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Hypothesis: The Role of Sterols in Autism Spectrum Disorder

Hypothesis: The Role of Sterols in Autism Spectrum Disorder Hindawi Publishing Corporation Autism Research and Treatment Volume 2011, Article ID 653570, 7 pages doi:10.1155/2011/653570 Review Article 1, 2 3, 4, 5 Ryan W. Y. Lee and Elaine Tierney Department of Neurology and Developmental Medicine, Kennedy Krieger Institute, 716 North Broadway Street, Baltimore, MD 21205, USA Department of Pediatrics, Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21287, USA Department of Psychiatry, Kennedy Krieger Institute, 716 North Broadway Street, Baltimore, MD 21205, USA Department of Psychiatry, Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MS 21287, USA Center for Genetic Disorders of Cognition and Behavior, Kennedy Krieger Institute, 716 North Broadway Street, Baltimore, MD 21205, USA Correspondence should be addressed to Elaine Tierney, tierney@kennedykrieger.org Received 27 September 2010; Revised 7 February 2011; Accepted 21 February 2011 Academic Editor: Roberto Canitano Copyright © 2011 R. W. Y. Lee and E. Tierney. 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. A possible role for sterols in the development of autism spectrum disorder (ASD) has not been proven, but studies in disorders of sterol biosynthesis, chiefly Smith-Lemli-Opitz syndrome (SLOS), enable hypotheses on a causal relationship to be discussed. Advances in genetic technology coupled with discoveries in membrane physiology have led to renewed interest for lipids in the nervous system. This paper hypothesizes on the role of sterol dysfunction in ASD through the framework of SLOS. Impaired sonic hedgehog patterning, alterations in membrane lipid rafts leading to abnormal synaptic plasticity, and impaired neurosteroid synthesis are discussed. Potential therapeutic agents include the development of neuroactive steroid-based agents and enzyme- specific drugs. Future investigations should reveal the specific mechanisms underlying sterol dysfunction in neurodevelopmental disorders by utilizing advanced imaging and molecular techniques. 1. Introduction of behavioral phenotypes such as ASD. Evidence supporting a role for sterols in the development of ASD was based on The autism spectrum describes a group of disorders with studies in disorders of sterol biosynthesis, chiefly SLOS [5–8]. early childhood onset, characterized by persistent core Furthermore, a study of 100 serum samples from the Autism deficits in socialization, language, and stereotypic and repet- Genetic Resource Exchange (AGRE) demonstrated that a itive behavior [1]. Over 50 years has passed since Leo Kanner subset (about 20%) of unrelated children from multiplex pioneered a description of infantile autism [2]. The defini- families with ASD had mild hypocholesterolemia (i.e., lower tion of autism has expanded to include a wide spectrum of than 100 mg/dL), which is in contrast to very low cholesterol clinically and biologically heterogeneous disorders, each with levels (<10mg/dL) oftenseeninsevereSLOScases [6]. variable degrees of core autistic feature expression, which we The findings of Tierney et al. were replicated when an now describe as autism spectrum disorder (ASD) [3]. The additional 100 AGRE subjects were tested by the same group estimated prevalence of ASD in the United States is 1 in 110 (unpublished data), but have not as yet been replicated by children [4]. The list of well-defined genetic disorders with other research teams. ASD continues to expand, with commonly studied examples Cholesterol serves many essential roles in the developing including fragile X syndrome, tuberous sclerosis, untreated nervous system. It is a structural component of myelin phenylketonuria (PKU), Rett syndrome, and Smith-Lemli- and membrane lipid rafts, serves as a substrate for neuros- Opitz syndrome (SLOS). Thus, studies involving relatively teroid formation, and facilitates hedgehog signaling [9, 10]. homogenous populations with well-described genetic disor- Impaired function of these activities is likely responsible for ders have begun to reveal the neurobiologic underpinnings the anatomic and neurobehavioral manifestations in SLOS. 2 Autism Research and Treatment Acetyl-CoA + acetoacetyl-CoA 3-Hydroxy-3-methylglutaryl-CoA Mevalonic acid Squalene Lanosterol DHCR7 HO HO HO Smith-Lemli-Opitz 8-DHC 7-DHC syndrome Cholesterol Hedgehog Vitamin D Myelin Lipid rafts Neurosteroids Bile acids signaling Figure 1: Effect of sterol precursor substitution in Smith-Lemli-Opitz syndrome. (Adapted with permission from Richard Kelley, M.D. and Forbes Porter, M.D.). Recent advances in gene