunction [115]. or dementia consistent with PDD [106]. Greater severity of GBA mutation also predicted the presence of cognitive impairment in PD patients; 56% of severe GBA mutation 5.3. Glucocerebrosidase as a LBD Biomarker. Because of carriers had cognitive impairment compared to 25% of mild the importance of mutant GBA function to PD and DLB 8 International Journal of Alzheimer’s Disease pathogenesis, the issue arises as to whether the measurement and IL-6 [123]. Because secreted CNS cytokines are readily of GBA activity, or a perhaps other related molecules, detected in CSF, they have been extensively examined as might be utilized as a biological marker. The activity of potential disease biomarkers. IL-1β,IL-2, IL-6, and TNF- peripherally secreted GBA was measured in plasma and α are all upregulated in PD brain, as well as in CSF CSF in a 10-month-old female with GD with the aim of from PD patients [124–126], and Chen et al. showed that monitoring the effect of experimental Cerezyme replacement plasma IL-6, but not IL-1β,TNF-α,or other acute phase therapy [116]. Baseline GBA activity was detected in both reactants, predicted risk for future PD in males [127]. In −6 −6 plasma (2.7 × 10 U/μL) and CSF (0.096 × 10 U/μL), terms of DLB, CSF IL-1β levels, which were relatively low, although CSF activity was several magnitudes lower than did not differ compared to AD or normal controls and plasma. Intravenous Cerezyme, a macrophage-targeted GBA, could not distinguish them apart. Comparable increases in rapidly raised the plasma activity within 1 hour and CSF CSF IL-6 levels were found in AD and DLB, but again not activity by 2.3-fold at 3 hours, both returning to baseline significantly different from each other to be of diagnostic after 24 hours. This study suggests the intriguing possibility value [128]. Indeed, the neuroinflammatory cytokines may that GBA activity, especially in CSF and plasma, might be be important as a pathogenetic response to CNS injury useful in monitoring the efficacy of novel therapies involving caused by accumulation of amyloidogenic proteins, but their CMA and lysosomal function. To extend this observation, role as biomarkers for the LBD, especially for DLB, is still Balducci et al. determined that multiple lysosomal hydro- unclear. lases, including GBA, are significantly decreased in the lumbar CSF of PD patients [117], perhaps supporting a 6.2. Neurofilament Proteins. Disorganization and breakdown more widespread lysosomal dysfunction in PD not limited in the cytoskeletal network occurs in various LBDs and to GBA alone. In this regard, other lysosomal enzymes such other neurodegenerative diseases, and as discussed, gamma- as mannosidase and β-hexosaminidase might be important synuclein and proteolytic degradation of the cytoskeleton additional biomarker targets for neurodegeneration. More- may be involved. As a result, a failure of normal axonal over, in DLB, AD, and FTD patients, lysosomal enzyme transport results from the accumulation of disrupted neu- activities in CSF demonstrated a very specific pattern of rofilament molecules within the neuropil, causing neuronal decrease, in which only DLB showed significant decreases demise [129]. Recently, a mutation in the NEFM gene encod- in CSF activity of α-mannosidase, β-mannosidase, GBA, ing the rod domain 2B of neurofilament M (NF-M) which galactosidase, and β-hexosaminidase, whereas in AD and causes aberrant NF assembly was identified in a single early- FTD, only CSF α-mannosidase activity was significantly onset PD patient [130]. It is recognized that in addition to diminished [118]. In DLB, CSF GBA activity showed the α-syn, three types of NF protein also comprise the structure greatest magnitude of decrease, reinforcing its importance of Lewy bodies [131]. Upon cell death or axonal damage, in the LBD, but also noteworthy is the fact that AD and accumulated neurofilament leaks into the extracellular space, FTD showed decreased α-mannosidase activity, suggesting subsequently appearing in CSF and perhaps other peripheral that this might be another important factor in lysosomal fluids. Elevated CSF NF protein was reported in MSA and dysfunction in neurodegeneration. Indeed, these promising PSP, but not in PD, and this was suggested to clinically candidates need to be investigated further to establish diag- aid in differentiating parkinsonian syndromes [132]. CSF nostic accuracy in terms of disease specificity and sensitivity NF protein was also measured in dementia, and although in cohorts of PD, DLB, and other dementing disorders. increased levels were observed in DLB, late-onset AD, and FTD, there were no differences among them [28]. Therefore, because cytoskeletal abnormalities are present in many 6. Miscellanous Candidate Biomarkers neurodegenerative dementias as well as in PD, NF protein may be more a reflection of nonspecific alterations in 6.1. Inflammatory Cytokines. Polymorphisms in proinflam- neuronal and axonal function, which does not appear to able matory cytokine genes including IL-1α,IL-1β,and TNF- to clinically separate DLB from other disorders. α are associated with increased risk in AD [119]. In PD, several case control genetic analyses have demonstrated that homozygous carriers of the IL-1β−511 and TNF-α−308 6.3. Brain Neurotransmitter Alterations in CSF and by Imaging promoter region variants have increased disease risk [120, Modalities. Severe cortical cholinergic deficits originating 121], and that earlier age at onset in PD was associated from deficiencies in the nucleus basalis of Meynert are with IL-1β−511 homozygosity at allele 1 [122]. But as characteristic of AD brain, but studies have shown that yet, no such genetic alterations in cytokines genes have cholinergic deficits are perhaps more severe in DLB brain been reported in DLB. Similar to Aβ-induced upregulation [5]. This suggests that measurement of cholinergic activity of inflammatory cytokines in AD, soluble secreted α-syn and/or acetylcholine (ACh) might be developed into a in the extracellular space in LBD might also induce the potential biomarker for the LBDs. Indeed, early attempts to production of a variety of neuroinflammatory mediators into quantify ACh or its major metabolite, choline, have shown the extracellular fluid. For instance, microglial activation in baseline levels to be low and perhaps difficult to measure response to stimulation by secreted α-syn from cultured cells accurately. In AD, CSF ACh was reported to be significantly and from overexpression in transgenic mouse models occurs lower than control levels [133], while in PD and Huntington’s in a dose-dependent manner, causing release TNF-α,IL-1β, disease patients, despite some cholinergic deficit, lumbar CSF International Journal of Alzheimer’s Disease 9 ACh and choline levels did not differ from normal [134]. No In the last decade, a series of Japanese studies consistently studies have directly examined CSF cholinergic levels in DLB demonstrated delayed heart to mediastinum ratio (H/M) of or LBDs, but recently, Shimada and colleagues employed I-MIBG uptake in DLB compared with AD and controls positron emission tomography (PET) mapping of brain ACh [143–146]. I-MIBG scintigraphy was found superior to activity in DLB and PDD patients and normal controls and brain perfusion SPECT imaging [147]. Estorch et al. further demonstrated a marked reduction in cholinergic activity showed that in dementia patients followed for four years in medial occipital cortex of DLB and PDD, greater than before “final diagnosis,” I-MIBG imaging distinguished that observed in PD patients without dementia [135]. Some DLB from other dementias with a sensitivity of 94%, correlation of mapped cholinergic activity with cognitive specificity of 96%, and a diagnostic accuracy of 95% [148]. decline measured by the Mini-Mental State Exam was also Finally, consistent with autonomic dysfunction in DLB, both found. Although preliminary, this has potential to be a more early and delayed H/M I-MIBG uptake were significantly practical and sensitive cholinergic biomarker for LBD. associated with the presence of orthostatic hypotension in Because of similar nigrostriatal loss to PD, a relative DLB patients and discriminated DLB from AD even in the dopaminergic deficiency also exists in DLB and LBDs. CSF absence of parkinsonism [149]. dopamine (DA) and its metabolites have been investigated previously in PD, and recently, Lunardi et al. showed differences in CSF DA and its metabolites, homovanillic 6.4.2. Other Structural and Functional Imaging Biomarkers. acid (HVA) and dihydroxyphenylacetic acid (DOPAC), in Various magnetic resonance (MR) imaging modalities have PD patients, demonstrating early-stage dopaminergic loss been explored in DLB and PDD, including volumetric and a correlation with the development of dyskinesia [136]. imaging, diffusion tensor imaging, and proton magnetic In DLB, HVA levels were significantly reduced compared resonance spectroscopy (reviewed in Watson et al.) [150], with AD, separating the disorders [137]. Similar to cholin- and although not directly useful as biomarkers at present, ergic activity, imaging modalities may also contribute to they have revealed insights in the pathobiology of LBDs. the assessment of dopaminergic function in the LBDs. In Using conventional MRI techniques such as voxel-based a small study, striatal DA uptake as measured by F- morphometry and region of interest analysis, some degree fluorodopa PET was decreased in both caudate and putamen of diffusion or focal frontal and parietal atrophy has been in DLB as compared with AD patients and controls [138]. observed [151]. Atrophy has been rated at 1.4% per year Also, DA transporter loss was determined across multi- in DLB brain [152], 1.31% per year in PDD, and 0.31% ple studies using I-2β-carbometoxy-3β-(4-iodophenyl)- per year in PD [153]. Not surprising is the fact that unlike N-(3-fluoropropyl) nortropane ligand with single-photon AD brain, medial temporal structures are relatively preserved emission computed tomography ( I-FP CIT SPECT) and in DLB and PDD, with global hippocampal loss at about demonstrated significant loss of caudate and putaminal 10–20% compared with controls and about 21–25% in AD DA transport compared with AD and control levels [139– [154]. Diffusion tensor imaging, an MR technique mapping 141]. A larger phase III, multicenter study of I-FP CIT brain microdiffusion of water in the direction of white matter SPECT in possible and probable DLB patients and non-DLB tracts, has shown decreased fractional anisotropy of water comparators (mostly AD) demonstrated a mean sensitivity movement in DLB in the precuneus and posterior cingulate of 77.7% for detecting clinically probable DLB, with a areas, perhaps highlighting their role in DLB pathogenesis specificity of 90.4% and 85.7% overall diagnostic accuracy [155]. 