Analysis of recent shared ancestry in a familial cohort identifies coding and noncoding autism spectrum disorder variants.

Autism spectrum disorder (ASD) is a collection of neurodevelopmental disorders characterized by deficits in social communication and restricted, repetitive patterns of behavior or interests. ASD is highly heritable, but genetically and phenotypically heterogeneous, reducing the power to identify causative genes. We performed whole genome sequencing (WGS) in an ASD cohort of 68 individuals from 22 families enriched for recent shared ancestry. We identified an average of 3.07 million variants per genome, of which an average of 112,512 were rare. We mapped runs of homozygosity (ROHs) in affected individuals and found an average genomic homozygosity of 9.65%, consistent with expectations for multiple generations of consanguineous unions. We identified potentially pathogenic rare exonic or splice site variants in 12 known (including KMT2C, SCN1A, SPTBN1, SYNE1, ZNF292) and 12 candidate (including CHD5, GRB10, PPP1R13B) ASD genes. Furthermore, we annotated noncoding variants in ROHs with brain-specific regulatory elements and identified putative disease-causing variants within brain-specific promoters and enhancers for 5 known ASD and neurodevelopmental disease genes (ACTG1, AUTS2, CTNND2, CNTNAP4, SPTBN4). We also identified copy number variants in two known ASD and neurodevelopmental disease loci in two affected individuals. In total we identified potentially etiological variants in known ASD or neurodevelopmental disease genes for ~61% (14/23) of affected individuals. We combined WGS with homozygosity mapping and regulatory element annotations to identify candidate ASD variants. Our analyses add to the growing number of ASD genes and variants and emphasize the importance of leveraging recent shared ancestry to map disease variants in complex neurodevelopmental disorders.

Huntington's disease genotype suppresses global manganese-responsive processes in pre-manifest and manifest YAC128 mice.

Manganese (Mn) is an essential micronutrient required for the proper function of several enzymes. Accumulating evidence demonstrates a selective decrease of bioavailable Mn in vulnerable cell types of Huntington's Disease (HD), an inherited progressive neurodegenerative disorder with no cure. Amelioration of underlying pathophysiology, such as alterations in Mn-dependent biology, may be therapeutic. We therefore sought to investigate global Mn-dependent and Mn-responsive biology following various Mn exposures in a mouse model of HD. YAC128 and wildtype (WT) littermate control mice received one of three different Mn exposure paradigms by subcutaneous injection of 50 mg kg-1 MnCl2·4(H2O) across two distinct HD disease stages. "Pre-manifest" (12-week old mice) mice received either a single (1 injection) or week-long (3 injections) exposure of Mn or vehicle (H2O) and were sacrificed at the pre-manifest stage. "Manifest" (32-week old) mice were sacrificed following either a week-long Mn or vehicle exposure during the manifest stage, or a 20-week-long chronic (2× weekly injections) exposure that began in the pre-manifest stage. Tissue Mn, mRNA, protein, and metabolites were measured in the striatum, the brain region most sensitive to neurodegeneration in HD. Across all Mn exposure paradigms, pre-manifest YAC128 mice exhibited a suppressed response to transcriptional and protein changes and manifest YAC128 mice showed a suppressed metabolic response, despite equivalent elevations in whole striatal Mn. We conclude that YAC128 mice respond differentially to Mn compared to WT as measured by global transcriptional, translational, and metabolomic changes, suggesting an impairment in Mn homeostasis across two different disease stages in YAC128 mice.

Microglia Activation and Gene Expression Alteration of Neurotrophins in the Hippocampus Following Early-Life Exposure to E-Cigarette Aerosols in a Murine Model.

Recent epidemiological data indicate that the popularity of electronic cigarettes (e-cigarettes), and consequently nicotine use, is rising in both adolescent and adult populations. As nicotine is a known developmental neurotoxin, these products present a potential threat for those exposed during early life stages. Despite this, few studies have evaluated the toxicity of e-cigarettes on the developing central nervous system. The goal of this study was to assess neurotoxicity resulting from early-life exposure to electronic cigarette aerosols in an in vivo model. Specifically, studies here focused on neuro-parameters related to neuroinflammation and neurotrophins. To accomplish this, pregnant and neonatal C57BL/6 mice were exposed to aerosols produced from classic tobacco flavor e-cigarette cartridges (with [13 mg/ml] and without nicotine) during gestation (∼3 weeks) and lactation (∼3 weeks) via whole-body inhalation. Exposure to e-cigarette aerosols with and without nicotine caused significant reductions in hippocampal gene expression of Ngfr and Bdnf, as well as in serum levels of cytokines IL-1ß, IL-2, and IL-6. Exposure to e-cigarette aerosols without nicotine enhanced expression of Iba-1, a specific marker of microglia, in the cornus ammonis 1 region of the hippocampus. Overall, our novel results indicate that exposure to e-cigarette aerosols, with and without nicotine, poses a considerable risk to the developing central nervous system. Consequently, e-cigarettes should be considered a potential public health threat, especially early in life, requiring further research and policy considerations.

