Quantitative Measurement of Tau Aggregation in Genetically Modified Rats with Neurodegeneration.

Animal models of neurodegenerative diseases have helped us to better understand the pathogenesis of neurodegenerative diseases. However, recent failure to translate pre-clinical model studies to the clinic urges us to develop more rigorous and faithful animal models in neurodegenerative diseases. As genetic manipulation of rats becomes much more accessible due to availability of CRISPR-Cas9 and other genomic editing toolboxes, rats have been emerging as a new model system for neurodegenerative diseases. Even though mouse models have been dominant over the last decades, rats may provide advantages over mice. Rats are more genetically and physiologically closer to humans than to mice. Also, certain rat models can represent deposition of tau, which is one of the key pathological features of Alzheimer's diseases and tauopathies. However, there is an unmet need for standardized, rigorous testing in rat models. We adopted two commonly used biochemical and immunofluorescence methods from mice and human postmortem brains to measure tau aggregation. Due to the intrinsic differences between mice and rats, e.g., size of rat brains, certain equipment is required for rat models to study tau pathologies. Along with specific tools, here we describe the detailed methods for rat models of neurodegenerative diseases.

GGA1 interacts with the endosomal Na+/H+ Exchanger NHE6 governing localization to the endosome compartment.

Mutations in the endosomal Na+/H+ exchanger (NHE6) cause Christianson syndrome (CS), an X-linked neurological disorder. Previous studies have shown that NHE6 functions in regulation of endosome acidification and maturation in neurons. Using yeast two-hybrid screening with the NHE6 carboxyl-terminus as bait, we identify Golgi-associated, Gamma adaptin ear containing, ARF binding protein 1 (GGA1) as an interacting partner for NHE6. We corroborated the NHE6-GGA1 interaction using co-immunoprecipitation (co-IP): using over-expressed constructs in mammalian cells; and co-IP of endogenously-expressed GGA1 and NHE6 from neuroblastoma cells, as well as from mouse brain. We demonstrate that GGA1 interacts with organellar NHEs (NHE6, NHE7 and NHE9) but not with cell-surface localized NHEs (NHE1 and NHE5). By constructing hybrid NHE1/NHE6 exchangers, we demonstrate that the cytoplasmic tail of NHE6 is necessary and sufficient for interactions with GGA1. We demonstrate the co-localization of NHE6 and GGA1 in cultured, primary hippocampal neurons, using super-resolution microscopy. We test the hypothesis that the interaction of NHE6 and GGA1 functions in the localization of NHE6 to the endosome compartment. Using subcellular fractionation experiments, we show that NHE6 is mis-localized in GGA1 knockout cells wherein we find less NHE6 in endosomes but more NHE6 transport to lysosomes, and more Golgi retention of NHE6 with increased exocytosis to the surface plasma membrane. Consistent with NHE6 mis-localization, and Golgi retention, we find the intra-luminal pH in Golgi to be alkalinized. Our study demonstrates a new interaction between NHE6 and GGA1 which functions in the localization of this intra-cellular NHE to the endosome compartment.

Christianson Syndrome across the Lifespan: An International Longitudinal Study in Children, Adolescents, and Adults.

