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Alterations in mitochondria-endoplasmic reticulum connectivity in human brain biopsies from idiopathic normal pressure hydrocephalus patients

Alterations in mitochondria-endoplasmic reticulum connectivity in human brain biopsies from... Idiopathic normal pressure hydrocephalus (iNPH) is a neuropathology with unknown cause characterised by gait impairment, cognitive decline and ventriculomegaly. These patients often present comorbidity with Alzheimer’sdisease (AD), including AD pathological hallmarks such as amyloid plaques mainly consisting of amyloid β-peptide and neurofibrillary tangles consisting of hyperphosphorylated tau protein. Even though some of the molecular mechanisms behind AD are well described, little is known about iNPH. Several studies have reported that mitochondria-endoplasmic reticulum contact sites (MERCS) regulate amyloid β-peptide metabolism and conversely that amyloid β-peptide can influence the number of MERCS. MERCS have also been shown to be dysregulated in several neurological pathologies including AD. In this study we have used transmission electron microscopy and show, for the first time, several mitochondria contact sites including MERCS in human brain biopsies. These unique human brain samples were obtained during neurosurgery from 14 patients that suffer from iNPH. Three of these 14 patients presented comorbidities with other dementias: one patient with AD, one with AD and vascular dementia and one patient with Lewy body dementia. Furthermore, we report that the numbers of MERCS are increased in biopsies obtained from patients diagnosed with dementia. Moreover, the presence of both amyloid plaques and neurofibrillary tangles correlates with decreased contact length between endoplasmic reticulum and mitochondria, while amyloid plaques alone do not seem to affect endoplasmic reticulum-mitochondria apposition. Interestingly, we report a significant positive correlation between the number of MERCS and ventricular cerebrospinal fluid amyloid β-peptide levels, as well as with increasing age of iNPH patients. Keywords: Brain biopsies, iNPH, Amyloid β-peptide,Tau,MERCS,MAM Introduction the symptoms in certain patients [15]. Interestingly, iNPH Idiopathic normal pressure hydrocephalus (iNPH) is a patients often present comorbidity with Alzheimer’sdisease neurological disease with unknown aetiology, characterised (AD) [17]. AD is characterized by two major hallmarks: by gait and cognitive impairment as well as enlarged cere- extracellular amyloid plaques, mainly consisting of amyloid bral ventricles (ventriculomegaly) [37]. The exact molecular β-peptide (Aβ), and intracellular neurofibrillary tangles mechanisms underlying this pathology are still unknown. (NFT), consisting of hyperphosphorylated tau (pTau) Currently, the only available treatment for iNPH, is the protein [29]. Due to the often co-existing lesions in these implementation of a surgical CSF shunt which alleviates two diseases, it is important to understand the specific mechanistic differences and similarities between AD and iNPH to better diagnose and treat patients. * Correspondence: maria.ankarcrona@ki.se Center for Alzheimer Research, Division of Neurogeriatrics, Department of Recent studies from our and other laboratories have Neurobiology, Care Sciences and Society, Karolinska Institutet, Novum 5th highlighted the role of mitochondria-endoplasmic reticulum floor, SE-141 57 Huddinge, Sweden (ER) contact sites (MERCS) in neurodegenerative disorders Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Leal et al. Acta Neuropathologica Communications (2018) 6:102 Page 2 of 9 [1, 5, 18, 28, 33]. MERCS are formed where the outer mito- was made three centimetres laterally from the midline chondrial membrane interacts with a specific sub-region of close to the coronal suture in anesthetized iNPH patients. ER that presents a lipid raft-like domain commonly known One to three cylindrical cortical biopsies (2-5 mm in as mitochondria associated membranes (MAM) [7]. diameter, 3-7 mm in length) were taken using disposable MERCS are involved in several cellular mechanisms like 14G biopsy needle (Temno™, BD, Franklin Lakes, NJ, 2+ Ca -shuttling from ER to mitochondria, phospholipid USA). The biopsy material was placed in fixative solution metabolism, autophagosome formation and Aβ metabolism (1% glutaraldehyde and 3.7% formaldehyde in sodium [22]. ChangesinMERCS have been shownina varietyof phosphate buffer) 10 min after collection. The samples diseases like AD, cancer, diabetes, obesity, Parkinson’s were kept in fixative solution 4–14 days and subsequently disease, traumatic brain injury and FTD/ALS. embedded into the paraffin. Consecutive 7 μmthick However, structural analysis of MERCS in human brain sections were stained with hematoxylin-eosin (HE) and material has so far not been performed. In this study we, immunohistochemistry (IHC) including pTau (MN-1020, for the first time, have visualized MERCS and other types clone AT8 IGH 135; Thermofisher) and Aβ (6F/3D, of mitochondria contacts in a unique material: brain biop- M0872; Dako). Stained sections were assessed under light sies from patients undergoing iNPH reversal surgery. Fur- microscopy at × 100 to × 200 magnifications. Cellular or thermore, as iNPH patients often have AD-related lesions, neuritic pTau-structures were identified and rated as nega- we saw this material as an opportunity to assess any poten- tive or positive. In Aβ-IHC stained sections, fleecy, diffuse tial connectionofthe pathological hallmarks seeninAD and dense plaques were assessed (Dr Rauramaa) and the and contact formation. We analysed MERCS in iNPH suf- staining results of Aβ semi quantitatively rated [31]. ferers by grouping patients based on their dementia diag- A total of 14 human biopsies from 14 patients were nose or presence of amyloid plaques and NFT at time of analysed (age range 71 to 86 years, average 77.3 years surgery. Interestingly, we detected an increased number of old, 28.6% males and 71.4% females). The same patients MERCS per cell profile in samples from patients diagnosed were stratified in two different ways: first in two groups with dementia (Lewy body dementia (LBD), vascular de- according to presence or absence of dementia diagnose mentia (VaD), AD). Positive correlations between the num- and second in three groups according to the presence of + + ber of MERCS per cell profile and age as well as ventricular amyloid plaques and NFT (Aβ /tau ), plaques only + − − − cerebrospinal fluid (CSF) Aβ42 levels were also detected. (Aβ /tau ) or negative staining (Aβ /tau ) (Table 1 and Additional file 1: Figure S1). Material and methods Human brain biopsies CSF sampling and analysis Human brain biopsies were obtained as previously CSF samples were obtained by lumbar puncture during described [26]. In brief, a right frontal 12-mm burr hole diagnostic tap-test at outpatient clinic or during insertion Table 1 Clinical data collected from iNPH patients # Gender Age Comorbidities MMSE CSF (lumbar) CSF (ventricular) in ng/L in ng/L Aβ42 p-Tau Total-Tau Aβ42 p-Tau Total-Tau − − Aβ /tau 1 F 75 NI 22 NA NA NA 1286,98 93,14 827,59 2 F 76 NI 23 655,03 20,88 90,38 655,03 31,56 316,31 3 F 77 NI 25 904,05 35,99 185,81 311,68 28,50 228,98 4 M 75 NI 19 1092,13 36,74 292,90 650,03 42,09 625,81 + − Aβ /tau 5 M 86 LBD/VaD 13 NA NA NA NA NA NA 6 F 79 NI 24 833,23 32,66 225,14 817,66 35,22 328,78 7 M 79 NI 19 489,59 25,66 225,62 124,05 46,87 1001,38 8 F 71 NI 20 785,34 24,49 128,72 618,75 123,54 3122,29 9 F 76 NI 23 463,30 36,63 218,42 326,77 74,43 1118,52 + + Aβ /tau 10 F 78 NI 23 611,61 38,54 228,68 502,86 52,52 505,93 11 F 77 NI 28 860,71 28,01 152,77 464,72 70,77 1619,43 12 F 74 NI 24 876,30 57,34 311,77 281,83 46,13 506,74 13 M 79 AD/VaD 14 436,77 29,93 176,40 282,76 111,23 2252,69 14 F 81 AD 15 695,54 52,00 470,42 569,76 82,00 1005,13 NA Not available, NI Non-identified Leal et al. Acta Neuropathologica Communications (2018) 6:102 Page 3 of 9 of intraventricular catheter. Low protein binding PP tubes Results were used. Samples were centrifuged, divided into 1 mL Organelle contact sites in human brain biopsies tubes and frozen at − 80 °C. CSF AD biomarkers (total Cellular organelles interact with each other through tau, pTau ,Aβ ) were measured at the University of membrane contact sites [14]. Here we have identified 181 1–42 Eastern Finland (UEF) Neurology using INNOTEST such contacts using TEM in fixed human brain biopsies. ELISA