123 99m [141]. I-FP CIT SPECT DA transporter imaging greatly Brain perfusion SPECT ( Tc-HMPAO SPECT) has enhanced diagnostic accuracy for DLB over clinical diagnosis been evaluated in its ability to diagnostically separate DLB alone when coupled with autopsy confirmation, raising from AD, and in AD, reduced relative cerebral blood sensitivity for DLB from 75% to 88% and specificity from flow (rCBF) in the frontal, and medial temporal regions 42% to 100% [139]. Furthermore, DA transporter loss in is characteristic, whereas in DLB, occipital hypoperfusion the caudate may also be inversely associated with depression, is often observed [156]. Colloby et al. applied statistical apathy, and delusions in DLB patients [142]. parametric mapping to SPECT imaging of DLB patients, more precisely showing large perfusion deficits in the left medial occipital gyrus and the bilateral central, inferior parietal, precuneate, superior frontal and cingulate regions 6.4. Miscellaneous Imaging Biomarkers in LBD on the brain, which are functionally consistent with frontal- 6.4.1. MIBG Scintigraphy as a DLB Biomarker. Autonomic executive and visuospatial deficits in DLB [157]. Across failure is a common clinical finding in LBD, including PD studies, sensitivity ranged from 65 to 85% and specificity and DLB, but not in non-LBD dementias, and therefore from 85–87%, which appears less robust as a potential it has been investigated as an alternative biomarker for imaging marker compared with other methods. the diagnostic separation of DLB from other dementias. Abnormal autonomic function can be determined using car- 123 123 diac I-meta-iodobenzyl guanidine ( I-MIBG) imaging, a 6.5. Other PD Genes and Their Protein Products as Possible technique which assesses cardiac sympathetic nerve function DLB Markers. Aside from α-syn and DJ-1, numerous other in both cardiac and neurological disorders by measuring mutations have been associated with familial early-onset PD the uptake of I-MIBG, a norepinephrine analogue [143]. and possibly LBD (Table 1). Among these gene products 10 International Journal of Alzheimer’s Disease are parkin (PARK 2), UCHL-1 (PARK 5), PINK1 (PTEN- the relationship among LRRK2, α-syn, and tau in PD, DLB, induced putative kinase 1; PARK 6), and LRRK2/dardarin and other LBD is also influenced by population differences. (PARK 8) [158]. Indeed, none of these mutations have These findings make LRRK2/dardarin an attractive candidate yet been associated with prototypic LBD pathology, and it for examination as a potential biomarker, and if identified in remains to be determined whether they actually represent CSF or peripheral fluids, they might be used with α-syn and LBDs or separate diseases with parkinsonian phenotype. tau as combined biomarkers. Furthermore, no studies have addressed their role as bio- Furthermore, emerging evidence is redefining the roles logical markers of disease, but since both synucleins and of PINK1 and parkin in PD pathogenesis. Because energy DJ-1 are detected in CSF and peripheral fluids, it seems generation is critical for cellular function, mammalian cells plausible that the protein products of other dominant genes are highly dependent on mitochondria [168]. Depolariza- in PD could be peripheral biomarker candidates for DLB tion and morphological defects characterize damaged or and other LBD. Parkin, UCHL-1, and PINK1 genes, like impaired mitochondria which are targeted for removal DJ-1, all encode proteins important in neuroprotection in through mitophagy, a highly specialized form of autophagy terms of maintaining protein homeostasis and preventing in which parkin and PINK1 play a crucial role (reviewed by stress-related cellular damage, and mutations in these genes Vives-Bauza and Przedborski) [169]. In this process, PINK1 cause a loss of these critical functions. Leucine-rich repeat cleavage is inhibited by the loss of mitochondrial membrane kinase 2 (LRRK2/dardarin), on the contrary, is linked with potential, causing its lengthening and the recruitment of autosomal-dominant late-onset PD, and mutations result in cytosolic parkin [170, 171]. Voltage-dependent anion chan- a toxic gain of function. nel 1 and other outer mitochondrial membrane proteins LRRK2/dardarin is a kinase consisting of multiple func- are then ubiquitinated in a parkin-dependent manner, and tional domains, and recent evidence suggests that physio- this in turn recruits the binding of adapter proteins such logically, its principal function may be to regulate neurite as p62 and histone deacetylase 6 to initiate autophago- outgrowth. Expression in cultured neurons of several LRRK2 some assembly around the damaged mitochondrion and mutations associated with familial PD, such as G2019S, subsequent removal [169]. Of relevance to PD, mutant increased kinase activity and significantly reduced neurite PINK1 and mutant parkin both cause motor dysfunction, outgrowth, whereas expression of a dominant-negative dopaminergic loss, and abnormal mitochondrial morphol- mutation, K1906M, markedly increased neurite length [159]. ogy in Drosophila [172]. In this paradigm, loss of function PD-associated mutations also generated tau-positive axonal PINK1 mutants are rescued by concurrent overexpression inclusions in cultured neurons, suggesting that LRRK2 may with wild-type parkin but not vice versa, indicating that be linked to abnormalities in tau. Indeed, expression of parkin specifically acts downstream of PINK1. Also, parkin mutant G2019S LRRK2 in Drosophila caused activation mutations have been shown to interfere with ubiquitination of the Drosophila GSK-3β homolog and promoted tau and the downstream steps in normal mitophagy [173]. Thus, hyperphosphorylation leading to microtubule fragmentation PD, and possibly related dementias, might be a result, to and dendritic pathology [160]. Similar tau hyperphosphory- some extent, of defective mitophagy due to loss of function lation was also present in transgenic mice expressing G2019S in PINK1 and parkin such as found in autosomal dominant LRRK2, and expression of both wild-type human LRRK2 early-onset PD. and G2019S mutant LRRK2 caused abnormal dopaminergic Although LRRK2, parkin, PINK1, and UCHL-1 have transmission [161]. LRRK2 may also interact with α-syn, not yet been identified in peripheral fluids, PINK1 and another dominantly inherited PD gene, to exert its effect. parkin may be a promising candidates. Unexpectedly, both Lin et al. showed that overexpression of LRRK2 with A53T PINK1 and parkin, which are normally cytosolic or tar- mutant α-syn in transgenic mice worsened neurodegenera- geted to mitochondria, were localized extracellularly in AD tion, while ablation of LRRK2 expression suppressed α-syn and multiple sclerosis brain, and colocalized with amyloid aggregation and pathology [162], and α-syn also activates plaques, reactive astrocytes, as well as amyloid-affected GSK-3β in mice causing tau hyperphosphorylation [163], vessels [174, 175]. This suggests that both PINK1 and parkin indicating that LRRK2, α-syn, and tau alterations may all be are actively released from neurons and glia in response to linked in the same pathway, perhaps with LRRK2 upstream injury and might be upregulated in CSF and peripheral fluids of these events. Although early, evidence has indicated that during neurodegeneration. Interestingly, given a role in LRRK2 is also a component of LB in PD and DLB brains mitophagy, they might also be a CSF or peripheral reflection [164], and that LRRK2 and α-syn interact in DLB brain of mitochondrial health and turnover. It remains to be seen and coimmunoprecipitate in cultured cells after oxidative whether these gene products can be detected in biological stress challenge [165], suggesting that the LRRK2 may also be fluids such as CSF as potential biomarkers in PD and LBD. important in DLB pathogenesis. Interestingly, genome-wide association studies (GWASs) in a European cohort demon- strated that LRRK2, α-syn, and tau are loci associated with 7. Unbiased Methods in PD risk [166], but examination of tau in a Japanese GWAS LBD Biomarker Discovery cohort failed to identify it as a PD risk locus [167], showing a population difference with regard to this locus. Certainly, 7.1. Genomics in PD and LBDs. As detailed above, traditional population differences might apply to all risk loci examined methods for molecular biomarker determination have been forPD and LBD, and itisimportant to determinewhether derived from targeted analyses of candidate genes/mutations International Journal of Alzheimer’s Disease 11 and corresponding proteins in brain and body fluids such as 156 candidate proteins involved in ubiquitin-proteasome CSF and blood, with the subsequent exploration of mecha- system and synaptic function, from which the heat shock nisms in cell culture and animal models. An emerging alter- cognate-71, a chaperone involved in neurodegenerative nate approach has been to evaluate genomes and proteomes disease, was identified and validated as a candidate target with regard to specific neurodegenerative diseases and their [182]. Abdi and colleagues carried out proteomic evaluation components in an unbiased manner to yield a number of of CSF from AD, PD, and DLB patients and normal control potential pathogenetic, therapeutic, and biomarker targets individuals, using chromatography, MS, and isobaric tagging for further validation. With regard to the genomic analysis for relative and absolute quantification (iTRAQ), identifying of the LBDs, gene expression profiling has proved to be numerous candidate proteins related to PD and DLB, such as a promising tool. Scherzer et al., for instance, examined lipoproteins ApoC1 and ApoH [183]. Lastly, using surface- transgenic Drosophila expressing the human α-syn gene and enhanced laser desorption/ionization-time of flight (SELDI- performed temporal profiling of resultant gene expression TOF) MS analysis of serum from DLB patients compared [176]. They demonstrated a number of changes, including a to AD, a combination of protein peaks provided the ability downregulation of phospholipase A2 and other lipid genes, to separate DLB from non-DLB cases, with a sensitivity of downregulation of several mitochondrial respiratory chain 83.3% and a specificity of 95.8% [184]. Given promising molecules, and alteration in membrane transport and energy findings, further exploration of the proteomics of the LBDs genes such as voltage-gated calcium channel and lysosomal is warranted, and perhaps consideration should be given ATPase, suggesting that mitochondrial integrity might be to determining whether combining various genomic and affected by α-syn overexpression. proteomic methods will be of value. In Parkinson’s disease brain, RNA from populations of mesencephalic dopaminergic neurons with and without 8. Conclusions LB were isolated by immunolaser capture microdissection, amplified by polymerase chain reaction and expressed [177]. Over the last decade, tremendous advances have been Interestingly, upregulation of the ubiquitin-specific protease made in understanding the pathogenetics of PD, PDD, 8 in LB-containing neurons indicated cellular damage and and DLB, which has revealed not only the genetic basis increased levels of ubiquitination in LB, whereas non- of these disorders, but also related mechanisms common LB-bearing neurons showed increased expression of novel to all the LBD. In parallel, these discoveries have been a cytoprotective genes such as bullous pemphigoid antigen 1, catalyst for translating and developing many of the involved an HSP-70-like gene (STCH) and Kelch-like 1. Although proteins into promising biomarkers for disease. A common promising, further genomic profiling studies in DLB, PDD, theme centers on genes that drive a complex network of and other LBD are needed to expand the range of novel gene synergistic and opposing cellular actions underlying path- targets for examination and validation. ogenesis. Aggregation of α-syn, the main constituent of intracellular LBs, results in toxic