Mutations in the X-linked endosomal Na+/H+ Exchanger 6 (NHE6) causes Christianson Syndrome (CS). In the largest study to date, we examine genetic diversity and clinical progression, including cerebellar degeneration, in CS into adulthood. Data were collected as part of the International Christianson Syndrome and NHE6 (SLC9A6) Gene Network Study. Forty-four individuals with 31 unique NHE6 mutations, age 2 to 32 years, were followed prospectively, herein reporting baseline, 1-year follow-up, and retrospective natural history. We present data on the CS phenotype with regard to physical growth, adaptive and motor regression, and across the lifespan, including information on mortality. Longitudinal data on body weight and height were examined using a linear mixed model: the rate of growth across development was slow and resulted in prominently decreased age-normed height and weight by adulthood. Adaptive functioning was longitudinally examined: a majority of adult (18+ years) participants lost gross and fine motor skills over a 1-year follow-up. Previously defined core diagnostic criteria for CS (present in >85%) - namely nonverbal status, intellectual disability, epilepsy, postnatal microcephaly, ataxia, hyperkinesia - were universally present in age 6 to 16; however, an additional core feature of high pain tolerance was added (present in 91%), and furthermore, evolution of symptoms were noted across the lifespan, such that postnatal microcephaly, ataxia and high pain threshold were often not apparent prior to age 6, and hyperkinesis decreased after age 16. While neurologic exams were consistent with cerebellar dysfunction, importantly, a majority of individuals (>50% older than 10) also had corticospinal tract abnormalities. Three participants died during the period of the study. In this large and longitudinal study of CS, we begin to define the trajectory of symptoms and the adult phenotype, thereby identifying critical targets for treatment.

Targeting NHE6 gene expression identifies lysosome and neurodevelopmental mechanisms in a haploid in vitro cell model.

Christianson syndrome (CS) is an X-linked disorder resulting from loss-of-function (LoF) mutations in SLC9A6 encoding the endosomal Na+/H+ exchanger 6 (NHE6). CS presents with developmental delay, seizures, intellectual disability, nonverbal status, postnatal microcephaly, and ataxia. To define transcriptome signatures of NHE6 LoF, we conducted in-depth RNA-sequencing (RNA-seq) analysis on a haploid NHE6 null cell model. CRIPSR/Cas9 genome editing introduced multiple LoF mutations into SLC9A6 in the near haploid human cell line Hap1. Isogenic, paired parental controls were also studied. NHE6 mutant cell lines were confirmed to have intra-endosomal over-acidification as was seen in other NHE6 null cells. RNA-seq analysis was performed by two widely used pipelines: HISAT2-StringTie-DEseq2 and STAR-HTseq-DEseq2. We identified 1056 differentially expressed genes in mutant NHE6 lines, including genes associated with neurodevelopment, synapse function, voltage-dependent calcium channels, and neuronal signaling. Weighted gene co-expression network analysis was then applied and identified a critical module enriched for genes governing lysosome function. By identifying significantly changed gene expression that is associated with lysosomal mechanisms in NHE6-null cells, our analyses suggest that loss of NHE6 function may converge on mechanisms implicated in lysosome-related neurologic disease. Further, this haploid cell model will serve as an important tool for translational science in CS.

Dynamic Measurement of Endosome-Lysosome Fusion in Neurons Using High-Content Imaging.

Endocytosis is a dynamic cellular process that actively transports particles into a cell. Late endosome fusion with the lysosome is a crucial step in the delivery of newly synthesized lysosomal proteins and endocytosed cargo for degradation. Disturbing this step in neurons is associated with neurological disorders. Thus, studying endosome-lysosome fusion in neurons will provide new insight into the mechanisms of these diseases and open new possibilities for therapeutic treatment. However, measuring endosome-lysosome fusion is challenging and time consuming, which limits the research in this area. Here we developed a high throughput method using pH-insensitive dye-conjugated dextrans and the Opera Phenix® High Content Screening System. By using this method, we successfully separated endosomes and lysosomes in neurons, and time-lapse images were collected to capture endosome-lysosome fusion events in hundreds of cells. Both assay set-up and analysis can be completed in an expeditious and efficient manner.

Live-Imaging Detection of Multivesicular Body-Plasma Membrane Fusion and Exosome Release in Cultured Primary Neurons.

Exosomes represent a class of extracellular vesicles (EVs) derived from the endocytic pathway that is important for cell-cell communication and implicated in the spread of pathogenic protein aggregates associated with neurological diseases. Exosomes are released extracellularly when multivesicular bodies (also known as late endosomes) fuse with the plasma membrane (PM). An important breakthrough in exosome research is the ability to capture MVB-PM fusion and exosome release simultaneously in individual cells using live-imaging microscopy techniques. Specifically, researchers have created a construct fusing CD63, a tetraspanin enriched in exosomes, with the pH-sensitive reporter pHluorin whereby CD63-pHluorin fluorescence is quenched in the acidic MVB lumen and only fluoresces when released into the less acidic extracellular environment. Here, we describe a method using this CD63-pHluorin construct to visualize MVB-PM fusion/exosome secretion in primary neurons using total internal reflection fluorescence (TIRF) microscopy.