kits (Fujirebio Europe, Ghent, Belgium). Several diverse organelle contacts were identified including: mitochondria-plasma membrane (PM) (Fig. 1a, left) [36], mitochondria-nucleus (Fig. 1a, middle left) MMSE [24], mitochondria-Golgi (Fig. 1a, middle right) [4] and The Mini-Mental State Examination (MMSE, range mitochondria-lysosome (Fig. 1a, right) [12]. However, 0–30) was used to evaluate patients’ cognitive function the most common membrane contact site observed were [8]. Patients were classified into three groups: no signifi- MERCS [35] (Fig. 1b). In fact, 12.8 ± 0.5% of the mito- cant cognitive impairment (27 ≤ MMSE ≤30), minor chondria profile surface was found to be in contact with cognitive impairment (23 ≤ MMSE ≤26) or moderate or ER and, on average, 45.9 ± 3.4% of mitochondria were in severe cognitive impairment (MMSE ≤22) [16]. contact with at least one stretch of ER. In accordance with previous studies [10], we also detected different types of MERCS in human brain in our TEM analysis. While in some electron micrographs only a part of ER is Transmission electron microscopy (TEM) and image in contact with mitochondria (Fig. 1b, top panels), other analysis show long extensions of interactions between the two Ultrathin sections from human biopsies were processed organelles (Fig. 1b, bottom left and middle panel). There using Leica Ultracut UCT (Leica, Vienna, Austria) and are also examples where just a branch of ER touches the contrasted with uranyl acetate and lead citrate. Sections outer mitochondrial membrane (Fig. 1b, bottom right were observed with a Tecnai 12 BioTWIN transmission panel). Moreover, as recently reported in mouse brain electron microscope (FEI Company, Eindhoven, The tissue, we also detected MERCS in the pre- and Netherlands) at 100 kV. Digital images were acquired with post-synaptic terminals in human brain (Fig. 1c, left and a Veleta camera (Olympus Soft imaging Solutions, GmbH, right, respectively) [38]. Münster, Germany) at a primary magnification of 20.500×. Pictures were acquired as before [6]. Briefly, 10 random Patients diagnosed with dementia and with lower MMSE cells were chosen per patient and, for each cell, pictures of showed increased number of MERCS all visible mitochondria were taken. In total, 140 cells were Several studies suggest that MERCS are dysregulated in analysed, including more than 800 MERCS and 2000 different neurological disorders [1, 13, 25, 33]. Here we mitochondria. The number of MERCS and mitochondria aimed to study MERCS in human brain biopsies as well as MERCS length and mitochondria perimeter collected from iNPH patients. Therefore, we stratified were obtained using iTEM FEI software (EMSIS GmbH, the clinical data in different groups. Among the 14 Muenster, Germany). MERCS were considered as such patients analysed, only three were diagnosed with at when the distance between ER and mitochondria was least one type of specific dementia (#5 LBD/VaD, #13 equal or bellow 30 nm. Values presented in Additional file VaD/AD, #14 AD). Interestingly, demented patients 1: Table S1 represent average values per cell profile per presented a higher number of MERCS per cell profile as patient. The overall quality of the tissue was very high and compared to non-demented (Fig. 2a) while no differ- representative images are shown. In particular, the struc- ences were observed in MERCS length per cell profile ture of mitochondria was very well preserved while the ER (Fig. 2b). In addition, patients with moderate or severe structures sometimes appeared dilated. cognitive impairment (MMSE ≤22) presented a higher number of MERCS per cell profile as well as MERCS Statistical analysis perimeter when compared to patients with mild Data were analysed using IBM SPSS Statistics 24 soft- cognitive impairment (23 ≤ MMSE ≤26) or no significant ware (IMB Corportation, New York, NY, USA). Data did cognitive impairment (27 ≤ MMSE ≤30) (Fig. 2c and d). not follow normal distribution and therefore samples Since only one patient presented MMSE ≥27 no statis- were compared two by two by non-parametric inde- tical analysis was performed. To confirm these data, we pendent test (Mann-Whitney U test). For correlation performed correlation studies between MMSE and studies the Pearson correlation coefficient (r) was used MERCS number and length (Additional file 1: Figure as our data was numeric and continuous. All values are S3). For both cases we saw that there was a negative cor- expressed as mean ± SEM, n = correspond to number of relation between MMSE and number (Additional file 1: patients, * p < 0.05 was considered to be significant. Figure S3a) and length (Additional file 1: Figure S3b) of Leal et al. Acta Neuropathologica Communications (2018) 6:102 Page 4 of 9 Fig. 1 Selected electron micrographs of human brain biopsies from iNPH patients. a Interaction of mitochondria with plasma membrane (PM), nucleus (n), Golgi and lisosomes (L) (from left to right). b Interactions between ER and mitochondria (m). Small stretches of ER in contact with mitochondria (top left and right), longer contacts of ER in contact with mitochondria (bottom panel, left and middle), and a point contact (bottom, right). c MERCS in pre-synaptic (left) and post-synaptic (right) density. Black arrow head – mitochondria-PM interaction, black arrow – mitochondria- nucleus interaction, # – mitochondria-lysosome interaction, white arrow – ER, white arrow head – MERCS, * - synapse. Scale bar = 500 nm MERCS. Since mitochondria surface area could influ- with thenumberand/orlengthofMERCS.Wefound that ence MERCS we also measured number of mitochondria the number of MERCS had a significant positive correlation profile and perimeter. No significant differences were with increasing patients’ age (r = 0.653, p = 0.011) (Fig. 3a). observed (Additional file 1: Figure S2a-d). Like mentioned before, iNPH patients often present comor- bidity with AD. We found that there was a significant posi- Number of MERCS correlates with age and ventricular CSF tive correlation between the number of MERCS and the Aβ42 levels levels of ventricular CSF Aβ42 (r = 0.713, p = 0.006) (Fig. As we had access to several clinical parameters collected 3b). Although, no significant correlations were detected from the iNPH patients (Table 1), we used Pearson’scorrel- between number of MERCS and lumbar CSF Aβ42 nor ation coefficient in order to identify possible correlations between MERCS, pTau and total tau levels. Leal et al. Acta Neuropathologica Communications (2018) 6:102 Page 5 of 9 ab cd Fig. 2 Patients diagnose with dementia and with lower MMSE present increased MERCS. Quantification of a number and b length of MERCS from the electron micrographs of iNPH patients’ biopsies according to dementia diagnose. Non-demented patients are #1 to #4 and #6 to #12, demented patients are #5, #13 and #14. Each point represent one iNPH patient. Quantification of c number and d length of MERCS from the electron micrographs of iNPH patients’ biopsies according to MMSE. MMSE scores represent: MMSE ≥27 – No significant cognitive impairment, 23 ≤ MMSE ≤26 – Minor cognitive impairment, MMSE ≤22 – moderate or severe cognitive impairment + + + − The presence of amyloid plaques and NFT correlates with in the Aβ /tau group as compared to the Aβ /tau and − − shorter MERCS in human brain Aβ /tau groups (Fig. 4b and Additional file 1:Table S1). Due to the fact that iNPH and AD can coexist histopatho- These data were further corroborated analysing the median + + logical analysis was performed, and brain biopsies divided of MERCS length of Aβ /tau samples (median = 118.5) − − + − into three groups according to the presence or absence of compared to Aβ /tau (median = 162.1) and Aβ /tau − − + − + + amyloid plaques and NFT (Aβ /tau ,Aβ /tau ,Aβ /tau ) (median = 151.1) samples. Meanwhile, the number of (Material and methods,Table 1 and Additional file 1: MERCS was similar between the three groups (Fig. 4a and Figure S1). The length of MERCS was significantly shorter Additional file 1: Table S1). Quantifications of the number ab Fig. 3 Number of contacts positively correlate with age and levels of Aβ42. Representation of the correlation between mitochondria-ER contacts a with age and b with ventricular levels of Aβ42. Linear regression was performed and the Pearson correlation coeficient (r) calculated. Each point represent one iNPH patient Leal et al. Acta Neuropathologica Communications (2018) 6:102 Page 6 of 9 ab Fig. 4 Patients with amyloid plaques and NFT