oligomers and protofibrils 7.2. Proteomic Profiling in PD and LBDs. As a complement to which not only act intracellularly, but also are actively and gene expression profiling and genomic methods, proteomic passively released into the extracellular environment causing profiling has also assumed a greater importance in biomarker damage to surrounding tissue. Proinflammatory cytokines discovery for neurodegeneration with relevance to the LBD. such as interleukins are also produced which perpetuates Advances in methodologies such as 2-dimensional gel elec- the inflammatory cascade. On the contrary, DJ-1, PINK1, trophoresis (2-D GE), liquid chromatography (LC), high- parkin, and perhaps others molecules are upregulated to resolution mass spectrometry (MS), and quantitative pro- oppose cellular protein misfolding and oxidative stress and teomics allow analysis of static or condition-dependent pro- maintain mitochondrial function, while autophagy mech- tein structure and function associated with PD and LBD in a anisms attempt to limit the toxic effect of synucleins and variety of sample types such as brain or body fluids (reviewed other toxins by lysosomal engulfment and digestion. Much in Shi et al. 2009) [178]. In mice treated with MPTP, a specific of this is reminiscent of a relatively new concept applied mitochondrial toxin, isotope-coded affinity tag assay of brain to infectious diseases and mechanical tissue injury termed tissue followed by MS analysis revealed 100 proteins with “damage-associated molecular patterning” (DAMP), which significantly altered levels including many mitochondrial and is an evolved system to recognize, contain, and repair damage metabolic molecules, βAPP and DJ-1 [179]. to cells and tissues. It is characterized by the abnormal Basso et al. first examined the proteome of the substantia release of molecules normally confined and operating within nigra from Parkinson’s disease brain and age-matched healthy cells or from foreign pathogenic agents, that when controls [180]. Using 2D GE and peptide fingerprinting, of released into the extracellular space activate receptors and the 44 expressed proteins, 9 proteins differed in PD versus pathways leading to inflammation and multiplying cellular controls, including oxidative and mitochondrial proteins damage (reviewed by Bianchi) [185]. In this regard, events such as peroxiredoxin II, mitochondrial complex III, calcium in the pathogenesis of PD, DLB, and related disorders may channel, and others. A subsequent study in PD brain showed represent a novel variation of the DAMP response, and in a decreased frontal cortex levels of mortalin, a novel mito- sense, biological fluid markers are therefore a measurement chondrial chaperone protein with roles in energy generation of DAMP activity as it relates to neurodegeneration. [181]. 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Bridging Molecular Genetics and Biomarkers in Lewy Body and Related Disorders

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Copyright © 2011 Gilbert J. Ho et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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SAGE-Hindawi Access to Research International Journal of Alzheimer’s Disease Volume 2011, Article ID 842475, 18 pages doi:10.4061/2011/842475 Review Article Bridging Molecular Genetics and Biomarkers in Lewy Body and Related Disorders 1, 2 1, 2 3 3 Gilbert J. Ho, Willie Liang, Masaaki Waragai, Kazunari Sekiyama, 1 3 Eliezer Masliah, and Makoto Hashimoto Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093-0624, USA The Center for Memory and Aging, Poway, CA 92064, USA Laboratory for Chemistry and Metabolism, Tokyo Metropolitan Institute for Neuroscience, Tokyo 183-8526, Japan Correspondence should be addressed to Gilbert J. Ho, giho@ucsd.edu Received 30 December 2010; Accepted 20 April 2011 Academic Editor: G. B. Frisoni Copyright © 2011 Gilbert J. Ho et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Recent advances have been made in defining the genetic and molecular basis of dementia with Lewy bodies (DLBs) and related neurodegenerative disorders such as Parkinson’s disease (PD) and Parkinson’s disease dementia (PDD) which comprise the spectrum of “Lewy body disorders” (LBDs). The genetic alterations and underlying disease mechanisms in the LBD overlap substantially, suggesting common disease mechanisms. As with the other neurodegenerative dementias, early diagnosis in LBD or even identification prior to symptom onset is key to developing effective therapeutic strategies, but this is dependent upon the development of robust, specific, and sensitive biomarkers as diagnostic tools and therapeutic endpoints. Recently identified mutations in the synucleins and other relevant genes in PD and DLB as well as related biomolecular pathways suggest candidate markers from biological fluids and imaging modalities that reflect the underlying disease mechanisms. In this context, several promising biomarkers for the LBD have already been identified and examined, while other intriguing possible candidates have recently emerged. Challenges remain in defining their correlation with pathological processes and their ability to detect DLB and related disorders, and perhaps a combined array of biomarkers may be needed to distinguish various LBDs. 1. Introduction of AD tau pathology, further complicating the diagnosis [2]. Approximately 20–40% of Parkinson’s disease (PD) Over the past decade, dementia with Lewy bodies (DLBs) patients also eventually develop a progressive dementing has arguably become the second most common form illness designated as Parkinson’s disease dementia (PDD) of neurodegenerative dementia behind Alzheimer’s disease characterized by a frontal-subcortical clinical presentation (AD). In addition to progressive decline in cognition, [3]. DLB/Diffuse Lewy body disease (DLBD), Lewy body DLB is characterized by fluctuations in cognition with variant of AD (LBV), and PDD comprise an emerging variations in attention and alertness, recurrent formed visual spectrum of clinical phenotypes from relatively pure motor hallucinations, visuospatial dysfunction, and spontaneous PD to the more predominant cognitive and behavioral disturbance observed in PDD and DLB, yet the reason for parkinsonism. Often, DLB patients also exhibit neuroleptic sensitivity, transient loss of consciousness, falls, and rapid eye the variability remains unknown. Despite the heterogeneity movement (REM-) sleep behavior disorder [1]. The clinical of their clinical phenotypes, a significant neuropathological overlap is observed among these diseases, hence the term separation of DLB from other similar disorders is often difficult resulting in poor diagnostic accuracy, but the relative “Lewy body disorders” (LBDs) to collectively describe con- temporal co-occurrence of parkinsonian features with the ditions in which Lewy bodies (LBs) and Lewy neurites (LNs) typical DLB cognitive and behavioral symptoms such as predominate as the hallmark histological lesions. Variation visuospatial disturbance strongly suggests the diagnosis. in the distribution of Lewy body pathology is present among Clinical presentation of DLB is also influenced by the amount LBDs, with more neocortical and limbic system LB in both 2 International Journal of Alzheimer’s Disease DLB and PDD compared to the brains of PD patients while the α-secretase pathway precludes Aβ formation by without cognitive symptoms and greater neuronal loss in cleaving βAPP at a site within the Aβ sequence. Genetic substantia nigra in PDD than DLB [4]. Yet, DLB and overlap analysis of early-onset familial AD cases revealed numerous disorders such as LBV, more so than PDD, have β-amyloid mutations in the βAPP gene as well as presenilin 1 (PS1; pathology and basal forebrain cholinergic deficit similar to chromosome 14) and presenilin 2 (PS2; chromosome 1) AD patients [5]. genes, all of which accelerate the processing of βAPP, leading As with other forms of dementia, the pathobiological to increased Aβ generation [12]. Specifically, the βAPP changes in LBD likely occur decades prior to the onset of KM670/671NL (Swedish) mutation affects the β-secretase clinical symptoms and correspond to widespread irreversible site, A692G (Flemish) mutation alters the α-secretase site, neurodegeneration [6, 7]. It is increasingly clear from the and both V717F (Indiana) and V717I (London) mutations AD therapeutic experience that by the time widespread affect the γ-secretase processing, leading to elevated Aβ neuronal injury ensues, symptomatic cholinergic treatments levels. Also important in the Notch developmental signaling are minimally effective at best, and disease-modifying thera- pathway which is analogous to βAPP processing, the pre- peutic approaches in trials have thus far proven ineffective senilins are thought to be a component of the γ-secretase at altering disease course or in rescuing diseased brain [8, enzyme complex, which suggests that missense mutations 9]. To demonstrate efficacy, any potential disease-modifying in the presenilins mechanistically lead to accelerated βAPP therapy in neurodegenerative dementia must be initiated processing to Aβ [13]. prior to the expression of the clinical phenotype during the Therefore, the abnormal proteolytic cleavage of βAPP initial molecular pathogenetic events before irreversible neu- leads to elevated brain Aβ deposition, and as a result, ronal damage has occurred. At present, accurately predicting diminished peripheral levels of Aβ. Reflecting a shift from those individuals at risk for developing neurodegenerative soluble Aβ to insoluble brain deposits, significant decreases dementia is challenging, and this places greater urgency on in CSF Aβ42 levels have been demonstrated in AD and developing earlier methods of disease detection. Critically more recently in DLB cases [14]. Parnetti et al. found that important is not only distinguishing the LBD from AD and DLB, compared with PD, PDD, and AD patients, showed the other forms of dementia, but also separating DLBs and other lowest CSF levels of Aβ42 and, when combined with CSF tau, LBDs. Although there are many promising candidates for differentiated DLB from PD and PDD, but not from AD [15]. LBD, no biomarkers have yet been validated for clinical Also, Spies and colleagues showed a greater decrease in Aβ40 diagnostic use, and thus many opportunities exist to develop in clinical DLB and vascular dementia patients compared such tests. Here, we highlight from the perspective of how with control levels and even with AD. Differentiation of major genetic discoveries in the LBD and their corresponding non-AD dementias such as vascular dementia and DLB biomolecular processes might translate into useful disease was improved by comparing the ratio of Aβ42 and Aβ40 markers in biological fluids. Because of many common [16]. More recently, the detection of amyloid in dementia pathogenetic features among the LBDs, the emerging genetic patients has been greatly enhanced by the use of amyloid- influences found in PD have readily been translated to DLBs binding agents such as Pittsburgh compound B [17], which and other LBDs, providing clues to rational approaches also demonstrated amyloid burden in DLB. An Australian for selecting future DLB and LBD biomarker targets for study reported more variable cortical PiB binding in DLB exploration. This paper will highlight several PD and DLB patients than in AD [18], whereas a subsequent examination genes and their protein products as candidates for biological of PiB binding in LBDs including DLB, PDD, and PD, disease markers (Table 1). compared with AD and normal patients, showed higher amyloid burden inDLB and ADthaninPDD, PD, or NC patients [19]. Amyloid load was highest in LBD patients in the parietal and posterior cingulate regions, corresponding 2. Amyloid and Tau in Lewy Body Disorders to visuospatial impairments on neuropsychological testing, 2.1. Amyloid Genetics and Biomarkers in LBD. Aβ,akey suggesting that amyloid deposition could partly contribute component of neuritic plaques in AD brain, is overproduced to the clinical presentation of LBDs. leading to various degrees of amyloid aggregation and synaptic and neuronal toxicity [10]. As indicated previously, amyloid pathology in the form of neuritic and diffuse 2.2. Tau Genetics and Biomarkers in LBD. Mutations in plaques can be also found in varying degrees in the brain the tau gene on chromosome 17 may also present with tissue of patients with DLB, which may interact with LB phenotypic features of PDD or DLB, but they differ patho- or synuclein pathology or influence the clinical features of logically from these disorders in that LBs are generally absent LBDs [4, 11]. The genetic mechanisms of Aβ overproduction [20]. Tau-bearing neurofibrillary tangles remain one of the in AD are well established; the βAPP gene (chromosome pathological hallmarks of AD but are also central to a diverse 21), the first identified AD susceptibility gene, encodes a group of disorders termed “tauopathies” which include pro- transmembranous protein ranging from 695 to 770 residues, gressive supranuclear palsy, corticobasal ganglionic degener- which undergoes a process of regulated intramembranous ation, frontotemporal dementia (FTD) with parkinsonism proteolysis ultimately releasing Aβ peptides, primarily Aβ42 linked to chromosome 17, and other disorders [21]. Tau and Aβ40, as well as other fragments. Aβ is generated by is a microtubule binding protein, which acts to stabilize the concerted action of β-secretase and γ-secretase complex, tubulin polymerization in microtubules critical for axonal International Journal of Alzheimer’s Disease 3 Table 1: Genetics and biomarkers in LBD. Biochemical Source of Gene defect Relevance to LB disorders marker biomarker APP: K670M/N671L and so forth. Aβ PS1: H163R and so forth PS2: Deposited in plaques CSF, plasma AD lesions N141I and so forth Tauopathy: P301L, N279K, K317M, Found in NFT in AD brain, released after Tau CSF and so forth neuronal damage Mutation →↑ α-syn aggregation. α-synuclein A53T, A30P (PD), G209A (DLB), CSF, skin LB component, toxic oligomers and (PARK1/4) E46K, triplication (PD & DLB) cells, platelets protofibrils PD/DLB Inhibit α-syn aggregation: mutant causes β-synuclein P123H, V70M (DLB) CSF lesions degeneration Amyloidogenic: affects neuronal and Ventricular γ-synuclein SNP in DLBD axonal cytoskeleton CSF K161N, W453Stop, 202-203delAG, Ubiquitin E3 ligase, LOF mutation in PD Parkin (PARK 2) ND M192L, K211N, and so forth alters mitophagy Neuronal deubiquitinating hydrolase; UCHL-1 I93M, S18Y (SNP) impaired synaptic and cognitive function ND Proteostasis/ (PARK 5) in AD & PD oxidative A168P, A217D, E417G, E240K, and Mitochondrial serine/threonine kinase; stress PINK 1 (PARK 6) ND so forth (PD) LOF mutation in PD alters mitophagy M26I, D149A, G78G, R98Q (PD), Redox-dependent chaperone; LOF DJ-1 (PARK7) CSF, plasma L166P (PD & DLB) mutation in PD Gain of function mutant in PD?DLB: G2019S, duplication, triplication LRRK2 (PARK 8) interacts with α-syn and tau, and with ND (PD) parkin in apoptotic cell death Disrupted NF → abnormal axonal Cytoskeletal NF NEFM (PD) CSF transport; released in cell damage Lysosomal 84 dupl G, IVS 2 + 1, N370S, L444P Gaucher’s disease, abnormal lysosomal GBA CSF, plasma dysfunction (PD) function/autophagy in PD α-syn-induced microglial activation →↑ IL-1α,IL-1β, Inflammation SNP: IL-1β−511,TNF-α−308 secretion of neuroinflammatory CSF IL-6, TNFα mediators CSF: cerebrospinal fluid; GBA: glucocerebrosidase; Aβ: β-amyloid; NF: neurofilament; ND: not yet determined; PD: Parkinson’s disease; DLB: Dementia with Lewy body; UCHL1: ubiquitin carboxy terminal hydrolase L1; PINK 1: PTEN-induced putative kinase 1; LRRK2: leucine-rich repeat kinase 2; LOF: loss of function; SNP: single nucleotide polymorphism. cytoskeletal integrity and function. In disease, tau protein Consequently, both total tau and hyperphosphorylated truncation at Glu 391 or hyperphosphorylation causes forms have been widely investigated and detected in CSF, microtubule destabilization and aggregation of unbound tau but not serum, by enzyme-linked immunosorbent assay into paired helical filaments (PHFs) leading to characteristic methods. In the differentiation of dementia types, Arai et al. tangle formations [22]. Unlike the tauopathies, no direct initially reported elevated total CSF tau levels in AD but not pathogenetic tau mutations have been identified in LBDs, in PD, but subsequently, they showed that total tau was also but tau pathology appears to be a consistent feature among increased in DLB at similar levels to AD [24]. Yet, others have neurodegenerative dementias including AD and LBDs, and found differences for both total and phospho-tau (p-tau) in given the pathological overlap, they might share similar differentiating DLB from AD [25], and levels of total tau and pathogenetic pathways (reviewed in Stoothoff and Johnson) p-tau 181 were significantly increased in autopsy-confirmed [23]. The Ser/Thr kinase and glycogen synthase kinase- DLB patients [26]. In clinically diagnosed dementia cases, 3β (GSK3β), in concert with other molecules such as fyn CSF p-tau 231 discriminated AD from non-AD dementias kinase, normally regulate tau function but with aberrant as a group, where levels were significantly higher in AD activation accelerate the hyperphosphorylation of tau in patients compared with DLB, FTD, vascular dementia, other neurodegenerative disease. Similarly, the cell cycle family disorders, and control subjects [27]. Separation of DLB kinase and cyclin-dependent kinase 5 (Cdk5/p35), active from AD, however, was less robust, provided that CSF p-tau during normal brain development and involved in regulatory 231 levels were also increased in DLB. Clinically diagnosed tau phosphorylation during mitosis, may also contribute to DLB cases also showed elevated levels of CSF p-tau 181 PHF formation. compared with controls [28], and Hampel et al. reported 4 International Journal of Alzheimer’s Disease that p-tau 181 provided the best discrimination of DLB Another mutation, E46K, was discovered in a Spanish family from AD yielding a sensitivity of 94% and specificity of 64% presenting with autosomal dominant DLB [48], and in [29]. In autopsy-confirmed DLB and AD patients, however, genetic studies of a large family with the spectrum of Lewy sensitivity decreased to 75% and specificity to 61%, with a body phenotype ranging from PD to DLB, α-syn gene diagnostic accuracy reported as 73% [30]. triplication was described, causing α-syn overproduction similar to the trisomy effect observed in Down syndrome patients [49]. 3. Synucleins: Genetics to Biomarkers in Autosomal dominant point mutations are shown to affect the Lewy Body Disorders the aggregative properties of α-syn, which has mechanistic implications for the pathogenesis of LBD. Compared to wild- 3.1. Pathogenetics of Synucleins in LBD type α-syn, biophysical analyses reveal that α-syn aggregation is folding state dependent, where A53T and A30P mutated 3.1.1. Functions of α-Synuclein. LBs are filamentous inclu- proteins cause increased aggregation only from the partially sions consisting primarily of the presynaptic protein α- folded intermediate state and not the monomeric state [50]. synuclein (α-syn), which might have several roles in vivo. A53T α-syn transgenic mice have increased oligomerization Studies demonstrate that it is localized to multiple neural of the protein in brain regions devoid of inclusions as well tissues, including high expression in neocortex and hip- as those areas with more abundant lesions and neurodegen- pocampus, and that expression increases during acquisition- eration, and consistent with prior biophysical findings, α-syn related synaptic plasticity [31]. Interaction with tubulin toxicity in these mice was dependent on the conformation of suggests α-syn could be a microtubule-associated protein intermediate species [51]. In fact, the E46K mutation, as well similar to tau [32, 33], and it is highly active in various as the others not only increase the tendency toward aggre- membrane lipid bilayers such as in presynaptic vesicles gation, but also promote formation of annular protofibrillar acting as a chaperone for soluble NSF attachment protein structures, causes pore formation in various membranes and receptor (SNARE) complex formation [34], in neuronal neuronal damage [52]. Golgi apparatus influencing protein trafficking [35]and in the inner membrane of neuronal mitochondrial [36]. 3.1.3. β-Synuclein Mutations in DLB. α-Syn is a member of Thesynucleinsmight actto preservemembranestability, a larger family of synuclein proteins which also includes β- provide antioxidant function, and assist with membrane synuclein (β-syn) and γ-synuclein (γ-syn). β-syn has recently turnover, although the actual role of synucleins remains been implicated in PD and DLB pathogenesis, but its precise elusive [37, 38]. Because of its association with LB and the role in disease is still emerging. Despite having strong homol- tendency to self-aggregate into pathological oligomers and ogy with α-syn, it is not clearly amyloidogenic, but is highly ultimately fibrillar structures [39], α-syn plays a central role localized to presynaptic sites in neocortex, hippocampus, in the pathogenesis of LBD, hence the alternate designation and thalamus like α-syn [53, 54]. Normal β-syn may act “synucleinopathies.” The degree of α-syn immunoreactivity as a biological negative regulator of α-syn. In bigenic α- in cortical LBs correlates with cognitive severity and disease syn/β-syn-overexpressing mice and in doubly transfected progression in PDD and DLB [4, 40]. Also, the protein can be cultured cells, β-syn ameliorated amyloidogenicity, neurode- recovered from filaments in purified Lewy bodies from PDD generative changes, and motor deficits induced by α-syn and DLB brain [41], and recombinant α-syn tends to form overexpression alone [55]. On the other hand, mutated β- Lewy body-like fibrillar structures in vitro [42]. syn leads to neuronal damage and disease and augments neurodegeneration, perhaps through a loss of its natural 3.1.2. α-Synuclein Mutations in PD and DLB. In the past regulator function. Two novel β-syn point mutations, P123H decade, tremendous advances have been made in under- and V70M, were found in highly conserved regions of the β- standing the genetic factors influencing the pathogenesis of syn gene in respective familial (P123H) and sporadic (V70M) Lewy body disorders. Compelling evidence for a genetic basis DLB index cases [56], where abundant LB pathology and for PD and DLB followed the discovery of mutations in the α-syn aggregation was present without β-syn aggregation. α-syn gene (PARK1/4) in patients with autosomal dominant P123H β-syn overexpression in transgenic mice resulted in familial Parkinson’s