technology and membrane biology 8-dehydrocholesterol (8-DHC) and often low serum total have contributed to a better understanding of the complex cholesterol. There is a broad range of cholesterol seen in mechanisms underlying impaired cognition and behavior SLOS (less than 10 mg/dL to greater than 200 mg/dL). It in cholesterol-deficient conditions. This paper hypothesizes remains uncertain whether morphologic and behavioral on the role of sterol dysfunction in ASD and proposes manifestations of SLOS are caused by decreased cholesterol future directions for targeted therapeutics. We hypothesize levels, increased 7-DHC, or both. SLOS is associated with that cholesterol dysfunction may lead to ASD by three ASD in 50–75% of cases [6, 18, 19]. To date, the neuro- mechanisms working in concert during brain development: biologic relationship between SLOS and ASD has not been (1) impaired sonic hedgehog patterning, (2) alterations explained. in membrane lipid raft structure and protein function Sonic Hedgehog (SHH) is a morphogen involved in the resulting in abnormal synaptic plasticity, and (3) impaired patterning of the nervous system and limbs, along with neurosteroid synthesis. other transcription factors and secreted proteins [20–25]. During embryonic development, SHH is covalently modified with both palmitate and cholesterol and secreted as part of a lipoprotein complex that regulates brain morphogenesis 2. Sonic Hedgehog and Cholesterol through the patched/smoothened signaling system [26– Dysfunction in SLOS 29]. SHH is secreted from the notochord and ventral Smith-Lemli-Opitz syndrome (SLOS) is an autosomal reces- floor plate cells and forms a concentration gradient along sive disorder of cholesterol biosynthesis caused by muta- the entire dorsal-ventral axis [29]. The posttranslational tions in the gene encoding 7-dehydrocholesterol reduc- effect of SHH after covalent modification by cholesterol tase (DHCR7) located on chromosome 11q12-13 [11, is the establishment of a morphogenic SHH concentration 12](Figure 1). SLOS has an estimated incidence among gradient that moves from the ventral (high concentration) individuals of European ancestry of 1 in 15,000 to 1 in to dorsal regions (lower concentration). Variations in the 60,000 births and a carrier frequency of 1 in 30 to 1 SHH gradient affect intracellular cell signaling systems and in 50 [13–17]. Individuals with SLOS have abnormally ultimately determine the expression of future cell types elevated plasma 7-dehydrocholesterol (7-DHC) or its isomer by sequential induction of transcription factors in ventral Autism Research and Treatment 3 Ectoderm Dorsal roof plate Nt V0 interneurons V1 interneurons V2 interneurons Motor neurons V3 neurons Ventral floor plate SHH Nc Figure 2: The sonic hedgehog gradient in embryonic neural patterning. SHH-regulated gradient defines neuronal subtypes during embryonic patterning. Sonic hedgehog (SHH) (yellow) is secreted from cells of notochord (Nc) and ventral floor plate to create a ventral- dorsal concentration gradient along the neural tube (Nt). Spatial organization of six progenitor-cell domains is established by the SHH gradient restricting the expression of various protein-marker profiles. The initiation of these markers at successive developmental time periods results in V0–V3 and motor neuron (MN) subtype patterning along the ventral midline in the neural tube. progenitor cells [29]. The formation of discrete cell precursor in the establishment and advancement of the SHH gradient domains in the neural tube as a result of the SHH mor- and its effects on transcription factors, may provide an phogenic front is one determinant of the structural fate of the explanation for the development of cognitive and behavioral maturing brain [30–32](Figure 2). In animal studies, during impairment in disorders with diffuse neural abnormalities, late embryonic and postnatal brain development, neural such as autism and SLOS. precursor and stem cell proliferation in dorsal neocortical, hippocampal, tectal, and cerebellar regions is regulated by SHH signaling [33, 34]. In humans, failure of midline 3. Membrane Lipid Rafts and ASD brain structures to form appropriately can result from a loss of SHH processing, as evidenced in holoprosencephaly Studies on cholesterol and lipid organization in disease [35]. Incomplete formation of midline structures including have led to progress in understanding the molecular basis the corpus callosum and cerebellum is the most common of neurologic disorders [40]. As a result, autism research neuroimaging abnormality found in individuals with SLOS involving sterols and other metabolites continues to gain [36]. Interestingly, reduction in corpus callosum size is popularity. For over a decade, lipid rafts or specialized mem- among the most common neuroimaging abnormality in brane microdomains have been investigated for their key autism and supports the aberrant connectivity hypothesis role in cellular communication [41, 42]. Rafts are dynamic that autism is a disorder of connectivity, involving inter- and structures enriched with cholesterol, sphingomyelin, and intrahemispheric communications with possible alterations phosphatidylcholine [43]. The primary raft subtype called of intracortical connections [37–39]. In both autism and caveolae comprised of scaffolding proteins (caveolin), is SLOS, it is uncertain whether callosal hypoplasia is due to distinguished by flask-shaped invaginations of the plasma a primary patterning defect or later dysfunction of neuronal membrane [44]. These platforms serve as signaling regions in cortical connectivity and axonal migration or both. clatharin-independent endocytosis, lipid homeostasis, signal We hypothesize that in SLOS, low cholesterol or elevated transduction, and tumorigenesis [45]. Caveolae are widely sterol precursors result in establishment of an abnormal expressed in brain endothelial cells, astrocytes, oligodendro- SHH gradient, which may alter the fate of cells in the cytes, Schwann cells, dorsal root ganglia, and hippocampal developing brain. Further studies are required to support neurons [46]. Lipid rafts play a critical role in many this hypothesis. While the hypothesis may be plausible for neurologic disorders including SLOS, Huntington disease, SLOS and certain cholesterol-dependent ASD, incomplete Alzheimer’s disease, Tangier disease, and Niemann-Pick formation of midline structures is present in numerous disease type C [40, 47, 48]. The essential role of cholesterol disorders of cognition and behavior without abnormal sterol in formation of lipid rafts and membrane organization is biosynthesis. In addition, there are many individuals with highlighted in studies of membrane physiology. Cholesterol ASD that do not have midline structural brain abnormalities. content is extremely important for cell membrane lateral For these reasons, multiple mechanisms are likely to arise as organization and protein function [49–51]. Samuli Ollila etiologies of the ASD phenotype. In sum, regional differences et al. [49] report that lipid membrane lateral pressure profiles 4 Autism Research and Treatment were significantly altered when cholesterol was replaced of SLOS [64]. Biochemical studies have demonstrated that with sterol precursors, desmosterol, 7-DHC, or ketosterol. neurosteroids possess pharmacologic properties applicable Furthermore, 7-DCH and 8-DHC have been shown to accu- to anesthesia and epilepsy [57, 65]. Benzodiazepines inhibit mulate in membrane lipid rafts of liver tissue in individuals the enzymes responsible for neurosteroid metabolism, per- with SLOS [52]. The accumulation of sterol precursors in haps due to shared pharmacologic action at the GABA rafts depletes cholesterol from structures such as hippocam- receptor [66]. Interestingly, some antidepressant agents pal membranes and limits ligand-binding activity of the such as fluoxetine have been found to increase circulating serotonin 1A receptor [53]. Functional changes at the cellular neurosteroid levels [67, 68]. The molecular effects of these level may be explained by studies showing that DHCR7- medications on the nervous system in SLOS have not been deficient neuronal cell lines downregulate genes critical to investigated. lipid synthesis such as sterol-regulatory element binding Since cholesterol does not cross the blood-brain barrier, protein 2 (SREB-2), SREBF chaperone, site-1 protease, fatty neurosteroids are synthesized with cholesterol de novo [69]. acid synthase, and squalene synthase [47]. Decreased DHCR7 For nearly a decade, it has been proposed that increased 7- has also been shown to alter expression of key molecules for DHC levels might inhibit neurosteroid formation or lead intracellular signaling and vesicular transport such as Egr1, to synthesis of an inhibitory analog in the brain [70]. Snx, and Adam19 [47]. These studies support a possible role Marcos et al. [64] studied urinary steroids and found that for abnormal neuronal cell membrane protein signaling in dehydrocholesterols provided the substrate for formation of DHCR7 mutations that lead to behavioral manifestations allopregnanolone and dehydroallopregnanolone in patients in SLOS. More studies are needed to determine if these with SLOS. While only providing evidence for extraneural mechanisms are involved in the human pathophysiology