Metals and Circadian Rhythms.

Circadian rhythms describe the behavioral and physiological changes that occur in living organisms in order to attune to a 24 hour cycle of day and night. The most striking aspect of circadian function is the sleep-wake cycle, however many other physiological processes are regulated in 24 hour oscillations, including blood pressure, body temperature, appetite, urine production, and the transcription and translation of thousands of circadian dependent genes. Circadian disruption and sleep disorders are strongly connected to neurodegenerative diseases including Parkinson's disease, Alzheimer's disease, and Huntington's disease as well as others. Metal exposures have been implicated in neurodegenerative diseases, in some cases involving metals that are essential micronutrients but are toxic at high levels of exposure (such as manganese, copper, and zinc), and in other cases involving metals that have no biological role but are toxic to living systems (such as lead, mercury, and aluminum). In this review, we examine the evidence for circadian and sleep disorders with exposures to these metals and review the literature for possible mechanisms. We suggest that giving the aging population, the prevalence of environmental exposures to metals, and the increasing prevalence of neurodegenerative disease in the aged, more research into the mechanisms of circadian disruption subsequent to metal exposures is warranted.

Untangling the Manganese-α-Synuclein Web.

Neurodegenerative diseases affect a significant portion of the aging population. Several lines of evidence suggest a positive association between environmental exposures, which are common and cumulative in a lifetime, and development of neurodegenerative diseases. Environmental or occupational exposure to manganese (Mn) has been implicated in neurodegeneration due to its ability to induce mitochondrial dysfunction, oxidative stress, and α-synuclein (α-Syn) aggregation. The role of the α-Syn protein vis-a-vis Mn is controversial, as it seemingly plays a duplicitous role in neuroprotection and neurodegeneration. α-Syn has low affinity for Mn, however an indirect interaction cannot be ruled out. In this review we will examine the current knowledge surrounding the interaction of α-Syn and Mn in neurodegenerative process.

Manganese and aging.

Manganese (Mn) is an essential metal that is required as a cofactor for many enzymes and is necessary for optimal biological function. Mn is abundant in the earth's crust and is present in soil and well water. Mn is also found in industrial settings, including mining, welding, and battery manufacture. Mn is also present in infant formula, parenteral nutrition, as well as pesticides and gasoline additives. A sufficient amount of Mn is obtained from most diets, and Mn deficiency is exceedingly rare. Excessive exposure to Mn in high doses can result in a condition known as manganism that results in psychological and emotional disturbances and motor symptoms that are reminiscent of Parkinson's disease, including gait disturbance, tremor, rigidity, and bradykinesia. Treatment for manganism is to remove the patient from Mn exposure, though symptoms are generally irreversible. The effects of exposure to Mn at lower doses are less clear. Little work has been done to evaluate the effects of chronic exposure to subclinical levels of Mn, especially in regard to lifelong exposures and the effects on the aging process. Mn is known to have effects on some of the same mechanistic processes that are altered in aging. This review will describe the general effects of Mn exposure and will focus on how Mn may be related to some of the mechanism of aging: neurogenesis, oxidative stress, and microglial activation and inflammation.

RNASeq in C. elegans Following Manganese Exposure.

Manganese is a metal that is required for optimal biological functioning of organisms. Absorption, cellular import and export, and excretion of manganese are all tightly regulated. While some genes involved in regulation, such as DMT-1 and ferroportin, are known, it is presumed that many more are involved and as yet unknown. Excessive exposure to manganese, usually in industrial settings such as mining or welding, can lead to neurotoxicity and a condition known as manganism that closely resembles Parkinson's disease. Elucidating transcriptional changes following manganese exposure could lead to the development of biomarkers for exposure. This unit presents a protocol for RNA sequencing in the worm Caenorhabditis elegans to assay for transcriptional changes following exposure to manganese. This protocol is adaptable to any environmental exposure in C. elegans. The protocol results in counts of gene transcripts in control versus exposed conditions and a ranked list of differentially expressed genes for further study.

Gene-wise association of variants in four lysosomal storage disorder genes in neuropathologically confirmed Lewy body disease.