present smaller contacts. Quantification of a number and b length of mitochondria-ER contacts − − from the electron micrographs of iNPH patients biopsies. c Representation of the % of distribution of contacts according to their length. Aβ /tau + − + + patients present no amyloid plaques nor NFT; Aβ /tau patients presents amyloid plaques but not NFT; and Aβ /tau patients present both amyloid plaques and NFT. Each point represent one iNPH patient of mitochondrial profiles and mitochondrial perimeter re- described membrane contact sites in human brain cells. vealed no significant differences between the three different Our study expands on the extensive work carried out by groups (Additional file 1: Figure S4a, b and Table S1). Wu and colleagues in which they assessed MERCS distribu- In a previous study by Area-Gomez et al. MERCS length tion in mouse brain tissue. Importantly, we similarly showed was categorized into three groups: punctate (50 nm), long the existence of MERCS in intact pre- and post-synaptic (50-200 nm) and very long (> 200 nm) [1]. We decided to terminals in this material, hence opening up potential group our data accordingly to assess the distribution of avenues for research on the role of these structures in these the contacts length analysed. We observed that while the important areas of neurons and glial cells [38]. − − Aβ /tau group present a higher percentage of very long Even though MERCS have been shown to be dysregu- contacts (40.5%) when compared to punctate contacts lated in a variety of neurodegenerative diseases, the mech- + + (26.9%), the Aβ /tau group showed the opposite pattern anisms behind this disruption and the role of MERCS in with less very long contacts (22.7%) and more punctate different pathologies is still largely unknown. Here, we contacts (50.3%) (Fig. 4c). These findings support the idea used iNPH patient samples to assess the relationship that the average length of MERCS is shorter in patients’ between MERCS structure and different clinical parame- samples with amyloid plaques and NFT. ters of these patients. Interestingly, iNPH patients with comorbities with AD, VaD/AD or LBD/VaD showed an Discussion increased number of MERCS per cell profile when In this study, we have characterized for the first time compared to non-demented patients. In line with these membrane contact sites in human brain biopsies. We show data low MMSE scores correlated with increased numbers several electron micrographs from TEM of a variety of of MERCS per cell profile. Curiously, the number and membrane contact sites, including mitochondria-PM, function of MERCS have been reported to be increased in mitochondria-nucleus, mitochondria-Golgi and mitochon- e.g. AD and to be decreased in e.g. frontal temporal dria-lysosome contacts. Due to the method used to collect dementia, showing the dubious dysregulation of these the samples and due to the ultrastructural similarities contacts in different diseases [7]. More biopsies of patients between different cell types it was not possible to identify diagnosed with dementia would be necessary to assert neither which cortical layer nor the type of cell analysed. whether these results represent a population’strend or Nevertheless, our data show the existence of previously just an artefact of our small sample set. Nevertheless, to Leal et al. Acta Neuropathologica Communications (2018) 6:102 Page 7 of 9 our knowledge this is the first time that a connection extracellularly by different mechanisms: cleavage by between late stages of dementia and an increased number neprilysin (on the cell membrane), transport across the of MERCS per cell profile has been observed and reported blood-brain-barrier, drainage into CSF via interstitial in human brain tissue. fluid bulk flow or by absorption of CSF into the lymph- The major risk factor for neurodegenerative diseases is atic and circulatory system [34]. Furthermore, it is ageing. Neurons are non-dividing post-mitotic cells and thought that while Aβ40 is mainly degraded intracellu- are particularly affected by noxious stimuli. Ageing neu- larly, Aβ42 is degraded extracellularly [11]. Surprisingly, rons experience increased oxidative stress, accumulation no correlation between lumbar CSF Aβ42 and MERCS of damaged proteins and energy imbalance. Due to their were found. Likewise, no significant correlations were substantial energetic demands and delicate physiology, observed between CSF total-tau or CSF pTau and the neurons are more sensitive to cell stress leading to number of MERCS per cell profile. Up to date only two deregulated homeostasis and death [21]. Interestingly, we publications have reported a connection between tau observed a positive correlation between age of the patients protein and MERCS. Perreault and colleagues showed analysed and the number of MERCS per cell profile. that the tau mutant JNLP3 increased the proximity Although mitochondria are commonly associated with between mitochondria and ER [23], and Cieri and ATP production they also have a major role in controlling colleagues showed that a form of truncated tau (caspase cell death processes. Both apoptosis and necrosis can be 3-cleaved 2N4RΔC20 tau) has the same effect [3]. Yet, 2+ triggered by changes in Ca levels in mitochondria. Influx the role of pathological pTau on ER-mitochondria 2+ of Ca has been shown to induce opening of the mito- dynamics remains largely unknown. So far, our data chondrial permeability transition pore which leads to loss suggests that increased levels of ventricular Aβ and of the mitochondrial membrane potential leading to cell number of MERCS are positively correlated, unlike tau 2+ death. Ca has also been described as a regulator of protein and MERCS. Further studies will be required to several mitochondrial dehydrogenases in the Krebs cycle confirm our findings and investigate further the role of including pyruvate dehydrogenase. In fact, increased levels Aβ and tau on MERCS dynamics. 2+ of Ca in mitochondria lead to phosphorylation of pyru- As already mentioned iNPH patients often show vate dehydrogenase reducing its activity and affecting ATP comorbidities with AD, including the respective levels. Since it has been shown that increased connectivity hallmarks, amyloid plaques and NFT. Even though some between ER and mitochondria leads to increased shuttling studies have shown that Aβ affects MERCS no reports 2+ of Ca from ER to mitochondria these changes could lead have revealed the effect of amyloid plaques and NFT in to neuronal death. [2, 7]. Therefore, we believe that the MERCS. Therefore, the relationship between these reported increase of MERCS per cell profile, both in hallmarks and MERCS in human brain remains elusive. demented patients and with increasing age, could contrib- We decided to categorize our samples according to the ute to synaptic loss and cognitive decline. presence or absence of amyloid plaques and NFT and Recently, we and others have shown in different investigate if amyloid plaques alone or together with models that Aβ causes an increased connectivity NFT had an impact on MERCS. Our data show that between mitochondria and ER [13, 27, 39]. We have also patients with both amyloid plaques and NFT presented observed that Aβ is formed in subcellular fractions shorter MERCS as compared with patients lacking these enriched in MAM and MERCS modulation leads to hallmarks or presenting just amyloid plaques (Fig. 4b changes in intra- and extracellular Aβ levels [18, 28]. and c). No changes in the number of MERCS per cell However, most of these studies have relied on in vitro profile were detected here. At a first glance, these experiments and mouse models with increased levels of findings may seem to be contradictory to the previous Aβ which do not always mimic perfectly the progression correlation results discussed above: simultaneous of the pathology as observed in humans. In concordance, presence of amyloid plaques and NFT cause decreased we report a positive correlation between ventricular connectivity between mitochondria and ER, while Aβ Aβ42 levels and number of MERCS in this study; (monomeric/oligomeric) cause increased contact between indicating an increased connectivity between the two the two organelles. However, here samples were grouped organelles. However, we should consider the limitation based on histological characterization of amyloid plaques of this correlation since we are comparing ventricular and NFT staining while soluble Aβ-levels were not