of synthesis of 7- and 8-dehydroallopragnanolones, there is a SLOS and other neurodevelopmental disorders. Rafts may high likelihood that abnormal synthesis occurs in the brain represent one of the many biologic substrates that shape given the low tissue specificity of 5α-reductase and 3α- neuronal networks in the brain. Recent data has shown hydroxysteroid dehydrogenase [64]. Currently, mouse model that reduction in cholesterol levels impair exocytosis of studies are investigating the prospect that reduced levels synaptic vesicles [54]. Numerous questions are surfacing of neurosteroids possessing anxiolytic properties, such as about the clinical manifestations of neuronal and glial mem- allopregnanolone, impact behavior in SLOS. brane alterations caused by altered lipid raft composition in humans. For example, it remains unknown whether membrane proteins important for synaptic plasticity such as 5. Targeted Therapeutics and Conclusions AMPA kainate, GABA , and NMDA receptors are affected by abnormal sterol levels or whether these abnormalities are Current treatment of SLOS involves endogenous cholesterol present either transiently or for longer periods in regions of supplementation in the form of crystallized purified choles- the developing brain for individuals with autism. Therefore, terol suspended in Ora-Plus, microencapsulated powdered we hypothesize that neuronal or glial expression of autism purified cholesterol (brandname SLOesterol), or egg yolks. candidate genes and their resulting membrane proteins may Several publications discuss the role of simvastatin therapy be altered in disorders of abnormal cholesterol homeostasis. [71–73]. Efficacy for either of these therapies remains unclear. Endogenous cholesterol biosynthesis is the primary mechanism for nervous system cholesterol homeostasis, making a role for extrinsic cholesterol in altering nervous 4. Neurosteroids and ASD system function questionable [47]. As we look ahead, Neurosteroids are steroid molecules produced by the central pharmacologic agents derived from neuroactive steroids or nervous system to rapidly augment neuronal excitabil- steroid analogues may provide targeted therapy for behav- ity through membrane-bound, ion-gated neurotransmitter ioral symptoms in SLOS and ASD. Currently, clinical trials receptors [55, 56]. While classic steroid hormones typically are examining the therapeutic effects of neurosteroids on exert endocrine function on the order of hours to days, mood disorders, schizophrenia, substance abuse, traumatic neuroactive steroids can act rapidly in a nontranscriptional brain injury, and cognitive disorders. Lipids such as 7- mechanism to produce behavioral effects in seconds to DHC may undergo perioxidation to form bioactive products minutes [56–59]. Neuroactive steroids are synthesized from called oxysterols that have been shown to reduce prolifer- cholesterol in neurons and glia or steroid precursors from ation of Neuro2a cells and induce cell differentiation [74]. peripheral tissues [60, 61]. Expression of steroidogenic Oxysterols have long been hypothesized in the pathology enzymes is developmentally regulated [62]. There are many of SLOS and remain a promising area for interventional different types of neurosteroids resulting in an array of trials to reduce oxygen free radicals [75–78]. Enzyme- functional diversity including positive allosteric modulation specific candidate drugs are being investigated in SLOS. of GABA and NMDA receptors, myelin formation, axonal Appropriate modulation of embryonic SHH patterning and guidance, and dendrite growth [55, 62, 63]. These molecular lipid rafts are not likely to be achieved until future studies activities enable moment-to-moment modulation of neu- elucidate the specific mechanisms and biologic substrates roendocrine functions and behavior. underlying brain development. These studies may be aided Because of their broad psychiatric characteristics, neu- by advances in functional neuroimaging and molecular rosteroids have been implicated in the behavioral profile imaging techniques. Furthermore, discussion on the ethics Autism Research and Treatment 5 involving embryologic or childhood neuromodulatory ther- [13] R. I. Kelley and R. C. H. Hennekam, “Smith-Lemli-Opitz Syndrome and other disorders of cholesterol biosynthesis,” in apy in patients with abnormal neural patterning should be The Metabolic and Molecular Basis of Inherited Disease,C.R. considered if technology advances toward such a therapeutic Scriver, A. L. Beaudet, W. S. Sly, and D. Valle, Eds., chapter option. 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Hypothesis: The Role of Sterols in Autism Spectrum Disorder