disease, and subsequently, mutations axonal damage, gliosis, profound memory, and behavioral were identified in patients with both sporadic and familial deficits [57]. These phenomena may involve α-syn, since DLBs. From a susceptibility marker on chromosome 4q21- bigenic mice overexpressing α-syn with P123H β-syn show 23 that segregated with the PD phenotype in Italian and greater deficits compared with monogenic mice and com- Greek kindreds, A53T [43] and A30P [44]were the first pared with P123H β-syn expressed with α-syn knockout, two missense mutations in α-syn associated with familial implying that the P123H mutation has a synergistic effect Parkinson’s disease. Clinical analysis of the Italian A53T with other synucleinopathies to cause neurodegeneration. mutation revealed phenotypic variability over the disease P123H as well as V70M β-syn mutations might also injure course with several individuals demonstrating moderate to neurons by disrupting normal lysosomal pathways and severe dementia [45]. Subsequently, a case of clinically and corresponding cellular autophagic processes [58]. pathologically well-characterized DLBD in the United States and a Greek proband of DLB with a family history of PD were 3.1.4. Association of γ-Synuclein with LBD. Unlike the oth- both determined to have the A53T α-syn mutation [46, 47]. er synuclein family members, γ-syn or persyn is largely International Journal of Alzheimer’s Disease 5 expressed in the cell bodies and axons of primary sensory PD and DLB patients [69, 70], and also from postmortem neurons, sympathetic neurons, and motor neurons as well CSF from DLB and other neurodegenerative diseases [71]. as in brain [59]. In cancer biology, γ-syn is associated with Comparative findings regarding differences in CSF α-syn abnormally altering cellular mitotic checkpoints in various levels among various neurodegenerative diseases, however, types of malignancies, making them more aggressively are difficult to interpret because of inconsistent observations. metastatic [60], but as far as neurodegeneration, it is the In PD, a smaller early study showed that no differences in most recent synuclein member to be linked to LBD neu- full-length CSF 19 kDa α-syn have been found in relation ropathology and the least well understood. Single-nucleotide to control individuals [69], but a recent effort using a polymorphisms in all three synucleins have been associated new Luminex assay in a larger sample controlling for with sporadic DLBD, most prominently γ-syn [61], and in extraneous influences showed significantly decreased levels sporadic PD, DLB, and LBV patients, γ-syn antibodies, as in PD compared to controls with 92% disease sensitivity well as β-syn and α-syn reveal unique hippocampal axonal and 58% specificity [72]. Elevated α-syn levels, however, pathology [62]. In vivo, γ-syn overexpression in trans- were foundin DLB, AD, andvasculardementia with no genic mice shows age- and dose-dependent neuronal loss differences among them [71]. Perhaps more intriguing, throughout the neuraxis, especially in spinal motor neurons, higher-molecular weight aggregated α-syn species in CSF where γ-syn-bearing inclusions, gliosis, and alterations in might be associated with PD and DLB. Reduced levels of heat shock protein and neurofilament structure are found a24kD α-syn-immunoreactive band were found in DLB [63], perhaps suggesting relevance to motor neuron disease CSF and correlated directly with declining cognition [73]. associated with dementia. In vitro evidence further supports Moreover, using a specific enzyme-linked immunosorbent a cytoskeletal role for γ-syn in maintaining neurofilament assay (ELISA), soluble aggregated α-syn oligomers in CSF structure; γ-syn overexpression in cultured neurons causes were significantly increased in PD patients compared against disruption of the neurofilament network by destabilizing the control subjects, AD and progressive supranuclear palsy, structural integrity of neurofilament-H allowing degradation and specificity ranged from approximately 85 to 87%, while by calcium-dependent proteases, which has implications for sensitivity was about 53–75% range [74]. neurodegeneration [64]. Plasma α-syn detected by immunoblotting was decreased in PD compared with age-matched control subjects, and those PD patients with age-at-onset prior to 55 years (early- 3.2. Synucleins as Biomarkers of LBD onset) had significantly lower levels than those with onset after 55 years of age (late-onset) [75]. In addition, soluble 3.2.1. Synucleins in the Extracellular Compartment. Synu- oligomeric α-syn detected by specific ELISA was significantly cleins are known as intracellular molecules, but they also elevated in plasma from PD. This test demonstrated a appear in extracellular and peripheral fluids from active specificity of approximately 85%, a sensitivity of 53%, and passive processes. Evidence suggests that turnover and and a positive predictive value of 0.818 [76]. Although secretion of these proteins might occur during normal measurement of plasma α-syn appears interesting as a cellular processing, releasing synucleins into extracellular biomarker, it was reported that skin cells and platelets are space and hence into peripheral sites. In transfected and un- also sources for α-syn, and their levels did not correlate transfected cultured neuroblastoma cells, 15 kDa α-syn is with disease presence or severity [77]. Moreover, red blood released into surrounding media [65], and furthermore, not cells are also a major source of α-syn [78], and thus, only monomeric α-syn but also aggregated forms are secreted plasma could be contaminated by α-syn not originating from in an unconventional exocytic manner into extracellular fluid brain, which might render interpretation of results difficult. in response to proteasomal and mitochondrial dysfunction One promising consideration for the future exploration [66]. Remarkably, Desplats et al. recently showed that of α-syn as an LBD biomarker will be the development neuronally secreted α-syn can also be taken in endocytically of novel imaging compounds and techniques, similar to by other neurons or glia as a means of transmitting pathology amyloid imaging, to specifically target and visualize α-syn [67]. Secreted α-syn interacts with various molecules such distribution in the PD and LBD brain. The availability of enzymes; in cultures, matrix metalloproteinase-3 cleaves such methods will be a significant advance in biomarkers for native α-syn to smaller proteolytic fragments that enhance synucleinopathies. its aggregative properties [68]. Whether β-syn and γ-syn also undergo unconventional exocytosis and secretion remains unknown, but given structural and functional similarity to 3.2.3. β-Syn and γ-Syn as Potential Biomarkers in Lewy α-syn, the possibility exists. Certainly, synaptic and axonal Body Disorders. Due to their increasing importance in LBD damage reflecting neurodegeneration may also allow release pathogenesis, β-syn and γ-syn, as much as α-syn, might of synucleins into the extracellular millieu and access to be excellent targets as peripheral markers of disease. As peripheral fluids such as CSF and blood. such, levels of these synucleins might be altered in the CSF of patients with PD/PDD and DLB, reflecting the 3.2.2. α-Synuclein as a PD and DLB Biomarker. Multiple underlying degenerative processes in brain. No studies to forms of α-syn are released into cerebrospinal fluid (CSF) date have examined β-syn levels in peripheral fluids in and other biological fluids. Full-length α-syn has been relation to neurodegenerative disease, but a small study recovered from lumbar CSF from living normal control, reported elevated postmortem ventricular CSF γ-syn levels 6 International Journal of Alzheimer’s Disease in DLB, AD, and vascular dementia patients, with the highest aggregation of α-syn and LB formation [80]or contributeto levels seen in DLB patients [71]. More detailed examination pathogenesis by other molecular pathways. of both β-syn and γ-syn as a peripheral disease markers DJ-1 is found in brain across a wide range of neurodegen- in well-characterized populations of PD, DLB, and other erative diseases including PD, FTD, AD, DLB, and LBVAD, disorders is warranted to determine their specificity and and demonstrates striking association with neuropil threads sensitivity in the synucleinopathies. and neurofibrillary pathology in neocortex and subcortical brain regions in these disorders [90]. Interestingly, this association with tau pathology was seen in DLB and LBV brains, suggesting that as a chaperone molecule, DJ-1 may 4. DJ-1 in the LewyBodyDisorders be involved in tangle formation, and the binding of DJ- 4.1. Functional Role of DJ-1 in Lewy Body Diseases. Recently, 1 with these lesions could abolish the normally protective DJ-1 (PARK 7) has emerged as a significant molecular target effect of DJ-1, enhancing oxidative neurotoxicity. Wang et al. of interest in LBD principally because of its genetic associa- observed that DJ-1 knockout mice have markedly abnormal tion with PD and its increasing importance in cellular oxida- hippocampal long-term depression accompanied by a less tive neuroprotection. Although its exact role is unknown, severe abnormality in long-term potentiation, which was multiple functions have been assigned to the DJ-1 protein. reversed by the D2/3 agonist quinpirole, indicating that DJ-1 Described by Nagakubo et al. as a mitogen-dependent has a role in dopamine-dependent signaling in hippocampal oncogene involved in Ras-related signaling pathways [79], plasticity [91]. This implies that DJ-1 may be important in it shares structural homology with the carboxy-terminal the maintenance of memory and cognition. domain of Escherichia coli HPII catalase and is reported to possess catalase activity which reduces oxidative stress in cultured cells [80]. It also binds to and regulates the PIAS 4.3. DJ-1 as a Potential Biomarker for Lewy Body Dis- SUMO-1 ligase and is itself posttranslationally modified by eases. Given its pathogenetic significance, DJ-1 could be a sumoylation [81, 82]. Of relevance to Lewy body formation candidate biological marker for DLB and LB and might and neurotoxicity, DJ-1 displays redox-dependent chaperone serve as a means of monitoring in vivo oxidative damage activity conferring proper protein folding and thermal sta- and protein misfolding. Although intracellular and mito- bility, which in fact, also inhibits α-syn aggregation [80]. The chondrial in localization, DJ-1 is presumed to be secreted overexpression of DJ-1 in rats protects nigral dopaminergic perhaps specifically under disease conditions which induce neurons against degeneration involving 6-hydroxydopamine, oxidative damage. Using semiquantitative immunoblotting, while mutant DJ-1 in mice causes abnormal dopamine we previously identified DJ-1 in CSF of sporadic PD patients, reuptake and susceptibility to 1-methyl- 4-phenyl-1,2,3,6- where levels were significantly elevated compared with tetrahydropyridine (MPTP) toxicity [83]. Deletion of DJ- controls. Levels were higher in the earlier stage PD cohort 1 homologs in Drosophila renders them sensitive to H O , (Hoehn-Yahr stages I-II) than in the more severe patients 2 2 paraquat, and rotenone toxicity [84]. (Hoehn-Yahr stages III-IV) [92]. Similarly, plasma DJ-1 levels in PD patients were markedly increased compared to controls, but unlike CSF, levels were