consid- CSF Aβ42 levels and MERCS number obtained from ered. Importantly, several studies show that oligomeric analysed organelles within the cell. Aβ has been shown species of Aβ, and not plaques per se, are the main driver to be cleared both inside and outside the cell. Inside the of toxicity in AD. In fact, there is a lack of correlation cell Aβ can be degraded by insulin degrading enzyme in between the plaque burden and the progression of AD [9, the cytosol and endosomes, and presequence peptidase 19]. Furthermore, there is substantial neuronal death in in the mitochondria [20]. Aβ can also be cleared regions lacking plaques, while plaques were found in Leal et al. Acta Neuropathologica Communications (2018) 6:102 Page 8 of 9 patients with no cognitive impairment [30, 32]. Our for helpful discussions; Gabriele Turacchi for help in the MERCS quantification and Marita Parviainen for patient management. present data suggests that in samples with amyloid These studies were supported grants from: Gun and Bertil Stohne’s plaques alone (NFT negative samples) MERCS are not af- Foundation, Gamla Tjänarinnor Foundation, Swedish Dementia Foundation, fected, while the levels of ventricular CSF Aβ42 correlate The Foundation for Geriatric Diseases at Karolinska Institutet and Kuopio University Hospital VTR Fund. Karolinska Institutet Doctoral Grant and Gun with the number of these contacts. Therefore, we postu- och Bertil Stohne’s Research Stipend to NSL, and Marie Skłodowska Curie ITN late that intracellular Aβ and amyloid plaques seem to grant SyDAD to GD. have different effects on MERCS, however further studies Availability of data and materials are required to elucidate the underlying mechanisms. The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Conclusions Authors’ contributions In summary, we show that iNPH patients diagnosed with Patient biopsies and clinical data were obtained by VL, TR, AK, MH, SKH, NP either AD, VaD or LBD present an increased number of and OPK. NSL, BS and MA designed the study. NSL, BS, MA and GD collected MERCS per cell profile. We also show that the number of the data. NSL analysed the data and the data interpretation performed by NSL, BS, GD, MA. NSL, GD and MA did the literature research as well as the MERCS positively correlates with age and levels of ven- writing of the manuscript. NSL and GD generated the figures. All authors tricular CSF Aβ42. In addition, the length of MERCS was had final approval of the submitted and published version. decreased in iNPH patients presenting both amyloid pla- Ethics approval and consent to participate ques and NFT. Together, these findings strengthen the hy- All procedures performed in studies involving human participants were in pothesis that MERCS affect cell homeostasis and could be accordance with the ethical standards of the institutional and/or national one of the players in the neurodegenerative process found research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The brain biopsy part was in different diseases like AD and iNPH. Future studies in approved by the Kuopio University Hospital Research Ethics Committee (5/ relevant models are needed to reveal the exact cellular 2008, 19.3.2008). mechanisms and can also be used to test to drug candi- dates correcting the ER-mitochondria interplay. Consent for publication All iNPH patients or their next-of-kin gave their written informed consent. Additional file Competing interests The authors declare that they have no competing interests. Additional file 1: Figure S1. Immuno-labelling of biopsies of frontal cortices of iNPH patients. Representative immunohistochemistry pictures Publisher’sNote of frontal cortices of patients analysed. Patients were divided in groups Springer Nature remains neutral with regard to jurisdictional claims in according to the presence or absence of amyloid plaques and NFT. Anti- published maps and institutional affiliations. Aβ antibody (6F/3D, M0872; Dako) (first column) and anti-p-Tau antibody (AT8) (second column) were used. The arrow indicate a NFT and the star Author details indicates neuropil threads. Scale bar = 500 μm. Table S1. Electron micros- Center for Alzheimer Research, Division of Neurogeriatrics, Department of copy measurements and respective averages. Figure S2. Mitochondria Neurobiology, Care Sciences and Society, Karolinska Institutet, Novum 5th number and perimeter are not significantly changed in patients diag- floor, SE-141 57 Huddinge, Sweden. Institute of Clinical Medicine – nosed with dementia. Quantification of a number and b perimeter of Neurosurgery, University of Eastern Finland, Kuopio, Finland. Department of mitochondria profiles from the electron micrographs of iNPH patients’ Neurosurgery, Kuopio University Hospital, Kuopio, Finland. Institute of biopsies according to dementia diagnose. Non-demented patients are #1 Clinical Medicine – Neurology, University of Eastern Finland, Kuopio, Finland. to #4 and #6 to #12, demented patients are #5, #13 and #14. Each point Department of Neurology, Kuopio University Hospital, Kuopio, Finland. represent one iNPH patient. Quantification of c number and d perimeter Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland. of mitochondria profiles from the electron micrographs of iNPH patients’ Institute of Clinical Medicine – Pathology, University of Eastern Finland, biopsies according to MMSE. MMSE scores represent: MMSE ≥27 – No Kuopio, Finland. Department of Pathology, Kuopio University Hospital, significant cognitive impairment, 23 ≤ MMSE ≤26 – Minor cognitive Kuopio, Finland. Unit of Clinical Neuroscience, Neurosurgery, University of impairment, MMSE ≤22 – moderate or severe cognitive impairment. Oulu, Oulu, Finland. Medical Research Center, Oulu University Hospital, Figure S3. Number and length of MERCS negatively with MMSE. Repre- Oulu, Finland. sentation of the correlation between MMSE and mitochondria-ER contact sites a number and b length. Linear regression was performed and the Received: 21 September 2018 Accepted: 21 September 2018 Pearson correlation coefficient (r) calculated. Each point represent one iNPH patient. Figure S4. Amyloid plaques and NFT have no effect in the number of mitochondria profile nor mitochondria perimeter. Quantifica- References tion of a number and b perimeter of mitochondria profiles from the elec- − − 1. Area-Gomez E, del Carmen Lara Castillo M, Tambini MD, Guardia-Laguarta C, tron micrographs of iNPH patients biopsies. Aβ /tau patients present no + − de Groof AJC, Madra M, Ikenouchi J, Umeda M, Bird TD, Sturley SL, Schon E amyloid plaques nor NFT; Aβ /tau patients presents amyloid plaques + + (2012) Upregulated function of mitochondria-associated ER membranes in but not NFT; and Aβ /tau patients present both amyloid plaques and Alzheimer disease. EMBO J 31:4106–4123. https://doi.org/10.1038/emboj. NFT. Each point represent a different iNPH patient. (PDF 16959 kb) 2012.202 2. Bravo-Sagua R, Rodriguez AE, Kuzmicic J, Gutierrez T, Lopez-Crisosto C, Quiroga Acknowledgments C, Díaz-Elizondo J, Chiong M, Gillette TG, Rothermel BA, Lavandero S (2013) The authors would like to thank: Dr. Kjell Hultenby, Eva Blomén and Eva Cell death and survival through the endoplasmic reticulum-mitochondrial axis. Idsund Jonsson (EMil, Dept Laboratory Medicine; Karolinska Institutet, Curr Mol Med. https://doi.org/10.2174/1566524011313020008 Huddinge, Sweden) for excellent help with transmission electron 3. Cieri D, Vicario M, Vallese F, D’Orsi B, Berto P, Grinzato A, Catoni C, De microscopy; Joana Braga Pereira, Konstantinos Poulakis and Emilia Stefani D, Rizzuto R, Brini M, Calì T (2018) Tau localises within mitochondrial Schwertner for statistic and MMSE stratification help; Catarina Moreira Pinho sub-compartments and its caspase cleavage affects ER-mitochondria Leal et al. Acta Neuropathologica Communications (2018) 6:102 Page 9 of 9 interactions and cellular ca 2+ handling. Biochim Biophys Acta - Mol Basis 22. Paillusson S, Stoica R, Gomez-Suaga P, Lau DHW, Mueller S, Miller T, Miller Dis. https://doi.org/10.1016/j.bbadis.2018.07.011 CCJ (2016) There’s something wrong with my MAM; the ER-mitochondria 4. 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Alterations in mitochondria-endoplasmic reticulum connectivity in human brain biopsies from idiopathic normal pressure hydrocephalus patients

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Springer Journals
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Copyright © 2018 by The Author(s).