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Copyright © 2011 Ryan W. Y. Lee and Elaine Tierney. 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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Hindawi Publishing Corporation Autism Research and Treatment Volume 2011, Article ID 653570, 7 pages doi:10.1155/2011/653570 Review Article 1, 2 3, 4, 5 Ryan W. Y. Lee and Elaine Tierney Department of Neurology and Developmental Medicine, Kennedy Krieger Institute, 716 North Broadway Street, Baltimore, MD 21205, USA Department of Pediatrics, Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21287, USA Department of Psychiatry, Kennedy Krieger Institute, 716 North Broadway Street, Baltimore, MD 21205, USA Department of Psychiatry, Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MS 21287, USA Center for Genetic Disorders of Cognition and Behavior, Kennedy Krieger Institute, 716 North Broadway Street, Baltimore, MD 21205, USA Correspondence should be addressed to Elaine Tierney, tierney@kennedykrieger.org Received 27 September 2010; Revised 7 February 2011; Accepted 21 February 2011 Academic Editor: Roberto Canitano Copyright © 2011 R. W. Y. Lee and E. Tierney. 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. A possible role for sterols in the development of autism spectrum disorder (ASD) has not been proven, but studies in disorders of sterol biosynthesis, chiefly Smith-Lemli-Opitz syndrome (SLOS), enable hypotheses on a causal relationship to be discussed. Advances in genetic technology coupled with discoveries in membrane physiology have led to renewed interest for lipids in the nervous system. This paper hypothesizes on the role of sterol dysfunction in ASD through the framework of SLOS. Impaired sonic hedgehog patterning, alterations in membrane lipid rafts leading to abnormal synaptic plasticity, and impaired neurosteroid synthesis are discussed. Potential therapeutic agents include the development of neuroactive steroid-based agents and enzyme- specific drugs. Future investigations should reveal the specific mechanisms underlying sterol dysfunction in neurodevelopmental disorders by utilizing advanced imaging and molecular techniques. 1. Introduction of behavioral phenotypes such as ASD. Evidence supporting a role for sterols in the development of ASD was based on The autism spectrum describes a group of disorders with studies in disorders of sterol biosynthesis, chiefly SLOS [5–8]. early childhood onset, characterized by persistent core Furthermore, a study of 100 serum samples from the Autism deficits in socialization, language, and stereotypic and repet- Genetic Resource Exchange (AGRE) demonstrated that a itive behavior [1]. Over 50 years has passed since Leo Kanner subset (about 20%) of unrelated children from multiplex pioneered a description of infantile autism [2]. The defini- families with ASD had mild hypocholesterolemia (i.e., lower tion of autism has expanded to include a wide spectrum of than 100 mg/dL), which is in contrast to very low cholesterol clinically and biologically heterogeneous disorders, each with levels (<10mg/dL) oftenseeninsevereSLOScases [6]. variable degrees of core autistic feature expression, which we The findings of Tierney et al. were replicated when an now describe as autism spectrum disorder (ASD) [3]. The additional 100 AGRE subjects were tested by the same group estimated prevalence of ASD in the United States is 1 in 110 (unpublished data), but have not as yet been replicated by children [4]. The list of well-defined genetic disorders with other research teams. ASD continues to expand, with commonly studied examples Cholesterol serves many essential roles in the developing including fragile X syndrome, tuberous sclerosis, untreated nervous system. It is a structural component of myelin phenylketonuria (PKU), Rett syndrome, and Smith-Lemli- and membrane lipid rafts, serves as a substrate for neuros- Opitz syndrome (SLOS). Thus, studies involving relatively teroid formation, and facilitates hedgehog signaling [9, 10]. homogenous populations with well-described genetic disor- Impaired function of these activities is likely responsible for ders have begun to reveal the neurobiologic underpinnings the anatomic and neurobehavioral manifestations in SLOS. 2 Autism Research and Treatment Acetyl-CoA + acetoacetyl-CoA 3-Hydroxy-3-methylglutaryl-CoA Mevalonic acid Squalene Lanosterol DHCR7 HO HO HO Smith-Lemli-Opitz 8-DHC 7-DHC syndrome Cholesterol Hedgehog Vitamin D Myelin Lipid rafts Neurosteroids Bile acids signaling Figure 1: Effect of sterol precursor substitution in Smith-Lemli-Opitz syndrome. (Adapted with permission from Richard Kelley, M.D. and