Moderators of Age of Diagnosis in > 20,000 Females with Autism in Two Large US Studies.

The objective of this study was to determine the clinical features that moderate a later age at ASD diagnosis in females in a large sample of females with ASD. Within two large and independent ASD datasets (> 20,000 females), females were first diagnosed with ASD 14-months later relative to males. This later age at diagnosis was moderated by a mild or atypical presentation, wherein repetitive behaviors were limited, IQ and language were broadly intact, and recognized symptoms emerged later in development. Females are at risk for a later age at ASD diagnosis and treatment implementation, and modification of early childhood ASD screening methods for females may be warranted.

17q12 deletion syndrome mouse model shows defects in craniofacial, brain and kidney development, and glucose homeostasis.

17q12 deletion (17q12Del) syndrome is a copy number variant (CNV) disorder associated with neurodevelopmental disorders and renal cysts and diabetes syndrome (RCAD). Using CRISPR/Cas9 genome editing, we generated a mouse model of 17q12Del syndrome on both inbred (C57BL/6N) and outbred (CD-1) genetic backgrounds. On C57BL/6N, the 17q12Del mice had severe head development defects, potentially mediated by haploinsufficiency of Lhx1, a gene within the interval that controls head development. Phenotypes included brain malformations, particularly disruption of the telencephalon and craniofacial defects. On the CD-1 background, the 17q12Del mice survived to adulthood and showed milder craniofacial and brain abnormalities. We report postnatal brain defects using automated magnetic resonance imaging-based morphometry. In addition, we demonstrate renal and blood glucose abnormalities relevant to RCAD. On both genetic backgrounds, we found sex-specific presentations, with male 17q12Del mice exhibiting higher penetrance and more severe phenotypes. Results from these experiments pinpoint specific developmental defects and pathways that guide clinical studies and a mechanistic understanding of the human 17q12Del syndrome. This mouse mutant represents the first and only experimental model to date for the 17q12 CNV disorder. This article has an associated First Person interview with the first author of the paper.

Loss of endosomal exchanger NHE6 leads to pathological changes in tau in human neurons.

Disruption of endolysosomal and autophagy-lysosomal systems is increasingly implicated in neurodegeneration. Sodium-proton exchanger 6 (NHE6) contributes to the maintenance of proper endosomal pH, and loss-of function mutations in the X-linked NHE6 lead to Christianson syndrome (CS) in males. Neurodegenerative features of CS are increasingly recognized, with postmortem and clinical data implicating a role for tau. We generated cortical neurons from NHE6 knockout (KO) and isogenic wild-type control human induced pluripotent stem cells. We report elevated phosphorylated and sarkosyl-insoluble tau in NHE6 KO neurons. We demonstrate that NHE6 KO leads to lysosomal and autophagy dysfunction involving reduced lysosomal number and protease activity, diminished autophagic flux, and p62 accumulation. Finally, we show that treatment with trehalose or rapamycin, two enhancers of autophagy-lysosomal function, each partially rescue this tau phenotype. We provide insight into the neurodegenerative processes underlying NHE6 loss of function and into the broader role of the endosome-lysosome-autophagy network in neurodegeneration.

Loss of mitochondrial enzyme GPT2 causes early neurodegeneration in locus coeruleus.