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Biomedicine; Neurosciences; Pathology; Neurology
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10.1186/s40478-018-0605-2
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Abstract

Idiopathic normal pressure hydrocephalus (iNPH) is a neuropathology with unknown cause characterised by gait impairment, cognitive decline and ventriculomegaly. These patients often present comorbidity with Alzheimer’sdisease (AD), including AD pathological hallmarks such as amyloid plaques mainly consisting of amyloid β-peptide and neurofibrillary tangles consisting of hyperphosphorylated tau protein. Even though some of the molecular mechanisms behind AD are well described, little is known about iNPH. Several studies have reported that mitochondria-endoplasmic reticulum contact sites (MERCS) regulate amyloid β-peptide metabolism and conversely that amyloid β-peptide can influence the number of MERCS. MERCS have also been shown to be dysregulated in several neurological pathologies including AD. In this study we have used transmission electron microscopy and show, for the first time, several mitochondria contact sites including MERCS in human brain biopsies. These unique human brain samples were obtained during neurosurgery from 14 patients that suffer from iNPH. Three of these 14 patients presented comorbidities with other dementias: one patient with AD, one with AD and vascular dementia and one patient with Lewy body dementia. Furthermore, we report that the numbers of MERCS are increased in biopsies obtained from patients diagnosed with dementia. Moreover, the presence of both amyloid plaques and neurofibrillary tangles correlates with decreased contact length between endoplasmic reticulum and mitochondria, while amyloid plaques alone do not seem to affect endoplasmic reticulum-mitochondria apposition. Interestingly, we report a significant positive correlation between the number of MERCS and ventricular cerebrospinal fluid amyloid β-peptide levels, as well as with increasing age of iNPH patients. Keywords: Brain biopsies, iNPH, Amyloid β-peptide,Tau,MERCS,MAM Introduction the symptoms in certain patients [15]. Interestingly, iNPH Idiopathic normal pressure hydrocephalus (iNPH) is a patients often present comorbidity with Alzheimer’sdisease neurological disease with unknown aetiology, characterised (AD) [17]. AD is characterized by two major hallmarks: by gait and cognitive impairment as well as enlarged cere- extracellular amyloid plaques, mainly consisting of amyloid bral ventricles (ventriculomegaly) [37]. The exact molecular β-peptide (Aβ), and intracellular neurofibrillary tangles mechanisms underlying this pathology are still unknown. (NFT), consisting of hyperphosphorylated tau (pTau) Currently, the only available treatment for iNPH, is the protein [29]. Due to the often co-existing lesions in these implementation of a surgical CSF shunt which alleviates two diseases, it is important to understand the specific mechanistic differences and similarities between AD and iNPH to better diagnose and treat patients. * Correspondence: maria.ankarcrona@ki.se Center for Alzheimer Research, Division of Neurogeriatrics, Department of Recent studies from our and other laboratories have Neurobiology, Care Sciences and Society, Karolinska Institutet, Novum 5th highlighted the role of mitochondria-endoplasmic reticulum floor, SE-141 57 Huddinge, Sweden (ER) contact sites (MERCS) in neurodegenerative disorders Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Leal et al. Acta Neuropathologica Communications (2018) 6:102 Page 2 of 9 [1, 5, 18, 28, 33]. MERCS are formed where the outer mito- was made three centimetres laterally from the midline chondrial membrane interacts with a specific sub-region of close to the coronal suture in anesthetized iNPH patients. ER that presents a lipid raft-like domain commonly known One to three cylindrical cortical biopsies (2-5 mm in as mitochondria associated membranes (MAM) [7]. diameter, 3-7 mm in length) were taken using disposable MERCS are involved in several cellular mechanisms like 14G biopsy needle (Temno™, BD, Franklin Lakes, NJ, 2+ Ca -shuttling from ER to mitochondria, phospholipid USA). The biopsy material was placed in fixative solution metabolism, autophagosome formation and Aβ metabolism (1% glutaraldehyde and 3.7% formaldehyde in sodium [22]. ChangesinMERCS have been shownina varietyof phosphate buffer) 10 min after collection. The samples diseases like AD, cancer, diabetes, obesity, Parkinson’s were kept in fixative solution 4–14 days and subsequently disease, traumatic brain injury and FTD/ALS. embedded into the paraffin. Consecutive 7 μmthick However, structural analysis of MERCS in human brain sections were stained with hematoxylin-eosin (HE) and material has so far not been performed. In this study we, immunohistochemistry (IHC) including pTau (MN-1020, for the first time, have visualized MERCS and other types clone AT8 IGH 135; Thermofisher) and Aβ (6F/3D, of mitochondria contacts in a unique material: brain biop- M0872; Dako). Stained sections were assessed under light sies from patients undergoing iNPH reversal surgery. Fur- microscopy at × 100 to × 200 magnifications. Cellular or thermore, as iNPH patients often have AD-related lesions, neuritic pTau-structures were identified and rated as nega- we saw this material as an opportunity to assess any poten- tive or positive. In Aβ-IHC stained sections, fleecy, diffuse tial connectionofthe pathological hallmarks seeninAD and dense plaques were assessed (Dr Rauramaa) and the and contact formation. We analysed MERCS in iNPH suf- staining results of Aβ semi quantitatively rated [31]. ferers by grouping patients based on their dementia diag- A total of 14 human biopsies from 14 patients were nose or presence of amyloid plaques and NFT at time of analysed (age range 71 to 86 years, average 77.3 years surgery. Interestingly, we detected an increased number of old, 28.6% males and 71.4% females). The same patients MERCS per cell profile in samples from patients diagnosed were stratified in two different ways: first in two groups with dementia (Lewy body dementia (LBD), vascular de- according to presence or absence of dementia diagnose mentia (VaD), AD). Positive correlations between the num- and second in three groups according to the presence of + + ber of MERCS per cell profile and age as well as ventricular amyloid plaques and NFT (Aβ /tau ), plaques only + − − − cerebrospinal fluid (CSF) Aβ42 levels were also detected. (Aβ /tau ) or negative staining (Aβ /tau ) (Table 1 and Additional file 1: Figure S1). Material and methods Human brain biopsies CSF sampling and analysis Human brain biopsies were obtained as previously CSF samples were obtained by lumbar puncture during described [26]. In brief, a right frontal 12-mm burr hole diagnostic tap-test at outpatient clinic or during insertion Table 1 Clinical data collected from iNPH patients # Gender Age Comorbidities MMSE CSF (lumbar) CSF (ventricular) in ng/L in ng/L Aβ42 p-Tau Total-Tau Aβ42 p-Tau Total-Tau − − Aβ /tau 1 F 75 NI 22 NA NA NA 1286,98 93,14 827,59 2 F 76 NI 23 655,03 20,88 90,38 655,03 31,56 316,31 3 F 77 NI 25 904,05 35,99 185,81 311,68 28,50 228,98 4 M 75 NI 19 1092,13 36,74 292,90 650,03 42,09 625,81 + − Aβ /tau 5 M 86 LBD/VaD 13 NA NA NA NA NA NA 6 F 79 NI 24 833,23 32,66 225,14 817,66 35,22 328,78 7 M 79 NI 19 489,59 25,66 225,62 124,05 46,87 1001,38 8 F 71 NI 20 785,34 24,49 128,72 618,75 123,54 3122,29 9 F 76 NI 23 463,30 36,63 218,42 326,77 74,43 1118,52 + + Aβ /tau 10 F 78 NI 23 611,61 38,54 228,68 502,86 52,52 505,93 11 F 77 NI 28 860,71 28,01 152,77 464,72 70,77 1619,43 12 F 74 NI 24 876,30 57,34 311,77 281,83 46,13 506,74 13 M 79 AD/VaD 14 436,77 29,93 176,40 282,76 111,23 2252,69 14 F 81 AD 15 695,54 52,00 470,42 569,76 82,00 1005,13 NA Not available, NI Non-identified Leal et al. Acta Neuropathologica Communications (2018) 6:102 Page 3 of 9 