relatively higher in late 4.2. DJ-1 Mutations and Possible Relevance to LBD. No less stage (III-IV) rather than early stage PD (I-II) [93]. The than 13 gene mutations have been identified in DJ-1 in reason for this difference between plasma and CSF DJ-1 is atypical younger-onset PD patients, but their significance to unknown, but we surmised previously that since CSF DJ-1 idiopathic late-onset PD remains uncertain. In autosomal originates from a central source produced mainly by reactive recessive early-onset PD from consanguineous families, a glia, early increases in CSF DJ-1 levels probably represent complete DJ-1 deletion in a Dutch family and a point an early protective response to damage, whereas plasma mutation L166P in an Italian case were identified [85]. DJ-1, like other plasma disease markers, likely represents When expressed in cultured cells, L166P appears to be a peripheral oxidative stress damage. In fact, DJ-1 is secreted loss-of-function mutation which leads to DJ-1 functional into blood in breast cancer, melanoma, familial amyloid instability, degradation by the proteasome system [86, 87], neuropathy, and stroke [94–96]. In the largest study to date, abnormal translocation of DJ-1 to mitochondria, and loss Hong et al. developed a more sensitive and quantitative of chaperone activity [80]. The importance of DJ-1 gene Luminex assay for CSF DJ-1 to complement immunoblotting alterations in dementia and DLB, however, is uncertain. One mass spectrometric and chromatographic analysis methods report found no impact on dementia risk of the DJ-1 14kb and found decreasing rather than increasing levels of DJ-1 deletion [88], and analysis of an insertion/deletion variant in PD CSF compared with control patients [72]. The 90% (g.168 185del) in DJ-1 in a larger sample of patients also disease sensitivity and 70% disease specificity for PD using showed no association with either PD or DLB compared to this method approaches minimal desired parameters for a control patients [89]. Given these early negative findings, the clinically useful biomarker for PD. Importantly, the study relevance of DJ-1 genetic mutations to DLB and other LBD is highlighted the fact that DJ-1 levels are greatly influenced not known. At present, no patient harboring a DJ-1 mutation by such variables as the extent of blood contamination and has come to autopsy, so the precise pathology is not known. patient age, which could account for some of the variability Although DJ-1 mutant cases may ultimately not be LBDs, it is across studies. Of note, DJ-1 is also subject to oxidative possible that alterations in DJ-1 may somehow influence the modifications in PD and AD brain tissue, and this might be International Journal of Alzheimer’s Disease 7 measured in peripheral fluids as well, as another monitor of mutation carriers [107]. These observations suggest a much oxidative damage [97]. CSF DJ-1 remains a promising and broader link between GBA mutations and the dementia perhaps clinically useful biomarker for PD, but as far as DLB phenotype of LBD. In fact, examination of GBA gene and other LBD, it is unknown whether CSF levels of DJ- alterations in DLB patients, with and without concomitant 1 are altered. Since plasma DJ-1 is increased in DLB, it is LBV-type AD pathology, showed that the majority of GBA hypothesized that CSF DJ-1 may also be elevated. Further mutationswere found in DLBpatientsratherthan inPD, investigation will be necessary to clarify the utility of DJ-1 with a mutation rate in DLB ranging from 18 to 23% as a biomarker in DLB and LBD. overall [108, 109]. The proportion of DLB patients with GBA mutations was higher in those with pure neocortical LB pathology compared to those with mixed LB and AD pathology and to those with predominantly brainstem LB. 5. Glucocerebrosidase as a Novel Biomarker for A significant association was also found between GBA Lewy Body Disorders mutation status and the presence of LB, indicating that 5.1. Glucocerebrosidase Mutations Influence PD and DLB. altered GBA might play a role in their formation and in Many clinicopathologic parallels can be drawn between synucleinopathy [108]. the lysosomal storage disorders, such as Niemann-Pick, Sandhoff’s, Tay-Sachs disease and others, and the age-related 5.2. Glucocerebrosidase and Chaperone-Mediated Autophagy neurodegenerative disorders, when considering the aberrant in LBD. Important in neurodegeneration, disrupted cellular accumulation of pathological substances (e.g., lysosomal proteostasis represents a state in which an imbalance exists sphingomyelin in Niemann-Pick disease versus synucleins between effective functioning of the innate cytoprotective in PD and DLB) and the phenotypes of neuronal loss machinery and excessive accumulation and aggregation of and cognitive deterioration found in both. Common to abnormally misfolded proteins, leading to neurotoxicity. It these diseases are abnormalities in lysosomal and autophagic is increasingly apparent that chaperone-mediated autophagy mechanisms as part of a larger disruption of cellular (CMA) and lysosomal degradation pathways are important proteostasis leading to abnormal storage/accumulation of in maintaining cellular proteostasis as part of a larger toxic materials and neuronal damage. In the past few network of cellular actions, with particular relevance for years, an altogether unexpected pathogenetic relationship neurodegenerative diseases. Recently, as evidence for CMA emerged between Gaucher’s disease (GD), a prototypic dysfunction in synucleinopathies, a significant decrease storage disease, and the synucleinopathies. Despite its overall in autophagy markers was reported in substantia nigra rarity, GD is the most common inherited lysosomal storage from PD brain [110]. Soluble forms of α-syn, including disease, especially in the Ashkenazi Jewish population. It is monomers, oligomers, and even protofibrils, are normally caused by autosomal recessive gene mutations in the gluco- cleared through the CMA/lysosomal degradation by inter- cerebrosidase (GBA) gene (chromosome 1q21), leading to acting with the chaperone, heat shock cognate-70, and either partial or complete deficiency of GBA, and hence, toxic becoming internalized into lysosomes via the Lamp-2a lysosomal accumulation of its substrate, glucosylceramide, in membrane receptor [111, 112]. Studies have indicated that multiple cell types including neurons [98]. Recent reports α-syn shares a common pentapeptide structure with other documented an increased incidence of PD in heterozygous lysosomal substrates, designating it as a target for removal relatives of patients with GD [99, 100], but interest in by this pathway [111], and the lysosomal structure is critical this phenomenon was propelled by the finding that GBA to maintaining the internal acidic environment, allowing mutations were in fact more common in PD patients of lysosomal hydrolases to degrade α-syn into peptides released Ashkenazi background compared with AD patients and PD into the cytosol [112]. Mutant GBA could therefore disrupt patients in the general population [101–103]. Moreover, lysosomal activity leading to abnormal accumulation of more severe GBA mutations such as 84 dupl G and IVS nondegraded α-syn, which then aggregates to toxic solu- 2 + 1 were associated with a greater degree of PD risk, ble oligomers and protofibrils. Also, abnormalities in the compared with less severe GBA mutations such as N370S ubiquitin-proteasome system (UPS) are present in AD and [104]. The relationship between PD and GBA has now been PD, and GBA alterations might secondarily overwhelm the replicated in much larger international studies with the most ability of UPS to remove accumulated α-syn, promoting common mutations being L444P and N370S, and about 28 aggregation and neurotoxicity [113]. Pathologically, in GD GBA mutations are presently recognized [105]. with parkinsonism, α-syn-positive inclusions were observed Interestingly, in a study of British patients with PD and in neurons in hippocampal CA2-4 regions, while cortical GBA mutations, all 17 carrier patients demonstrated abun- synuclein pathology was identified in other GD cases [114]. dant α-syn neuropathology with Braak stage 5-6 severity and Further, parkin, an E3 ubiquitin ligase also implicated common neocortical LB pathology. Clinically, these patients in PD, has been shown to affect the stability of mutant had earlier age at onset, and hallucinations were present GBA and increase its degradation causing further lysosomal in 45% of patients, while 48% had cognitive impairment dysfunction [115]. or dementia consistent with PDD [106]. Greater severity of GBA mutation also predicted the presence of cognitive impairment in PD patients; 56% of severe GBA mutation 5.3. Glucocerebrosidase as a LBD Biomarker. Because of carriers had cognitive impairment compared to 25% of mild the importance of mutant GBA function to PD and DLB 8 International Journal of Alzheimer’s Disease pathogenesis, the issue arises as to whether the measurement and IL-6 [123]. Because secreted CNS cytokines are readily of GBA activity, or a perhaps other related molecules, detected in CSF, they have been extensively examined as might be utilized as a biological marker. The activity of potential disease biomarkers. IL-1β,IL-2, IL-6, and TNF- peripherally secreted GBA was measured in plasma and α are all upregulated in PD brain, as well as in CSF CSF in a 10-month-old female with GD with the aim of from PD patients [124–126], and Chen et al. showed that monitoring the effect of experimental Cerezyme replacement plasma IL-6, but not IL-1β,TNF-α,or other acute phase therapy [116]. Baseline GBA activity was detected in both reactants, predicted risk for future PD in males [127]. In −6 −6 plasma (2.7 × 10 U/μL) and CSF (0.096 × 10 U/μL), terms of DLB, CSF IL-1β levels, which were relatively low, although CSF activity was several magnitudes lower than did not differ compared to AD or normal controls and plasma. Intravenous Cerezyme, a macrophage-targeted GBA, could not distinguish them apart. Comparable increases in rapidly raised the plasma activity within 1 hour and CSF CSF IL-6 levels were found in AD and DLB, but again not activity by 2.3-fold at 3 hours, both returning to baseline significantly different from each other to be of diagnostic after 24 hours. This study suggests the intriguing possibility value [128]. Indeed, the neuroinflammatory cytokines may that GBA activity, especially in CSF and plasma, might be be important as a pathogenetic response to CNS injury useful in monitoring the efficacy of novel therapies involving caused by accumulation of amyloidogenic proteins, but their CMA and lysosomal function. To extend this observation, role as biomarkers for the LBD, especially for DLB, is still Balducci et al. determined that multiple lysosomal hydro- unclear. lases, including GBA, are significantly decreased in the lumbar CSF of PD patients [117], perhaps supporting a 6.2. Neurofilament Proteins. Disorganization and breakdown more widespread lysosomal dysfunction in PD not limited in the cytoskeletal network occurs in various LBDs and to GBA alone. In this regard, other lysosomal enzymes such other neurodegenerative diseases, and as discussed, gamma- as mannosidase and β-hexosaminidase might be important synuclein and proteolytic degradation of the cytoskeleton additional biomarker targets for neurodegeneration. More- may be involved. As a