Locus coeruleus (LC) is among the first brain areas to degenerate in Alzheimer's disease and Parkinson's disease; however, the underlying causes for the vulnerability of LC neurons are not well defined. Here we report a novel mechanism of degeneration of LC neurons caused by loss of the mitochondrial enzyme glutamate pyruvate transaminase 2 (GPT2). GPT2 Deficiency is a newly-recognized childhood neurometabolic disorder. The GPT2 enzyme regulates cell growth through replenishment of tricarboxylic acid (TCA) cycle intermediates and modulation of amino acid metabolism. In Gpt2-null mice, we observe an early loss of tyrosine hydroxylase (TH)-positive neurons in LC and reduced soma size at postnatal day 18. Gpt2-null LC shows selective positive Fluoro-Jade C staining. Neuron loss is accompanied by selective, prominent microgliosis and astrogliosis in LC. We observe reduced noradrenergic projections to and norepinephrine levels in hippocampus and spinal cord. Whole cell recordings in Gpt2-null LC slices show reduced soma size and abnormal action potentials with altered firing kinetics. Strikingly, we observe early decreases in phosphorylated S6 in Gpt2-null LC, preceding prominent p62 aggregation, increased LC3B-II to LC3B-I ratio, and neuronal loss. These data are consistent with a possible mechanism involving deficiency in protein synthesis and cell growth, associated subsequently with abnormal autophagy and neurodegeneration. As compared to the few genetic animal models with LC degeneration, loss of LC neurons in Gpt2-null mice is developmentally the earliest. Early neuron loss in LC in a model of human neurometabolic disease provides important clues regarding the metabolic vulnerability of LC and may lead to new therapeutic targets.

Parental age and autism severity in the Rhode Island Consortium for Autism Research and Treatment (RI-CART) study.

Advanced parental age at offspring birth has been associated with autism spectrum disorder (ASD). The objective of the current study was to investigate associations between parental age at birth and autism severity. The Rhode Island Consortium for Autism Research and Treatment (RI-CART) study represents a community-based sample with a range of autism severity, including participants with and without ASD. This study involved participants (n = 1178) enrolled in RI-CART with available mother and father ages at birth. Primary data points included the age of mother and father at the participant's birth and results from the Autism Diagnostic Observation Schedule - Second Edition (ADOS-2). Mothers were 1.7 years older at the time of birth of the child with ASD, as compared to mothers of offspring without ASD. Fathers of children with ASD were 1.6 years older at the time of birth than fathers of children without ASD. The age of both parents at offspring birth displayed a positive, statistically significant association with overall ASD severity and the severity of restricted/repetitive behaviors. This finding was driven by the association between parental age and the severity of compulsions or rituals. Intelligence and adaptive functioning did not moderate the relationship between parental age and ASD severity. This study extends prior research to show that advanced parental age at birth is associated with the severity as well as the presence of ASD in offspring.

Mitochondrial enzyme GPT2 regulates metabolic mechanisms required for neuron growth and motor function in vivo.

The metabolic needs for postnatal growth of the human nervous system are vast. Recessive loss-of-function mutations in the mitochondrial enzyme glutamate pyruvate transaminase 2 (GPT2) in humans cause postnatal undergrowth of brain, and cognitive and motor disability. We demonstrate that GPT2 governs critical metabolic mechanisms in neurons required for neuronal growth and survival. These metabolic processes include neuronal alanine synthesis and anaplerosis, the replenishment of tricarboxylic acid (TCA) cycle intermediates. We performed metabolomics across postnatal development in Gpt2-null mouse brain to identify the trajectory of dysregulated metabolic pathways: alterations in alanine occur earliest; followed by reduced TCA cycle intermediates and reduced pyruvate; followed by elevations in glycolytic intermediates and amino acids. Neuron-specific deletion of GPT2 in mice is sufficient to cause motor abnormalities and death pre-weaning, a phenotype identical to the germline Gpt2-null mouse. Alanine biosynthesis is profoundly impeded in Gpt2-null neurons. Exogenous alanine is necessary for Gpt2-null neuronal survival in vitro but is not needed for Gpt2-null astrocytes. Dietary alanine supplementation in Gpt2-null mice enhances animal survival and improves the metabolic profile of Gpt2-null brain but does not alone appear to correct motor function. In surviving Gpt2-null animals, we observe smaller upper and lower motor neurons in vivo. We also observe selective death of lower motor neurons in vivo with worsening motor behavior with age. In conclusion, these studies of the pathophysiology of GPT2 Deficiency have identified metabolic mechanisms that are required for neuronal growth and that potentially underlie selective neuronal vulnerabilities in motor neurons.