OBJECTIVE: Variants in GBA are associated with Lewy Body (LB) pathology. We investigated whether variants in other lysosomal storage disorder (LSD) genes also contribute to disease pathogenesis. METHODS: We performed a genetic analysis of four LSD genes including GBA, HEXA, SMPD1, and MCOLN1 in 231 brain autopsies. Brain autopsies included neuropathologically defined LBD without Alzheimer Disease (AD) changes (n = 59), AD without significant LB pathology (n = 71), Alzheimer disease and lewy body variant (ADLBV) (n = 68), and control brains without LB or AD neuropathology (n = 33). Sequencing of HEXA, SMPD1, MCOLN1 and GBA followed by 'gene wise' genetic association analysis was performed. To determine the functional effect, a biochemical analysis of GBA in a subset of brains was also performed. GCase activity was measured in a subset of brain samples (n = 64) that included LBD brains, with or without GBA mutations, and control brains. A lipidomic analysis was also performed in brain autopsies (n = 67) which included LBD (n = 34), ADLBV (n = 3), AD (n = 4), PD (n = 9) and control brains (n = 17), comparing GBA mutation carriers to non-carriers. RESULTS: In a 'gene-wise' analysis, variants in GBA, SMPD1 and MCOLN1 were significantly associated with LB pathology (p range: 0.03-4.14 x10(-5)). Overall, the mean levels of GCase activity were significantly lower in GBA mutation carriers compared to non-carriers (p<0.001). A significant increase and accumulation of several species for the lipid classes, ceramides and sphingolipids, was observed in LBD brains carrying GBA mutations compared to controls (p range: p<0.05-p<0.01). INTERPRETATION: Our study indicates that variants in GBA, SMPD1 and MCOLN1 are associated with LB pathology. Biochemical data comparing GBA mutation carrier to non-carriers support these findings, which have important implications for biomarker development and therapeutic strategies.

Genetic factors and manganese-induced neurotoxicity.

Manganese (Mn), is a trace metal required for normal physiological processes in humans. Mn levels are tightly regulated, as high levels of Mn result in accumulation in the brain and cause a neurological disease known as manganism. Manganism shares many similarities with Parkinson's disease (PD), both at the physiological level and the cellular level. Exposure to high Mn-containing environments increases the risk of developing manganism. Mn is absorbed primarily through the intestine and then released in the blood. Excessive Mn is secreted in the bile and excreted in feces. Mn enters and exits cells through a number of non-specific importers localized on the cell membrane. Mutations in one of the Mn exporters, SLC30A10 (solute carrier family 30, member 10), result in Mn induced toxicity with liver impairments and neurological dysfunction. Four PD genes have been identified in connection to regulation of Mn toxicity, shedding new light on potential links between manganism and PD.

Identification of a potential susceptibility locus for macular telangiectasia type 2.

Macular Telangiectasia type 2 (MacTel) is a relatively rare macular disease of adult onset presenting with distortions in the visual field and leading to progressive loss of visual acuity. For the purpose of a gene mapping study, several pedigrees were ascertained with multiple affected family members. Seventeen families with a total of 71 individuals (including 45 affected or possibly affected) were recruited at clinical centers in 7 countries under the auspices of the MacTel Project. The disease inheritance was consistent with autosomal dominant segregation with reduced penetrance. Genome-wide linkage analysis was performed, followed by analysis of recombination breakpoints. Linkage analysis identified a single peak with multi-point LOD score of 3.45 on chromosome 1 at 1q41-42 under a dominant model. Recombination mapping defined a minimal candidate region of 15.6 Mb, from 214.32 (rs1579634; 219.96 cM) to 229.92 Mb (rs7542797; 235.07 cM), encompassing the 1q41-42 linkage peak. Sanger sequencing of the top 14 positional candidates genes under the linkage peak revealed no causal variants in these pedigrees.

Analysis of candidate genes for macular telangiectasia type 2.

PURPOSE: To find the gene(s) responsible for macular telangiectasia type 2 (MacTel) by a candidate-gene screening approach. METHODS: Candidate genes were selected based on the following criteria: those known to cause or be associated with diseases with phenotypes similar to MacTel, genes with known function in the retinal vasculature or macular pigment transport, genes that emerged from expression microarray data from mouse models designed to mimic MacTel phenotype characteristics, and genes expressed in the retina that are also related to diabetes or hypertension, which have increased prevalence in MacTel patients. Probands from eight families with at least two affected individuals were screened by direct sequencing of 27 candidate genes. Identified nonsynonymous variants were analyzed to determine whether they co-segregate with the disease in families. Allele frequencies were determined by TaqMan analysis of the large MacTel and control cohorts. RESULTS: We identified 23 nonsynonymous variants in 27 candidate genes in at least one proband. Of these, eight were known single nucleotide polymorphisms (SNPs) with allele frequencies of >0.05; these variants were excluded from further analyses. Three previously unidentified missense variants, three missense variants with reported disease association, and five rare variants were analyzed for segregation and/or allele frequencies. No variant fulfilled the criteria of being causal for MacTel. A missense mutation, p.Pro33Ser in frizzled homolog (Drosophila) 4 (FZD4), previously suggested as a disease-causing variant in familial exudative vitreoretinopathy, was determined to be a rare benign polymorphism. CONCLUSIONS: We have ruled out the exons and flanking intronic regions in 27 candidate genes as harboring causal mutations for MacTel.