of intraventricular catheter. Low protein binding PP tubes Results were used. Samples were centrifuged, divided into 1 mL Organelle contact sites in human brain biopsies tubes and frozen at − 80 °C. CSF AD biomarkers (total Cellular organelles interact with each other through tau, pTau ,Aβ ) were measured at the University of membrane contact sites [14]. Here we have identified 181 1–42 Eastern Finland (UEF) Neurology using INNOTEST such contacts using TEM in fixed human brain biopsies. ELISA kits (Fujirebio Europe, Ghent, Belgium). Several diverse organelle contacts were identified including: mitochondria-plasma membrane (PM) (Fig. 1a, left) [36], mitochondria-nucleus (Fig. 1a, middle left) MMSE [24], mitochondria-Golgi (Fig. 1a, middle right) [4] and The Mini-Mental State Examination (MMSE, range mitochondria-lysosome (Fig. 1a, right) [12]. However, 0–30) was used to evaluate patients’ cognitive function the most common membrane contact site observed were [8]. Patients were classified into three groups: no signifi- MERCS [35] (Fig. 1b). In fact, 12.8 ± 0.5% of the mito- cant cognitive impairment (27 ≤ MMSE ≤30), minor chondria profile surface was found to be in contact with cognitive impairment (23 ≤ MMSE ≤26) or moderate or ER and, on average, 45.9 ± 3.4% of mitochondria were in severe cognitive impairment (MMSE ≤22) [16]. contact with at least one stretch of ER. In accordance with previous studies [10], we also detected different types of MERCS in human brain in our TEM analysis. While in some electron micrographs only a part of ER is Transmission electron microscopy (TEM) and image in contact with mitochondria (Fig. 1b, top panels), other analysis show long extensions of interactions between the two Ultrathin sections from human biopsies were processed organelles (Fig. 1b, bottom left and middle panel). There using Leica Ultracut UCT (Leica, Vienna, Austria) and are also examples where just a branch of ER touches the contrasted with uranyl acetate and lead citrate. Sections outer mitochondrial membrane (Fig. 1b, bottom right were observed with a Tecnai 12 BioTWIN transmission panel). Moreover, as recently reported in mouse brain electron microscope (FEI Company, Eindhoven, The tissue, we also detected MERCS in the pre- and Netherlands) at 100 kV. Digital images were acquired with post-synaptic terminals in human brain (Fig. 1c, left and a Veleta camera (Olympus Soft imaging Solutions, GmbH, right, respectively) [38]. Münster, Germany) at a primary magnification of 20.500×. Pictures were acquired as before [6]. Briefly, 10 random Patients diagnosed with dementia and with lower MMSE cells were chosen per patient and, for each cell, pictures of showed increased number of MERCS all visible mitochondria were taken. In total, 140 cells were Several studies suggest that MERCS are dysregulated in analysed, including more than 800 MERCS and 2000 different neurological disorders [1, 13, 25, 33]. Here we mitochondria. The number of MERCS and mitochondria aimed to study MERCS in human brain biopsies as well as MERCS length and mitochondria perimeter collected from iNPH patients. Therefore, we stratified were obtained using iTEM FEI software (EMSIS GmbH, the clinical data in different groups. Among the 14 Muenster, Germany). MERCS were considered as such patients analysed, only three were diagnosed with at when the distance between ER and mitochondria was least one type of specific dementia (#5 LBD/VaD, #13 equal or bellow 30 nm. Values presented in Additional file VaD/AD, #14 AD). Interestingly, demented patients 1: Table S1 represent average values per cell profile per presented a higher number of MERCS per cell profile as patient. The overall quality of the tissue was very high and compared to non-demented (Fig. 2a) while no differ- representative images are shown. In particular, the struc- ences were observed in MERCS length per cell profile ture of mitochondria was very well preserved while the ER (Fig. 2b). In addition, patients with moderate or severe structures sometimes appeared dilated. cognitive impairment (MMSE ≤22) presented a higher number of MERCS per cell profile as well as MERCS Statistical analysis perimeter when compared to patients with mild Data were analysed using IBM SPSS Statistics 24 soft- cognitive impairment (23 ≤ MMSE ≤26) or no significant ware (IMB Corportation, New York, NY, USA). Data did cognitive impairment (27 ≤ MMSE ≤30) (Fig. 2c and d). not follow normal distribution and therefore samples Since only one patient presented MMSE ≥27 no statis- were compared two by two by non-parametric inde- tical analysis was performed. To confirm these data, we pendent test (Mann-Whitney U test). For correlation performed correlation studies between MMSE and studies the Pearson correlation coefficient (r) was used MERCS number and length (Additional file 1: Figure as our data was numeric and continuous. All values are S3). For both cases we saw that there was a negative cor- expressed as mean ± SEM, n = correspond to number of relation between MMSE and number (Additional file 1: patients, * p < 0.05 was considered to be significant. Figure S3a) and length (Additional file 1: Figure S3b) of Leal et al. Acta Neuropathologica Communications (2018) 6:102 Page 4 of 9 Fig. 1 Selected electron micrographs of human brain biopsies from iNPH patients. a Interaction of mitochondria with plasma membrane (PM), nucleus (n), Golgi and lisosomes (L) (from left to right). b Interactions between ER and mitochondria (m). Small stretches of ER in contact with mitochondria (top left and right), longer contacts of ER in contact with mitochondria (bottom panel, left and middle), and a point contact (bottom, right). c MERCS in pre-synaptic (left) and post-synaptic (right) density. Black arrow head – mitochondria-PM interaction, black arrow – mitochondria- nucleus interaction, # – mitochondria-lysosome interaction, white arrow – ER, white arrow head – MERCS, * - synapse. Scale bar = 500 nm MERCS. Since mitochondria surface area could influ- with thenumberand/orlengthofMERCS.Wefound that ence MERCS we also measured number of mitochondria the number of MERCS had a significant positive correlation profile and perimeter. No significant differences were with increasing patients’ age (r = 0.653, p = 0.011) (Fig. 3a). observed (Additional file 1: Figure S2a-d). Like mentioned before, iNPH patients often present comor- bidity with AD. We found that there was a significant posi- Number of MERCS correlates with age and ventricular CSF tive correlation between the number of MERCS and the Aβ42 levels levels of ventricular CSF Aβ42 (r = 0.713, p = 0.006) (Fig. As we had access to several clinical parameters collected 3b). Although, no significant correlations were detected from the iNPH patients (Table 1), we used Pearson’scorrel- between number of MERCS and lumbar CSF Aβ42 nor ation coefficient in order to identify possible correlations between MERCS, pTau and total tau levels. Leal et al. Acta Neuropathologica Communications (2018) 6:102 Page 5 of 9 ab cd Fig. 2 Patients diagnose with dementia and with lower MMSE present increased MERCS. Quantification of a number and b length of MERCS from the electron micrographs of iNPH patients’ biopsies according to dementia diagnose. Non-demented patients are #1 to #4 and #6 to #12, demented patients are #5, #13 and #14. Each point represent one iNPH patient. Quantification of c number and d length of MERCS from the electron micrographs of iNPH patients’ biopsies according to MMSE. MMSE scores represent: MMSE ≥27 – No significant cognitive impairment, 23 ≤ MMSE ≤26 – Minor cognitive impairment, MMSE ≤22 – moderate or severe cognitive impairment + + + − The presence of amyloid plaques and NFT correlates with in the Aβ /tau group as compared to the Aβ /tau and − − shorter MERCS in human brain Aβ /tau groups (Fig. 4b and Additional file 1:Table S1). Due to the fact that iNPH and AD can coexist histopatho- These data were further corroborated