Forbes Porter, M.D.). Recent advances in gene technology and membrane biology 8-dehydrocholesterol (8-DHC) and often low serum total have contributed to a better understanding of the complex cholesterol. There is a broad range of cholesterol seen in mechanisms underlying impaired cognition and behavior SLOS (less than 10 mg/dL to greater than 200 mg/dL). It in cholesterol-deficient conditions. This paper hypothesizes remains uncertain whether morphologic and behavioral on the role of sterol dysfunction in ASD and proposes manifestations of SLOS are caused by decreased cholesterol future directions for targeted therapeutics. We hypothesize levels, increased 7-DHC, or both. SLOS is associated with that cholesterol dysfunction may lead to ASD by three ASD in 50–75% of cases [6, 18, 19]. To date, the neuro- mechanisms working in concert during brain development: biologic relationship between SLOS and ASD has not been (1) impaired sonic hedgehog patterning, (2) alterations explained. in membrane lipid raft structure and protein function Sonic Hedgehog (SHH) is a morphogen involved in the resulting in abnormal synaptic plasticity, and (3) impaired patterning of the nervous system and limbs, along with neurosteroid synthesis. other transcription factors and secreted proteins [20–25]. During embryonic development, SHH is covalently modified with both palmitate and cholesterol and secreted as part of a lipoprotein complex that regulates brain morphogenesis 2. Sonic Hedgehog and Cholesterol through the patched/smoothened signaling system [26– Dysfunction in SLOS 29]. SHH is secreted from the notochord and ventral Smith-Lemli-Opitz syndrome (SLOS) is an autosomal reces- floor plate cells and forms a concentration gradient along sive disorder of cholesterol biosynthesis caused by muta- the entire dorsal-ventral axis [29]. The posttranslational tions in the gene encoding 7-dehydrocholesterol reduc- effect of SHH after covalent modification by cholesterol tase (DHCR7) located on chromosome 11q12-13 [11, is the establishment of a morphogenic SHH concentration 12](Figure 1). SLOS has an estimated incidence among gradient that moves from the ventral (high concentration) individuals of European ancestry of 1 in 15,000 to 1 in to dorsal regions (lower concentration). Variations in the 60,000 births and a carrier frequency of 1 in 30 to 1 SHH gradient affect intracellular cell signaling systems and in 50 [13–17]. Individuals with SLOS have abnormally ultimately determine the expression of future cell types elevated plasma 7-dehydrocholesterol (7-DHC) or its isomer by sequential induction of transcription factors in ventral Autism Research and Treatment 3 Ectoderm Dorsal roof plate Nt V0 interneurons V1 interneurons V2 interneurons Motor neurons V3 neurons Ventral floor plate SHH Nc Figure 2: The sonic hedgehog gradient in embryonic neural patterning. SHH-regulated gradient defines neuronal subtypes during embryonic patterning. Sonic hedgehog (SHH) (yellow) is secreted from cells of notochord (Nc) and ventral floor plate to create a ventral- dorsal concentration gradient along the neural tube (Nt). Spatial organization of six progenitor-cell domains is established by the SHH gradient restricting the expression of various protein-marker profiles. The initiation of these markers at successive developmental time periods results in V0–V3 and motor neuron (MN) subtype patterning along the ventral midline in the neural tube. progenitor cells [29]. The formation of discrete cell precursor in the establishment and advancement of the SHH gradient domains in the neural tube as a result of the SHH mor- and its effects on transcription factors, may provide an phogenic front is one determinant of the structural fate of the explanation for the development of cognitive and behavioral maturing brain [30–32](Figure 2). In animal studies, during impairment in disorders with diffuse neural abnormalities, late embryonic and postnatal brain development, neural such as autism and SLOS. precursor and stem cell proliferation in dorsal neocortical, hippocampal, tectal, and cerebellar regions is regulated by SHH signaling [33, 34]. In humans, failure of midline 3. Membrane Lipid Rafts and ASD brain structures to form appropriately can result from a loss of SHH processing, as evidenced in holoprosencephaly Studies on cholesterol and lipid organization in disease [35]. Incomplete formation of midline structures including have led to progress in understanding the molecular basis the corpus callosum and cerebellum is the most common of neurologic disorders [40]. As a result, autism research neuroimaging abnormality found in individuals with SLOS involving