ESCRT disruption provides evidence against signaling functions for synaptic exosomes.

Extracellular vesicles (EVs) are released by many cell types including neurons, carrying cargoes involved in signaling and disease. It is unclear whether EVs promote intercellular signaling or serve primarily to dispose of unwanted materials. We show that loss of multivesicular endosome-generating ESCRT (endosomal sorting complex required for transport) machinery disrupts release of EV cargoes from Drosophila motor neurons. Surprisingly, ESCRT depletion does not affect the signaling activities of the EV cargo Synaptotagmin-4 (Syt4) and disrupts only some signaling activities of the EV cargo Evenness Interrupted (Evi). Thus, these cargoes may not require intercellular transfer via EVs, and instead may be conventionally secreted or function cell autonomously in the neuron. We find that EVs are phagocytosed by glia and muscles, and that ESCRT disruption causes compensatory autophagy in presynaptic neurons, suggesting that EVs are one of several redundant mechanisms to remove cargoes from synapses. Our results suggest that synaptic EV release serves primarily as a proteostatic mechanism for certain cargoes.

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.

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.

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 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.

Complex Neurological Phenotype in Female Carriers of NHE6 Mutations.

Mutations in NHE6 (also termed SLC9A6) cause the X-linked neurological disorder Christianson syndrome (CS) in males. The purpose of this study was to examine the phenotypic spectrum of female carriers of NHE6 mutations. Twenty female carriers from 9 pedigrees were enrolled, ranging from approximately age 2 to 65. A subset of female carriers was assessed using standardized neuropsychological measures. Also, the association of NHE6 expression with markers of brain age was evaluated using 740 participants in the Religious Orders Study (ROS) and Rush Memory and Aging Project (MAP). A majority, but not all, female carriers demonstrated a deficit in at least one neurocognitive domain (85%). A recognizable neuropsychological profile emerged, revealing impairments in visuospatial function, attention, and executive function. Common neuropsychiatric diagnoses included: intellectual disability/developmental delay (20%), learning difficulties (31%), speech/language delays (30%), and attention-deficit/hyperactivity disorder (20%). Notable neurological diagnoses in aging CS female carriers include corticobasal degeneration and atypical parkinsonism. In postmortem brains from the ROS/MAP dataset of normal and pathological aging, decreased NHE6 expression was correlated with greater tau deposition. Our study provides an examination of the phenotypic range in female carriers of NHE6 mutations. The findings indicate that NHE6-related disease in females represents a new neurogenetic condition.

Mixed Neurodevelopmental and Neurodegenerative Pathology in Nhe6-Null Mouse Model of Christianson Syndrome.

Christianson syndrome (CS) is an X-linked disorder resulting from loss-of-function mutations in SLC9A6, which encodes the endosomal Na+/H+ exchanger 6 (NHE6). Symptoms include early developmental delay, seizures, intellectual disability, nonverbal status, autistic features, postnatal microcephaly, and progressive ataxia. Neuronal development is impaired in CS, involving defects in neuronal arborization and synaptogenesis, likely underlying diminished brain growth postnatally. In addition to neurodevelopmental defects, some reports have supported neurodegenerative pathology in CS with age. The objective of this study was to determine the nature of progressive changes in the postnatal brain in Nhe6-null mice. We examined the trajectories of brain growth and atrophy in mutant mice from birth until very old age (2 yr). We report trajectories of volume changes in the mutant that likely reflect both brain undergrowth as well as tissue loss. Reductions in volume are first apparent at 2 mo, particularly in the cerebellum, which demonstrates progressive loss of Purkinje cells (PCs). We report PC loss in two distinct Nhe6-null mouse models. More widespread reductions in tissue volumes, namely, in the hippocampus, striatum, and cortex, become apparent after 2 mo, largely reflecting delays in growth with more limited tissue losses with aging. Also, we identify pronounced glial responses, particularly in major fiber tracts such as the corpus callosum, where the density of activated astrocytes and microglia are substantially increased. The prominence of the glial response in axonal tracts suggests a primary axonopathy. Importantly, therefore, our data support both neurodevelopmental and degenerative mechanisms in the pathobiology of CS.

Discovery of Rare Mutations in Autism: Elucidating Neurodevelopmental Mechanisms.