analysing the median + + logical analysis was performed, and brain biopsies divided of MERCS length of Aβ /tau samples (median = 118.5) − − + − into three groups according to the presence or absence of compared to Aβ /tau (median = 162.1) and Aβ /tau − − + − + + amyloid plaques and NFT (Aβ /tau ,Aβ /tau ,Aβ /tau ) (median = 151.1) samples. Meanwhile, the number of (Material and methods,Table 1 and Additional file 1: MERCS was similar between the three groups (Fig. 4a and Figure S1). The length of MERCS was significantly shorter Additional file 1: Table S1). Quantifications of the number ab Fig. 3 Number of contacts positively correlate with age and levels of Aβ42. Representation of the correlation between mitochondria-ER contacts a with age and b with ventricular levels of Aβ42. Linear regression was performed and the Pearson correlation coeficient (r) calculated. Each point represent one iNPH patient Leal et al. Acta Neuropathologica Communications (2018) 6:102 Page 6 of 9 ab Fig. 4 Patients with amyloid plaques and NFT present smaller contacts. Quantification of a number and b length of mitochondria-ER contacts − − from the electron micrographs of iNPH patients biopsies. c Representation of the % of distribution of contacts according to their length. Aβ /tau + − + + patients present no amyloid plaques nor NFT; Aβ /tau patients presents amyloid plaques but not NFT; and Aβ /tau patients present both amyloid plaques and NFT. Each point represent one iNPH patient of mitochondrial profiles and mitochondrial perimeter re- described membrane contact sites in human brain cells. vealed no significant differences between the three different Our study expands on the extensive work carried out by groups (Additional file 1: Figure S4a, b and Table S1). Wu and colleagues in which they assessed MERCS distribu- In a previous study by Area-Gomez et al. MERCS length tion in mouse brain tissue. Importantly, we similarly showed was categorized into three groups: punctate (50 nm), long the existence of MERCS in intact pre- and post-synaptic (50-200 nm) and very long (> 200 nm) [1]. We decided to terminals in this material, hence opening up potential group our data accordingly to assess the distribution of avenues for research on the role of these structures in these the contacts length analysed. We observed that while the important areas of neurons and glial cells [38]. − − Aβ /tau group present a higher percentage of very long Even though MERCS have been shown to be dysregu- contacts (40.5%) when compared to punctate contacts lated in a variety of neurodegenerative diseases, the mech- + + (26.9%), the Aβ /tau group showed the opposite pattern anisms behind this disruption and the role of MERCS in with less very long contacts (22.7%) and more punctate different pathologies is still largely unknown. Here, we contacts (50.3%) (Fig. 4c). These findings support the idea used iNPH patient samples to assess the relationship that the average length of MERCS is shorter in patients’ between MERCS structure and different clinical parame- samples with amyloid plaques and NFT. ters of these patients. Interestingly, iNPH patients with comorbities with AD, VaD/AD or LBD/VaD showed an Discussion increased number of MERCS per cell profile when In this study, we have characterized for the first time compared to non-demented patients. In line with these membrane contact sites in human brain biopsies. We show data low MMSE scores correlated with increased numbers several electron micrographs from TEM of a variety of of MERCS per cell profile. Curiously, the number and membrane contact sites, including mitochondria-PM, function of MERCS have been reported to be increased in mitochondria-nucleus, mitochondria-Golgi and mitochon- e.g. AD and to be decreased in e.g. frontal temporal dria-lysosome contacts. Due to the method used to collect dementia, showing the dubious dysregulation of these the samples and due to the ultrastructural similarities contacts in different diseases [7]. More biopsies of patients between different cell types it was not possible to identify diagnosed with dementia would be necessary to assert neither which cortical layer nor the type of cell analysed. whether these results represent a population’strend or Nevertheless, our data show the existence of previously just an artefact of our small sample set. Nevertheless, to Leal et al. Acta Neuropathologica Communications (2018) 6:102 Page 7 of 9 our knowledge this is the first time that a connection extracellularly by different mechanisms: cleavage by between late stages of dementia and an increased number neprilysin (on the cell membrane), transport across the of MERCS per cell profile has been observed and reported blood-brain-barrier, drainage into CSF via interstitial in human brain tissue. fluid bulk flow or by absorption of CSF into the lymph- The major risk factor for neurodegenerative diseases is atic and circulatory system [34]. Furthermore, it is ageing. Neurons are non-dividing post-mitotic cells and thought that while Aβ40 is mainly degraded intracellu- are particularly affected by noxious stimuli. Ageing neu- larly, Aβ42 is degraded extracellularly [11]. Surprisingly, rons experience increased oxidative stress, accumulation no correlation between lumbar CSF Aβ42 and MERCS of damaged proteins and energy imbalance. Due to their were found. Likewise, no significant correlations were substantial energetic demands and delicate physiology, observed between CSF total-tau or CSF pTau and the neurons are more sensitive to cell stress leading to number of MERCS per cell profile. Up to date only two deregulated homeostasis and death [21]. Interestingly, we publications have reported a connection between tau observed a positive correlation between age of the patients protein and MERCS. Perreault and colleagues showed analysed and the number of MERCS per cell profile. that the tau mutant JNLP3 increased the proximity Although mitochondria are commonly associated with between mitochondria and ER [23], and Cieri and ATP production they also have a major role in controlling colleagues showed that a form of truncated tau (caspase cell death processes. Both apoptosis and necrosis can be 3-cleaved 2N4RΔC20 tau) has the same effect [3]. Yet, 2+ triggered by changes in Ca levels in mitochondria. Influx the role of pathological pTau on ER-mitochondria 2+ of Ca has been shown to induce opening of the mito- dynamics remains largely unknown. So far, our data chondrial permeability transition pore which leads to loss suggests that increased levels of ventricular Aβ and of the mitochondrial membrane potential leading to cell number of MERCS are positively correlated, unlike tau 2+ death. Ca has also been described as a regulator of protein and MERCS. Further studies will be required to several mitochondrial dehydrogenases in the Krebs cycle confirm our findings and investigate further the role of including pyruvate dehydrogenase. In fact, increased levels Aβ and tau on MERCS dynamics. 