sterols and other metabolites continues to gain [36]. Interestingly, reduction in corpus callosum size is popularity. For over a decade, lipid rafts or specialized mem- among the most common neuroimaging abnormality in brane microdomains have been investigated for their key autism and supports the aberrant connectivity hypothesis role in cellular communication [41, 42]. Rafts are dynamic that autism is a disorder of connectivity, involving inter- and structures enriched with cholesterol, sphingomyelin, and intrahemispheric communications with possible alterations phosphatidylcholine [43]. The primary raft subtype called of intracortical connections [37–39]. In both autism and caveolae comprised of scaffolding proteins (caveolin), is SLOS, it is uncertain whether callosal hypoplasia is due to distinguished by flask-shaped invaginations of the plasma a primary patterning defect or later dysfunction of neuronal membrane [44]. These platforms serve as signaling regions in cortical connectivity and axonal migration or both. clatharin-independent endocytosis, lipid homeostasis, signal We hypothesize that in SLOS, low cholesterol or elevated transduction, and tumorigenesis [45]. Caveolae are widely sterol precursors result in establishment of an abnormal expressed in brain endothelial cells, astrocytes, oligodendro- SHH gradient, which may alter the fate of cells in the cytes, Schwann cells, dorsal root ganglia, and hippocampal developing brain. Further studies are required to support neurons [46]. Lipid rafts play a critical role in many this hypothesis. While the hypothesis may be plausible for neurologic disorders including SLOS, Huntington disease, SLOS and certain cholesterol-dependent ASD, incomplete Alzheimer’s disease, Tangier disease, and Niemann-Pick formation of midline structures is present in numerous disease type C [40, 47, 48]. The essential role of cholesterol disorders of cognition and behavior without abnormal sterol in formation of lipid rafts and membrane organization is biosynthesis. In addition, there are many individuals with highlighted in studies of membrane physiology. Cholesterol ASD that do not have midline structural brain abnormalities. content is extremely important for cell membrane lateral For these reasons, multiple mechanisms are likely to arise as organization and protein function [49–51]. Samuli Ollila etiologies of the ASD phenotype. In sum, regional differences et al. [49] report that lipid membrane lateral pressure profiles 4 Autism Research and Treatment were significantly altered when cholesterol was replaced of SLOS [64]. Biochemical studies have demonstrated that with sterol precursors, desmosterol, 7-DHC, or ketosterol. neurosteroids possess pharmacologic properties applicable Furthermore, 7-DCH and 8-DHC have been shown to accu- to anesthesia and epilepsy [57, 65]. Benzodiazepines inhibit mulate in membrane lipid rafts of liver tissue in individuals the enzymes responsible for neurosteroid metabolism, per- with SLOS [52]. The accumulation of sterol precursors in haps due to shared pharmacologic action at the GABA rafts depletes cholesterol from structures such as hippocam- receptor [66]. Interestingly, some antidepressant agents pal membranes and limits ligand-binding activity of the such as fluoxetine have been found to increase circulating serotonin 1A receptor [53]. Functional changes at the cellular neurosteroid levels [67, 68]. The molecular effects of these level may be explained by studies showing that DHCR7- medications on the nervous system in SLOS have not been deficient neuronal cell lines downregulate genes critical to investigated. lipid synthesis such as sterol-regulatory element binding Since cholesterol does not cross the blood-brain barrier, protein 2 (SREB-2), SREBF chaperone, site-1 protease, fatty neurosteroids are synthesized with cholesterol de novo [69]. acid synthase, and squalene synthase [47]. Decreased DHCR7 For nearly a decade, it has been proposed that increased 7- has also been shown to alter expression of key molecules for DHC levels might inhibit neurosteroid formation or lead intracellular signaling and vesicular transport such as Egr1, to synthesis of an inhibitory analog in the brain [70]. Snx, and Adam19 [47]. These studies support a possible role Marcos et al. [64] studied urinary steroids and found that for abnormal neuronal cell membrane protein signaling in dehydrocholesterols provided the substrate for formation of DHCR7 mutations that lead to behavioral manifestations allopregnanolone and dehydroallopregnanolone in patients in SLOS. More studies are needed to determine if these with SLOS. While only providing evidence for extraneural