Transplantation of reprogrammed embryonic stem cells improves visual function in a mouse model for retinitis pigmentosa.

BACKGROUND: To study whether C57BL/6J-Tyr/J (C2J) mouse embryonic stem (ES) cells can differentiate into retinal pigment epithelial (RPE) cells in vitro and then restore retinal function in a model for retinitis pigmentosa: Rpe65/Rpe65 C57BL6 mice. METHODS: Yellow fluorescent protein (YFP)-labeled C2J ES cells were induced to differentiate into RPE-like structures on PA6 feeders. RPE-specific markers are expressed from differentiated cells in vitro. After differentiation, ES cell-derived RPE-like cells were transplanted into the subretinal space of postnatal day 5 Rpe65/Rpe65 mice. Live imaging of YFP-labeled C2J ES cells demonstrated survival of the graft. Electroretinograms (ERGs) were performed on transplanted mice to evaluate the functional outcome of transplantation. RESULTS: RPE-like cells derived from ES cells sequentially express multiple RPE-specific markers. After transplantation, YFP-labeled cells can be tracked with live imaging for as long as 7 months. Although more than half of the mice were complicated with retinal detachments or tumor development, one fourth of the mice showed increased electroretinogram responses in the transplanted eyes. Rpe65/Rpe65 mice transplanted with RPE-like cells showed significant visual recovery during a 7-month period, whereas those injected with saline, PA6 feeders, or undifferentiated ES cells showed no rescue. CONCLUSIONS: ES cells can differentiate, morphologically, and functionally, into RPE-like cells. Based on these findings, differentiated ES cells have the potential for the development of new therapeutic approaches for RPE-specific diseases such as certain forms of retinitis pigmentosa and macular degeneration. Nevertheless, stringent control of retinal detachment and teratoma development will be necessary before initiation of treatment trials.

Wnt signaling in angiogenesis.

Although progress has been made in understanding the role of growth factors and their receptors in angiogenesis, little is known about how the Wnt family of growth factors function in the vasculature. Wnts are multifunctional factors that act through the frizzled receptors to regulate proliferation, apoptosis, branching morphogenesis, inductive processes, and cell polarity. All of these processes must occur as developing vascular structures are formed and maintained. Recent evidence has linked the Wnt/Frizzled signaling pathway to proper vascular growth in murine and human retina. Here we review the literature describing the angiogenic functions for Wnt signaling and focus on a newly discovered angiogenic factor, Norrin, which acts through the Wnt receptor, Frizzled4.

Wnt5a signaling induces proliferation and survival of endothelial cells in vitro and expression of MMP-1 and Tie-2.

Wnts are lipid-modified secreted glycoproteins that regulate diverse biological processes. We report that Wnt5a, which functions in noncanonical Wnt signaling, has activity on endothelial cells. Wnt5a is endogenously expressed in human primary endothelial cells and is expressed in murine vasculature at several sites in mouse embryos and tissues. Expression of exogenous Wnt5a in human endothelial cells promoted angiogenesis. Wnt5a induced noncanonical Wnt signaling in endothelial cells, as measured by Dishevelled and ERK1/2 phosphorylation, and inhibition of canonical Wnt signaling, a known property of Wnt5a. Wnt5a induced endothelial cell proliferation and enhanced cell survival under serum-deprived conditions. The Wnt5a-mediated proliferation was blocked by Frizzled-4 extracellular domain. Wnt5a expression enhanced capillary-like network formation, whereas reduction of Wnt5a expression decreased network formation. Reduced Wnt5a expression inhibited endothelial cell migration. Screening for Wnt5a-regulated genes in cultured endothelial cells identified several encoding angiogenic regulators, including matrix metalloproteinase-1, an interstitial collagenase, and Tie-2, a receptor for angiopoietins. Thus, Wnt5a acts through noncanonical Wnt signaling to promote angiogenesis.