2+ of Ca in mitochondria lead to phosphorylation of pyru- As already mentioned iNPH patients often show vate dehydrogenase reducing its activity and affecting ATP comorbidities with AD, including the respective levels. Since it has been shown that increased connectivity hallmarks, amyloid plaques and NFT. Even though some between ER and mitochondria leads to increased shuttling studies have shown that Aβ affects MERCS no reports 2+ of Ca from ER to mitochondria these changes could lead have revealed the effect of amyloid plaques and NFT in to neuronal death. [2, 7]. Therefore, we believe that the MERCS. Therefore, the relationship between these reported increase of MERCS per cell profile, both in hallmarks and MERCS in human brain remains elusive. demented patients and with increasing age, could contrib- We decided to categorize our samples according to the ute to synaptic loss and cognitive decline. presence or absence of amyloid plaques and NFT and Recently, we and others have shown in different investigate if amyloid plaques alone or together with models that Aβ causes an increased connectivity NFT had an impact on MERCS. Our data show that between mitochondria and ER [13, 27, 39]. We have also patients with both amyloid plaques and NFT presented observed that Aβ is formed in subcellular fractions shorter MERCS as compared with patients lacking these enriched in MAM and MERCS modulation leads to hallmarks or presenting just amyloid plaques (Fig. 4b changes in intra- and extracellular Aβ levels [18, 28]. and c). No changes in the number of MERCS per cell However, most of these studies have relied on in vitro profile were detected here. At a first glance, these experiments and mouse models with increased levels of findings may seem to be contradictory to the previous Aβ which do not always mimic perfectly the progression correlation results discussed above: simultaneous of the pathology as observed in humans. In concordance, presence of amyloid plaques and NFT cause decreased we report a positive correlation between ventricular connectivity between mitochondria and ER, while Aβ Aβ42 levels and number of MERCS in this study; (monomeric/oligomeric) cause increased contact between indicating an increased connectivity between the two the two organelles. However, here samples were grouped organelles. However, we should consider the limitation based on histological characterization of amyloid plaques of this correlation since we are comparing ventricular and NFT staining while soluble Aβ-levels were not consid- CSF Aβ42 levels and MERCS number obtained from ered. Importantly, several studies show that oligomeric analysed organelles within the cell. Aβ has been shown species of Aβ, and not plaques per se, are the main driver to be cleared both inside and outside the cell. Inside the of toxicity in AD. In fact, there is a lack of correlation cell Aβ can be degraded by insulin degrading enzyme in between the plaque burden and the progression of AD [9, the cytosol and endosomes, and presequence peptidase 19]. Furthermore, there is substantial neuronal death in in the mitochondria [20]. Aβ can also be cleared regions lacking plaques, while plaques were found in Leal et al. Acta Neuropathologica Communications (2018) 6:102 Page 8 of 9 patients with no cognitive impairment [30, 32]. Our for helpful discussions; Gabriele Turacchi for help in the MERCS quantification and Marita Parviainen for patient management. present data suggests that in samples with amyloid These studies were supported grants from: Gun and Bertil Stohne’s plaques alone (NFT negative samples) MERCS are not af- Foundation, Gamla Tjänarinnor Foundation, Swedish Dementia Foundation, fected, while the levels of ventricular CSF Aβ42 correlate The Foundation for Geriatric Diseases at Karolinska Institutet and Kuopio University Hospital VTR Fund. Karolinska Institutet Doctoral Grant and Gun with the number of these contacts. Therefore, we postu- och Bertil Stohne’s Research Stipend to NSL, and Marie Skłodowska Curie ITN late that intracellular Aβ and amyloid plaques seem to grant SyDAD to GD. have different effects on MERCS, however further studies Availability of data and materials are required to elucidate the underlying mechanisms. The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Conclusions Authors’ contributions In summary, we show that iNPH patients diagnosed with Patient biopsies and clinical data were obtained by VL, TR, AK, MH, SKH, NP either AD, VaD or LBD present an increased number of and OPK. NSL, BS and MA designed the study. NSL, BS, MA and GD collected MERCS per cell profile. We also show that the number of the data. NSL analysed the data and the data interpretation performed by NSL, BS, GD, MA. NSL, GD and MA did the literature research as well as the MERCS positively correlates with age and levels of ven- writing of the manuscript. NSL and GD generated the figures. All authors tricular CSF Aβ42. In addition, the length of MERCS was had final approval of the submitted and published version. decreased in iNPH patients presenting both amyloid pla- Ethics approval and consent to participate ques and NFT. Together, these findings strengthen the hy- All procedures performed in studies involving human participants were in pothesis that MERCS affect cell homeostasis and could be accordance with the ethical standards of the institutional and/or national one of the players in the neurodegenerative process found research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The brain biopsy part was in different diseases like AD and iNPH. Future studies in approved by the Kuopio University Hospital Research Ethics Committee (5/ relevant models are needed to reveal the exact cellular 2008, 19.3.2008). mechanisms and can also be used to test to drug candi- dates correcting the ER-mitochondria interplay. Consent for publication All iNPH patients or their next-of-kin gave their written informed consent. Additional file Competing interests The authors declare that they have no competing interests. Additional file 1: Figure S1. Immuno-labelling of biopsies of frontal cortices of iNPH patients. Representative immunohistochemistry pictures Publisher’sNote of frontal cortices of patients analysed. Patients were divided in groups Springer Nature remains neutral with regard to jurisdictional claims in according to the presence or absence of amyloid plaques and NFT. Anti- published maps and institutional affiliations. Aβ antibody (6F/3D, M0872; Dako) (first column) and anti-p-Tau antibody (AT8) (second column) were used. The arrow indicate a NFT and the star Author details indicates neuropil threads. Scale bar = 500 μm. Table S1. Electron micros- Center for Alzheimer Research, Division of Neurogeriatrics, Department of copy measurements and respective averages. Figure S2. Mitochondria Neurobiology, Care Sciences and Society, Karolinska Institutet, Novum 5th number and perimeter are not significantly changed in patients diag- floor, SE-141 57 Huddinge, Sweden. Institute of Clinical Medicine – nosed with dementia. Quantification of a number and b perimeter of Neurosurgery, University of Eastern Finland, Kuopio, Finland. Department of mitochondria profiles from the electron micrographs of iNPH patients’ Neurosurgery, Kuopio University Hospital, Kuopio, Finland. Institute of biopsies according to dementia diagnose. Non-demented patients are #1 Clinical Medicine – Neurology, University of Eastern Finland, Kuopio, Finland. to #4 and #6 to #12, demented patients are #5, #13 and #14. Each point Department of Neurology, Kuopio University Hospital, Kuopio, Finland. represent one iNPH patient. Quantification of c number and d perimeter Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland. of mitochondria profiles from the electron micrographs of iNPH patients’ Institute of Clinical Medicine – Pathology, University of Eastern Finland, biopsies according to MMSE. MMSE scores represent: MMSE ≥27 – No Kuopio, Finland. Department of Pathology, Kuopio University Hospital, significant cognitive impairment, 23 ≤ MMSE ≤26 – Minor cognitive Kuopio, Finland. Unit of Clinical Neuroscience, Neurosurgery, University of impairment, MMSE ≤22 – moderate or severe cognitive impairment. Oulu, Oulu, Finland. Medical Research Center, Oulu University Hospital, Figure S3. Number and length of MERCS negatively with MMSE. Repre- Oulu, Finland. sentation of the correlation between MMSE and mitochondria-ER contact sites a number and b length. Linear regression was performed and the Received: 21 September 2018 Accepted: 21 September 2018 Pearson correlation coefficient (r) calculated. Each point represent one iNPH patient. Figure S4. Amyloid plaques and NFT have no effect in the number of mitochondria profile nor mitochondria perimeter. Quantifica- References tion of a number and b perimeter of mitochondria profiles from the elec- − − 1. 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