mechanisms are involved in the human pathophysiology of synthesis of 7- and 8-dehydroallopragnanolones, there is a SLOS and other neurodevelopmental disorders. Rafts may high likelihood that abnormal synthesis occurs in the brain represent one of the many biologic substrates that shape given the low tissue specificity of 5α-reductase and 3α- neuronal networks in the brain. Recent data has shown hydroxysteroid dehydrogenase [64]. Currently, mouse model that reduction in cholesterol levels impair exocytosis of studies are investigating the prospect that reduced levels synaptic vesicles [54]. Numerous questions are surfacing of neurosteroids possessing anxiolytic properties, such as about the clinical manifestations of neuronal and glial mem- allopregnanolone, impact behavior in SLOS. brane alterations caused by altered lipid raft composition in humans. For example, it remains unknown whether membrane proteins important for synaptic plasticity such as 5. Targeted Therapeutics and Conclusions AMPA kainate, GABA , and NMDA receptors are affected by abnormal sterol levels or whether these abnormalities are Current treatment of SLOS involves endogenous cholesterol present either transiently or for longer periods in regions of supplementation in the form of crystallized purified choles- the developing brain for individuals with autism. Therefore, terol suspended in Ora-Plus, microencapsulated powdered we hypothesize that neuronal or glial expression of autism purified cholesterol (brandname SLOesterol), or egg yolks. candidate genes and their resulting membrane proteins may Several publications discuss the role of simvastatin therapy be altered in disorders of abnormal cholesterol homeostasis. [71–73]. Efficacy for either of these therapies remains unclear. Endogenous cholesterol biosynthesis is the primary mechanism for nervous system cholesterol homeostasis, making a role for extrinsic cholesterol in altering nervous 4. Neurosteroids and ASD system function questionable [47]. As we look ahead, Neurosteroids are steroid molecules produced by the central pharmacologic agents derived from neuroactive steroids or nervous system to rapidly augment neuronal excitabil- steroid analogues may provide targeted therapy for behav- ity through membrane-bound, ion-gated neurotransmitter ioral symptoms in SLOS and ASD. Currently, clinical trials receptors [55, 56]. While classic steroid hormones typically are examining the therapeutic effects of neurosteroids on exert endocrine function on the order of hours to days, mood disorders, schizophrenia, substance abuse, traumatic neuroactive steroids can act rapidly in a nontranscriptional brain injury, and cognitive disorders. Lipids such as 7- mechanism to produce behavioral effects in seconds to DHC may undergo perioxidation to form bioactive products minutes [56–59]. Neuroactive steroids are synthesized from called oxysterols that have been shown to reduce prolifer- cholesterol in neurons and glia or steroid precursors from ation of Neuro2a cells and induce cell differentiation [74]. peripheral tissues [60, 61]. Expression of steroidogenic Oxysterols have long been hypothesized in the pathology enzymes is developmentally regulated [62]. There are many of SLOS and remain a promising area for interventional different types of neurosteroids resulting in an array of trials to reduce oxygen free radicals [75–78]. Enzyme- functional diversity including positive allosteric modulation specific candidate drugs are being investigated in SLOS. of GABA and NMDA receptors, myelin formation, axonal Appropriate modulation of embryonic SHH patterning and guidance, and dendrite growth [55, 62, 63]. These molecular lipid rafts are not likely to be achieved until future studies activities enable moment-to-moment modulation of neu- elucidate the specific mechanisms and biologic substrates roendocrine functions and behavior. underlying brain development. These studies may be aided Because of their broad psychiatric characteristics, neu- by advances in functional neuroimaging and molecular rosteroids have been implicated in the behavioral profile imaging techniques. Furthermore, discussion on the ethics Autism Research and Treatment 5 involving embryologic or childhood neuromodulatory ther- [13] R. I. Kelley and R. C. H. Hennekam, “Smith-Lemli-Opitz Syndrome and other disorders of cholesterol biosynthesis,” in apy in patients with abnormal neural patterning should be The Metabolic and Molecular Basis of Inherited Disease,C.R. considered if technology advances toward such a therapeutic Scriver, A. L. Beaudet, W. S. Sly, and D. Valle, Eds., chapter option. 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