result, a failure of normal axonal over, in DLB, AD, and FTD patients, lysosomal enzyme transport results from the accumulation of disrupted neu- activities in CSF demonstrated a very specific pattern of rofilament molecules within the neuropil, causing neuronal decrease, in which only DLB showed significant decreases demise [129]. Recently, a mutation in the NEFM gene encod- in CSF activity of α-mannosidase, β-mannosidase, GBA, ing the rod domain 2B of neurofilament M (NF-M) which galactosidase, and β-hexosaminidase, whereas in AD and causes aberrant NF assembly was identified in a single early- FTD, only CSF α-mannosidase activity was significantly onset PD patient [130]. It is recognized that in addition to diminished [118]. In DLB, CSF GBA activity showed the α-syn, three types of NF protein also comprise the structure greatest magnitude of decrease, reinforcing its importance of Lewy bodies [131]. Upon cell death or axonal damage, in the LBD, but also noteworthy is the fact that AD and accumulated neurofilament leaks into the extracellular space, FTD showed decreased α-mannosidase activity, suggesting subsequently appearing in CSF and perhaps other peripheral that this might be another important factor in lysosomal fluids. Elevated CSF NF protein was reported in MSA and dysfunction in neurodegeneration. Indeed, these promising PSP, but not in PD, and this was suggested to clinically candidates need to be investigated further to establish diag- aid in differentiating parkinsonian syndromes [132]. CSF nostic accuracy in terms of disease specificity and sensitivity NF protein was also measured in dementia, and although in cohorts of PD, DLB, and other dementing disorders. increased levels were observed in DLB, late-onset AD, and FTD, there were no differences among them [28]. Therefore, because cytoskeletal abnormalities are present in many 6. Miscellanous Candidate Biomarkers neurodegenerative dementias as well as in PD, NF protein may be more a reflection of nonspecific alterations in 6.1. Inflammatory Cytokines. Polymorphisms in proinflam- neuronal and axonal function, which does not appear to able matory cytokine genes including IL-1α,IL-1β,and TNF- to clinically separate DLB from other disorders. α are associated with increased risk in AD [119]. In PD, several case control genetic analyses have demonstrated that homozygous carriers of the IL-1β−511 and TNF-α−308 6.3. Brain Neurotransmitter Alterations in CSF and by Imaging promoter region variants have increased disease risk [120, Modalities. Severe cortical cholinergic deficits originating 121], and that earlier age at onset in PD was associated from deficiencies in the nucleus basalis of Meynert are with IL-1β−511 homozygosity at allele 1 [122]. But as characteristic of AD brain, but studies have shown that yet, no such genetic alterations in cytokines genes have cholinergic deficits are perhaps more severe in DLB brain been reported in DLB. Similar to Aβ-induced upregulation [5]. This suggests that measurement of cholinergic activity of inflammatory cytokines in AD, soluble secreted α-syn and/or acetylcholine (ACh) might be developed into a in the extracellular space in LBD might also induce the potential biomarker for the LBDs. Indeed, early attempts to production of a variety of neuroinflammatory mediators into quantify ACh or its major metabolite, choline, have shown the extracellular fluid. For instance, microglial activation in baseline levels to be low and perhaps difficult to measure response to stimulation by secreted α-syn from cultured cells accurately. In AD, CSF ACh was reported to be significantly and from overexpression in transgenic mouse models occurs lower than control levels [133], while in PD and Huntington’s in a dose-dependent manner, causing release TNF-α,IL-1β, disease patients, despite some cholinergic deficit, lumbar CSF International Journal of Alzheimer’s Disease 9 ACh and choline levels did not differ from normal [134]. No In the last decade, a series of Japanese studies consistently studies have directly examined CSF cholinergic levels in DLB demonstrated delayed heart to mediastinum ratio (H/M) of or LBDs, but recently, Shimada and colleagues employed I-MIBG uptake in DLB compared with AD and controls positron emission tomography (PET) mapping of brain ACh [143–146]. I-MIBG scintigraphy was found superior to activity in DLB and PDD patients and normal controls and brain perfusion SPECT imaging [147]. Estorch et al. further demonstrated a marked reduction in cholinergic activity showed that in dementia patients followed for four years in medial occipital cortex of DLB and PDD, greater than before “final diagnosis,” I-MIBG imaging distinguished that observed in PD patients without dementia [135]. Some DLB from other dementias with a sensitivity of 94%, correlation of mapped cholinergic activity with cognitive specificity of 96%, and a diagnostic accuracy of 95% [148]. decline measured by the Mini-Mental State Exam was also Finally, consistent with autonomic dysfunction in DLB, both found. Although preliminary, this has potential to be a more early and delayed H/M I-MIBG uptake were significantly practical and sensitive cholinergic biomarker for LBD. associated with the presence of orthostatic hypotension in Because of similar nigrostriatal loss to PD, a relative DLB patients and discriminated DLB from AD even in the dopaminergic deficiency also exists in DLB and LBDs. CSF absence of parkinsonism [149]. dopamine (DA) and its metabolites have been investigated previously in PD, and recently, Lunardi et al. showed differences in CSF DA and its metabolites, homovanillic 6.4.2. Other Structural and Functional Imaging Biomarkers. acid (HVA) and dihydroxyphenylacetic acid (DOPAC), in Various magnetic resonance (MR) imaging modalities have PD patients, demonstrating early-stage dopaminergic loss been explored in DLB and PDD, including volumetric and a correlation with the development of dyskinesia [136]. imaging, diffusion tensor imaging, and proton magnetic In DLB, HVA levels were significantly reduced compared resonance spectroscopy (reviewed in Watson et al.) [150], with AD, separating the disorders [137]. Similar to cholin- and although not directly useful as biomarkers at present, ergic activity, imaging modalities may also contribute to they have revealed insights in the pathobiology of LBDs. the assessment of dopaminergic function in the LBDs. In Using conventional MRI techniques such as voxel-based a small study, striatal DA uptake as measured by F- morphometry and region of interest analysis, some degree fluorodopa PET was decreased in both caudate and putamen of diffusion or focal frontal and parietal atrophy has been in DLB as compared with AD patients and controls [138]. observed [151]. Atrophy has been rated at 1.4% per year Also, DA transporter loss was determined across multi- in DLB brain [152], 1.31% per year in PDD, and 0.31% ple studies using I-2β-carbometoxy-3β-(4-iodophenyl)- per year in PD [153]. Not surprising is the fact that unlike N-(3-fluoropropyl) nortropane ligand with single-photon AD brain, medial temporal structures are relatively preserved emission computed tomography ( I-FP CIT SPECT) and in DLB and PDD, with global hippocampal loss at about demonstrated significant loss of caudate and putaminal 10–20% compared with controls and about 21–25% in AD DA transport compared with AD and control levels [139– [154]. Diffusion tensor imaging, an MR technique mapping 141]. A larger phase III, multicenter study of I-FP CIT brain microdiffusion of water in the direction of white matter SPECT in possible and probable DLB patients and non-DLB tracts, has shown decreased fractional anisotropy of water comparators (mostly AD) demonstrated a mean sensitivity movement in DLB in the precuneus and posterior cingulate of 77.7% for detecting clinically probable DLB, with a areas, perhaps highlighting their role in DLB pathogenesis specificity of 90.4% and 85.7% overall diagnostic accuracy [155]. 123 99m [141]. I-FP CIT SPECT DA transporter imaging greatly Brain perfusion SPECT ( Tc-HMPAO SPECT) has enhanced diagnostic accuracy for DLB over clinical diagnosis been evaluated in its ability to diagnostically separate DLB alone when coupled with autopsy confirmation, raising from AD, and in AD, reduced relative cerebral blood sensitivity for DLB from 75% to 88% and specificity from flow (rCBF) in the frontal, and medial temporal regions 42% to 100% [139]. Furthermore, DA transporter loss in is characteristic, whereas in DLB, occipital hypoperfusion the caudate may also be inversely associated with depression, is often observed [156]. Colloby et al. applied statistical apathy, and delusions in DLB patients [142]. parametric mapping to SPECT imaging of DLB patients, more precisely showing large perfusion deficits in the left medial occipital gyrus and the bilateral central, inferior parietal, precuneate, superior frontal and cingulate regions 6.4. Miscellaneous Imaging Biomarkers in LBD on the brain, which are functionally consistent with frontal- 6.4.1. MIBG Scintigraphy as a DLB Biomarker. Autonomic executive and visuospatial deficits in DLB [157]. Across failure is a common clinical finding in LBD, including PD studies, sensitivity ranged from 65 to 85% and specificity and DLB, but not in non-LBD dementias, and therefore from 85–87%, which appears less robust as a potential it has been investigated as an alternative biomarker for imaging marker compared with other methods. the diagnostic separation of DLB from other dementias. Abnormal autonomic function can be determined using car- 123 123 diac I-meta-iodobenzyl guanidine ( I-MIBG) imaging, a 6.5. Other PD Genes and Their Protein Products as Possible technique which assesses cardiac sympathetic nerve function DLB Markers. Aside from α-syn and DJ-1, numerous other in both cardiac and neurological disorders by measuring mutations have been associated with familial early-onset PD the uptake of I-MIBG, a norepinephrine analogue [143]. and possibly LBD (Table 1). Among these gene products 10 International Journal of Alzheimer’s Disease are parkin (PARK 2), UCHL-1 (PARK 5), PINK1 (PTEN- the relationship among LRRK2, α-syn, and tau in PD, DLB, induced putative kinase 1; PARK 6), and LRRK2/dardarin and other LBD is also influenced by population differences. (PARK 8) [158]. Indeed, none of these mutations have These findings make LRRK2/dardarin an attractive candidate yet been associated with prototypic LBD pathology, and it for examination as a potential biomarker, and if identified in remains to be determined whether they actually represent CSF or peripheral fluids, they might be used with α-syn and LBDs or separate diseases with parkinsonian phenotype. tau as combined biomarkers. Furthermore, no studies have addressed their role as bio- Furthermore, emerging evidence is redefining the roles logical markers of disease, but since both synucleins and of PINK1 and parkin in PD pathogenesis. Because energy DJ-1 are detected in CSF and peripheral fluids, it seems generation is critical for cellular function, mammalian cells plausible that the protein products of other dominant genes are highly dependent on mitochondria [168]. Depolariza- in PD could be peripheral biomarker candidates for DLB tion and morphological defects characterize damaged or and other LBD. Parkin, UCHL-1, and PINK1 genes, like impaired mitochondria which are targeted for removal DJ-1, all encode