Early lysosome defects precede neurodegeneration with amyloid-ß and tau aggregation in NHE6-null rat brain.

Loss-of-function mutations in the X-linked endosomal Na+/H+ exchanger 6 (NHE6) cause Christianson syndrome in males. Christianson syndrome involves endosome dysfunction leading to early cerebellar degeneration, as well as later-onset cortical and subcortical neurodegeneration, potentially including tau deposition as reported in post-mortem studies. In addition, there is reported evidence of modulation of amyloid-ß levels in experimental models wherein NHE6 expression was targeted. We have recently shown that loss of NHE6 causes defects in endosome maturation and trafficking underlying lysosome deficiency in primary mouse neurons in vitro. For in vivo studies, rat models may have an advantage over mouse models for the study of neurodegeneration, as rat brain can demonstrate robust deposition of endogenously-expressed amyloid-ß and tau in certain pathological states. Mouse models generally do not show the accumulation of insoluble, endogenously-expressed (non-transgenic) tau or amyloid-ß. Therefore, to study neurodegeneration in Christianson syndrome and the possibility of amyloid-ß and tau pathology, we generated an NHE6-null rat model of Christianson syndrome using CRISPR-Cas9 genome-editing. Here, we present the sequence of pathogenic events in neurodegenerating NHE6-null male rat brains across the lifespan. NHE6-null rats demonstrated an early and rapid loss of Purkinje cells in the cerebellum, as well as a more protracted neurodegenerative course in the cerebrum. In both the cerebellum and cerebrum, lysosome deficiency is an early pathogenic event, preceding autophagic dysfunction. Microglial and astrocyte activation also occur early. In the hippocampus and cortex, lysosome defects precede loss of pyramidal cells. Importantly, we subsequently observed biochemical and in situ evidence of both amyloid-ß and tau aggregation in the aged NHE6-null hippocampus and cortex (but not in the cerebellum). Tau deposition is widely distributed, including cortical and subcortical distributions. Interestingly, we observed tau deposition in both neurons and glia, as has been reported in Christianson syndrome post-mortem studies previously. In summary, this experimental model is among very few examples of a genetically modified animal that exhibits neurodegeneration with deposition of endogenously-expressed amyloid-ß and tau. This NHE6-null rat will serve as a new robust model for Christianson syndrome. Furthermore, these studies provide evidence for linkages between endolysosome dysfunction and neurodegeneration involving protein aggregations, including amyloid-ß and tau. Therefore these studies may provide insight into mechanisms of more common neurodegenerative disorders, including Alzheimer's disease and related dementias.

Autism severity aggregates with family psychiatric history in a community-based autism sample.

The purpose of this study was to examine family psychiatric history in individuals with autism spectrum disorder (ASD) and its association with clinical presentation. Participants were 798 individuals with a clinical diagnosis of ASD, confirmed by the Autism Diagnostic Observation Schedule, Second Edition (ADOS-2), enrolled in Rhode Island Consortium for Autism Research and Treatment, a statewide research registry. Prior research suggests a specific behavioral phenotype in individuals with ASD who have family members with psychiatric diagnoses, including higher IQ and less severe language impairment. However, studies have not specifically investigated autism severity. We hypothesized that increased psychiatric family history would be associated with increased autism severity symptoms. Results show a strong association of increased burden of first-degree family psychiatric history with higher autism symptom severity as measured by Social Responsiveness Scale, Second Edition (SRS-2), but not with ADOS-2 severity scores, IQ, or adaptive functioning. These findings support the importance of investigating the contribution of psychiatric family history toward clinical ASD presentation. LAY SUMMARY: This study explored how family psychiatric history is related to clinical presentation of Autism Spectrum Disorder (ASD). Higher amounts of first-degree family psychiatric history was associated with higher autism symptom severity as measured by the Social Responsiveness Scale, Second Edition (SRS-2). The contribution of psychiatric family history requires ongoing investigation.

Loss of Christianson Syndrome Na+/H+ Exchanger 6 (NHE6) Causes Abnormal Endosome Maturation and Trafficking Underlying Lysosome Dysfunction in Neurons.

Loss-of-function mutations in endosomal Na+/H+ exchanger 6 (NHE6) cause the X-linked neurologic disorder Christianson syndrome. Patients exhibit symptoms associated with both neurodevelopmental and neurodegenerative abnormalities. While loss of NHE6 has been shown to overacidify the endosome lumen, and is associated with endolysosome neuropathology, NHE6-mediated mechanisms in endosome trafficking and lysosome function have been understudied. Here, we show that NHE6-null mouse neurons demonstrate worsening lysosome function with time in culture, likely as a result of defective endosome trafficking. NHE6-null neurons exhibit overall reduced lysosomal proteolysis despite overacidification of the endosome and lysosome lumen. Akin to Nhx1 mutants in Saccharomyces cerevisiae, we observe decreased endosome-lysosome fusion in NHE6-null neurons. Also, we find premature activation of pH-dependent cathepsin D (CatD) in endosomes. While active CatD is increased in endosomes, CatD activation and CatD protein levels are reduced in the lysosome. Protein levels of another mannose 6-phosphate receptor (M6PR)-dependent enzyme, ß-N-acetylglucosaminidase, were also decreased in lysosomes of NHE6-null neurons. M6PRs accumulate in late endosomes, suggesting defective M6PR recycling and retromer function in NHE6-null neurons. Finally, coincident with decreased endosome-lysosome fusion, using total internal reflection fluorescence, we also find a prominent increase in fusion between endosomal multivesicular bodies and the plasma membrane, indicating enhanced exosome secretion from NHE6-null neurons. In summary, in addition to overacidification of endosomes and lysosomes, loss of NHE6 leads to defects in endosome maturation and trafficking, including enhanced exosome release, contributing to lysosome deficiency and potentially leading to neurodegenerative disease.SIGNIFICANCE STATEMENT Loss-of-function mutations in the endosomal Na+/H+ exchanger 6 (NHE6) cause Christianson syndrome, an X-linked neurologic disorder. Loss of NHE6 has been shown to overacidify endosomes; however, endosome trafficking mechanisms have been understudied, and the mechanisms leading to neurodegeneration are largely unknown. In NHE6-null mouse neurons in vitro, we find worsening lysosome function with days in culture. Notably, pH-dependent lysosome enzymes, such as cathepsin D, have reduced activity in lysosomes yet increased, precocious activity in endosomes in NHE6-null neurons. Further, endosomes show reduced fusion to lysosomes, and increased fusion to the plasma membrane with increased exosome release. This study identifies new mechanisms involving defective endosome maturation and trafficking that impair lysosome function in Christianson syndrome, likely contributing to neurodegeneration.

Human iPSC lines from a Christianson syndrome patient with NHE6 W523X mutation, a biologically-related control, and CRISPR/Cas9 gene-corrected isogenic controls.

Loss-of-function mutations in Na+/H + exchanger 6 (NHE6) (also termed SLC9A6) cause the X-linked neurogenetic disorder Christianson syndrome (CS). Using peripheral blood mononuclear cells, we developed induced pluripotent stem cell (iPSC) lines from a patient with the NHE6 nonsense mutation c.1569G > A (p.(W523X)) and diagnosed with CS and from a biologically-related control. Using CRISPR/Cas9 gene editing, we generated two isogenic control lines in which the c.1569G > A mutation was corrected. All lines were verified by DNA sequencing and for NHE6 protein expression, pluripotency, and differentiation potential. These lines will serve as a valuable resource for both basic and translational studies in CS.

Generation of pathogenic TPP1 mutations in human stem cells as a model for neuronal ceroid lipofuscinosis type 2 disease.