proteins important in neuroprotection in through mitophagy, a highly specialized form of autophagy terms of maintaining protein homeostasis and preventing in which parkin and PINK1 play a crucial role (reviewed by stress-related cellular damage, and mutations in these genes Vives-Bauza and Przedborski) [169]. In this process, PINK1 cause a loss of these critical functions. Leucine-rich repeat cleavage is inhibited by the loss of mitochondrial membrane kinase 2 (LRRK2/dardarin), on the contrary, is linked with potential, causing its lengthening and the recruitment of autosomal-dominant late-onset PD, and mutations result in cytosolic parkin [170, 171]. Voltage-dependent anion chan- a toxic gain of function. nel 1 and other outer mitochondrial membrane proteins LRRK2/dardarin is a kinase consisting of multiple func- are then ubiquitinated in a parkin-dependent manner, and tional domains, and recent evidence suggests that physio- this in turn recruits the binding of adapter proteins such logically, its principal function may be to regulate neurite as p62 and histone deacetylase 6 to initiate autophago- outgrowth. Expression in cultured neurons of several LRRK2 some assembly around the damaged mitochondrion and mutations associated with familial PD, such as G2019S, subsequent removal [169]. Of relevance to PD, mutant increased kinase activity and significantly reduced neurite PINK1 and mutant parkin both cause motor dysfunction, outgrowth, whereas expression of a dominant-negative dopaminergic loss, and abnormal mitochondrial morphol- mutation, K1906M, markedly increased neurite length [159]. ogy in Drosophila [172]. In this paradigm, loss of function PD-associated mutations also generated tau-positive axonal PINK1 mutants are rescued by concurrent overexpression inclusions in cultured neurons, suggesting that LRRK2 may with wild-type parkin but not vice versa, indicating that be linked to abnormalities in tau. Indeed, expression of parkin specifically acts downstream of PINK1. Also, parkin mutant G2019S LRRK2 in Drosophila caused activation mutations have been shown to interfere with ubiquitination of the Drosophila GSK-3β homolog and promoted tau and the downstream steps in normal mitophagy [173]. Thus, hyperphosphorylation leading to microtubule fragmentation PD, and possibly related dementias, might be a result, to and dendritic pathology [160]. Similar tau hyperphosphory- some extent, of defective mitophagy due to loss of function lation was also present in transgenic mice expressing G2019S in PINK1 and parkin such as found in autosomal dominant LRRK2, and expression of both wild-type human LRRK2 early-onset PD. and G2019S mutant LRRK2 caused abnormal dopaminergic Although LRRK2, parkin, PINK1, and UCHL-1 have transmission [161]. LRRK2 may also interact with α-syn, not yet been identified in peripheral fluids, PINK1 and another dominantly inherited PD gene, to exert its effect. parkin may be a promising candidates. Unexpectedly, both Lin et al. showed that overexpression of LRRK2 with A53T PINK1 and parkin, which are normally cytosolic or tar- mutant α-syn in transgenic mice worsened neurodegenera- geted to mitochondria, were localized extracellularly in AD tion, while ablation of LRRK2 expression suppressed α-syn and multiple sclerosis brain, and colocalized with amyloid aggregation and pathology [162], and α-syn also activates plaques, reactive astrocytes, as well as amyloid-affected GSK-3β in mice causing tau hyperphosphorylation [163], vessels [174, 175]. This suggests that both PINK1 and parkin indicating that LRRK2, α-syn, and tau alterations may all be are actively released from neurons and glia in response to linked in the same pathway, perhaps with LRRK2 upstream injury and might be upregulated in CSF and peripheral fluids of these events. Although early, evidence has indicated that during neurodegeneration. Interestingly, given a role in LRRK2 is also a component of LB in PD and DLB brains mitophagy, they might also be a CSF or peripheral reflection [164], and that LRRK2 and α-syn interact in DLB brain of mitochondrial health and turnover. It remains to be seen and coimmunoprecipitate in cultured cells after oxidative whether these gene products can be detected in biological stress challenge [165], suggesting that the LRRK2 may also be fluids such as CSF as potential biomarkers in PD and LBD. important in DLB pathogenesis. Interestingly, genome-wide association studies (GWASs) in a European cohort demon- strated that LRRK2, α-syn, and tau are loci associated with 7. Unbiased Methods in PD risk [166], but examination of tau in a Japanese GWAS LBD Biomarker Discovery cohort failed to identify it as a PD risk locus [167], showing a population difference with regard to this locus. Certainly, 7.1. Genomics in PD and LBDs. As detailed above, traditional population differences might apply to all risk loci examined methods for molecular biomarker determination have been forPD and LBD, and itisimportant to determinewhether derived from targeted analyses of candidate genes/mutations International Journal of Alzheimer’s Disease 11 and corresponding proteins in brain and body fluids such as 156 candidate proteins involved in ubiquitin-proteasome CSF and blood, with the subsequent exploration of mecha- system and synaptic function, from which the heat shock nisms in cell culture and animal models. An emerging alter- cognate-71, a chaperone involved in neurodegenerative nate approach has been to evaluate genomes and proteomes disease, was identified and validated as a candidate target with regard to specific neurodegenerative diseases and their [182]. Abdi and colleagues carried out proteomic evaluation components in an unbiased manner to yield a number of of CSF from AD, PD, and DLB patients and normal control potential pathogenetic, therapeutic, and biomarker targets individuals, using chromatography, MS, and isobaric tagging for further validation. With regard to the genomic analysis for relative and absolute quantification (iTRAQ), identifying of the LBDs, gene expression profiling has proved to be numerous candidate proteins related to PD and DLB, such as a promising tool. Scherzer et al., for instance, examined lipoproteins ApoC1 and ApoH [183]. Lastly, using surface- transgenic Drosophila expressing the human α-syn gene and enhanced laser desorption/ionization-time of flight (SELDI- performed temporal profiling of resultant gene expression TOF) MS analysis of serum from DLB patients compared [176]. They demonstrated a number of changes, including a to AD, a combination of protein peaks provided the ability downregulation of phospholipase A2 and other lipid genes, to separate DLB from non-DLB cases, with a sensitivity of downregulation of several mitochondrial respiratory chain 83.3% and a specificity of 95.8% [184]. Given promising molecules, and alteration in membrane transport and energy findings, further exploration of the proteomics of the LBDs genes such as voltage-gated calcium channel and lysosomal is warranted, and perhaps consideration should be given ATPase, suggesting that mitochondrial integrity might be to determining whether combining various genomic and affected by α-syn overexpression. proteomic methods will be of value. In Parkinson’s disease brain, RNA from populations of mesencephalic dopaminergic neurons with and without 8. Conclusions LB were isolated by immunolaser capture microdissection, amplified by polymerase chain reaction and expressed [177]. Over the last decade, tremendous advances have been Interestingly, upregulation of the ubiquitin-specific protease made in understanding the pathogenetics of PD, PDD, 8 in LB-containing neurons indicated cellular damage and and DLB, which has revealed not only the genetic basis increased levels of ubiquitination in LB, whereas non- of these disorders, but also related mechanisms common LB-bearing neurons showed increased expression of novel to all the LBD. In parallel, these discoveries have been a cytoprotective genes such as bullous pemphigoid antigen 1, catalyst for translating and developing many of the involved an HSP-70-like gene (STCH) and Kelch-like 1. Although proteins into promising biomarkers for disease. A common promising, further genomic profiling studies in DLB, PDD, theme centers on genes that drive a complex network of and other LBD are needed to expand the range of novel gene synergistic and opposing cellular actions underlying path- targets for examination and validation. ogenesis. Aggregation of α-syn, the main constituent of intracellular LBs, results in toxic oligomers and protofibrils 7.2. Proteomic Profiling in PD and LBDs. As a complement to which not only act intracellularly, but also are actively and gene expression profiling and genomic methods, proteomic passively released into the extracellular environment causing profiling has also assumed a greater importance in biomarker damage to surrounding tissue. Proinflammatory cytokines discovery for neurodegeneration with relevance to the LBD. such as interleukins are also produced which perpetuates Advances in methodologies such as 2-dimensional gel elec- the inflammatory cascade. On the contrary, DJ-1, PINK1, trophoresis (2-D GE), liquid chromatography (LC), high- parkin, and perhaps others molecules are upregulated to resolution mass spectrometry (MS), and quantitative pro- oppose cellular protein misfolding and oxidative stress and teomics allow analysis of static or condition-dependent pro- maintain mitochondrial function, while autophagy mech- tein structure and function associated with PD and LBD in a anisms attempt to limit the toxic effect of synucleins and variety of sample types such as brain or body fluids (reviewed other toxins by lysosomal engulfment and digestion. Much in Shi et al. 2009) [178]. In mice treated with MPTP, a specific of this is reminiscent of a relatively new concept applied mitochondrial toxin, isotope-coded affinity tag assay of brain to infectious diseases and mechanical tissue injury termed tissue followed by MS analysis revealed 100 proteins with “damage-associated molecular patterning” (DAMP), which significantly altered levels including many mitochondrial and is an evolved system to recognize, contain, and repair damage metabolic molecules, βAPP and DJ-1 [179]. to cells and tissues. It is characterized by the abnormal Basso et al. first examined the proteome of the substantia release of molecules normally confined and operating within nigra from Parkinson’s disease brain and age-matched healthy cells or from foreign pathogenic agents, that when controls [180]. Using 2D GE and peptide fingerprinting, of released into the extracellular space activate receptors and the 44 expressed proteins, 9 proteins differed in PD versus pathways leading to inflammation and multiplying cellular controls, including oxidative and mitochondrial proteins damage (reviewed by Bianchi) [185]. In this regard, events such as peroxiredoxin II, mitochondrial complex III, calcium in the pathogenesis of PD, DLB, and related disorders may channel, and others. A subsequent study in PD brain showed represent a novel variation of the DAMP response, and in a decreased frontal cortex levels of mortalin, a novel mito- sense, biological fluid markers are therefore a measurement chondrial chaperone protein with roles in energy generation of DAMP activity as it relates to neurodegeneration. [181]. 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