Neuronal ceroid lipofuscinosis type 2 (CLN2 disease) is an autosomal recessive neurodegenerative disorder generally with onset at 2 to 4 years of age and characterized by seizures, loss of vision, progressive motor and mental decline, and premature death. CLN2 disease is caused by loss-of-function mutations in the tripeptidyl peptidase 1 (TPP1) gene leading to deficiency in TPP1 enzyme activity. Approximately 60% of patients have one of two pathogenic variants (c.509-1G > C or c.622C > T [p.(Arg208*)]). In order to generate a human stem cell model of CLN2 disease, we used CRISPR/Cas9-mediated knock-in technology to introduce these mutations in a homozygous state into H9 human embryonic stem cells. Heterozygous lines of the c.622C > T (p.(Arg208*)) mutation were also generated, which included a heterozygous mutant with a wild-type allele and different compound heterozygous coding mutants resulting from indels on one allele. We describe the methodology that led to the generation of the lines and provide data on the initial validation and characterization of these CLN2 disease models. Notably, both mutant lines (c.509-1G > C and c.622C > T [p.(Arg208*)]) in the homozygous state were shown to have reduced or absent protein, respectively, and deficiency of TPP1 enzyme activity. These models, which we have made available for wide-spread sharing, will be useful for future studies of molecular and cellular mechanisms underlying CLN2 disease and for therapeutic development.

Human neurons from Christianson syndrome iPSCs reveal mutation-specific responses to rescue strategies.

Christianson syndrome (CS), an X-linked neurological disorder characterized by postnatal attenuation of brain growth (postnatal microcephaly), is caused by mutations in SLC9A6, the gene encoding endosomal Na+/H+ exchanger 6 (NHE6). To hasten treatment development, we established induced pluripotent stem cell (iPSC) lines from patients with CS representing a mutational spectrum, as well as biologically related and isogenic control lines. We demonstrated that pathogenic mutations lead to loss of protein function by a variety of mechanisms: The majority of mutations caused loss of mRNA due to nonsense-mediated mRNA decay; however, a recurrent, missense mutation (the G383D mutation) had both loss-of-function and dominant-negative activities. Regardless of mutation, all patient-derived neurons demonstrated reduced neurite growth and arborization, likely underlying diminished postnatal brain growth in patients. Phenotype rescue strategies showed mutation-specific responses: A gene transfer strategy was effective in nonsense mutations, but not in the G383D mutation, wherein residual protein appeared to interfere with rescue. In contrast, application of exogenous trophic factors (BDNF or IGF-1) rescued arborization phenotypes across all mutations. These results may guide treatment development in CS, including gene therapy strategies wherein our data suggest that response to treatment may be dictated by the class of mutation.

Homozygous deletions implicate non-coding epigenetic marks in Autism spectrum disorder.

More than 98% of the human genome is made up of non-coding DNA, but techniques to ascertain its contribution to human disease have lagged far behind our understanding of protein coding variations. Autism spectrum disorder (ASD) has been mostly associated with coding variations via de novo single nucleotide variants (SNVs), recessive/homozygous SNVs, or de novo copy number variants (CNVs); however, most ASD cases continue to lack a genetic diagnosis. We analyzed 187 consanguineous ASD families for biallelic CNVs. Recessive deletions were significantly enriched in affected individuals relative to their unaffected siblings (17% versus 4%, p < 0.001). Only a small subset of biallelic deletions were predicted to result in coding exon disruption. In contrast, biallelic deletions in individuals with ASD were enriched for overlap with regulatory regions, with 23/28 CNVs disrupting histone peaks in ENCODE (p < 0.009). Overlap with regulatory regions was further demonstrated by comparisons to the 127-epigenome dataset released by the Roadmap Epigenomics project, with enrichment for enhancers found in primary brain tissue and neuronal progenitor cells. Our results suggest a novel noncoding mechanism of ASD, describe a powerful method to identify important noncoding regions in the human genome, and emphasize the potential significance of gene activation and regulation in cognitive and social function.