Autism spectrum disorder (ASD) is a group of highly genetic neurodevelopmental disorders characterized by language, social, cognitive, and behavioral abnormalities. ASD is a complex disorder with a heterogeneous etiology. The genetic architecture of autism is such that a variety of different rare mutations have been discovered, including rare monogenic conditions that involve autistic symptoms. Also, de novo copy number variants and single nucleotide variants contribute to disease susceptibility. Finally, autosomal recessive loci are contributing to our understanding of inherited factors. We will review the progress that the field has made in the discovery of these rare genetic variants in autism. We argue that mutation discovery of this sort offers an important opportunity to identify neurodevelopmental mechanisms in disease. The hope is that these mechanisms will show some degree of convergence that may be amenable to treatment intervention.

Genetic and phenotypic diversity of NHE6 mutations in Christianson syndrome.

OBJECTIVE: Recently, Christianson syndrome (CS) has been determined to be caused by mutations in the X-linked Na(+) /H(+) exchanger 6 (NHE6). We aimed to determine the diagnostic criteria and mutational spectrum for CS. METHODS: Twelve independent pedigrees (14 boys, age = 4-19 years) with mutations in NHE6 were administered standardized research assessments, and mutations were characterized. RESULTS: The mutational spectrum was composed of 9 single nucleotide variants, 2 indels, and 1 copy number variation deletion. All mutations were protein-truncating or splicing mutations. We identified 2 recurrent mutations (c.1498 c>t, p.R500X; and c.1710 g>a, p.W570X). Otherwise, all mutations were unique. In our study, 7 of 12 mutations (58%) were de novo, in contrast to prior literature wherein mutations were largely inherited. We also report prominent neurological, medical, and behavioral symptoms. All CS participants were nonverbal and had intellectual disability, epilepsy, and ataxia. Many had prior diagnoses of autism and/or Angelman syndrome. Other neurologic symptoms included eye movement abnormalities (79%), postnatal microcephaly (92%), and magnetic resonance imaging evidence of cerebellar atrophy (33%). Regression was noted in 50%, with recurrent presentations involving loss of words and/or the ability to walk. Medical symptoms, particularly gastrointestinal symptoms, were common. Height and body mass index measures were below normal ranges in most participants. Behavioral symptoms included hyperkinetic behavior (100%), and a majority exhibited high pain threshold. INTERPRETATION: This is the largest cohort of independent CS pedigrees reported. We propose diagnostic criteria for CS. CS represents a novel neurogenetic disorder with general relevance to autism, intellectual disability, Angelman syndrome, epilepsy, and regression.

Clinical characteristics of children with autism spectrum disorder and co-occurring epilepsy.

OBJECTIVES: To estimate the prevalence of epilepsy in children with Autism Spectrum Disorder (ASD) and to determine the demographic and clinical characteristics of children with ASD and epilepsy in a large patient population. METHODS: Cross-sectional study using four samples of children with ASD for a total of 5,815 participants with ASD. The prevalence of epilepsy was estimated from a population-based sample. Children with and without epilepsy were compared on demographic and clinical characteristics. Multivariate logistic regression was used to examine the association between demographic and clinical characteristics and epilepsy. RESULTS: The average prevalence of epilepsy in children with ASD 2-17 years was 12.5%; among children aged 13 years and older, 26% had epilepsy. Epilepsy was associated with older age, lower cognitive ability, poorer adaptive and language functioning, a history of developmental regression and more severe ASD symptoms. The association between epilepsy and the majority of these characteristics appears to be driven by the lower IQ of participants with epilepsy. In a multivariate regression model, only age and cognitive ability were independently associated with epilepsy. Children age 10 or older had 2.35 times the odds of being diagnosed with epilepsy (p<.001) and for a one standard deviation increase in IQ, the odds of having epilepsy decreased by 47% (p<.001). CONCLUSION: This is among the largest studies to date of patients with ASD and co-occurring epilepsy. Based on a representative sample of children with ASD, the average prevalence of epilepsy is approximately 12% and reaches 26% by adolescence. Independent associations were found between epilepsy and older age and lower cognitive ability. Other risk factors, such as poor language and developmental regression, are not associated with epilepsy after controlling for IQ. These findings can help guide prognosis and alert clinicians to patients with ASD who are at increased risk for epilepsy.

Family functioning deficits in bipolar disorder and ADHD in youth.

BACKGROUND: Rates of diagnosis and treatment for bipolar disorder (BD) in youth continue to rise. Researchers and clinicians experience difficulty differentiating between BD in youth and other conditions that are commonly comorbid or share similar clinical features with BD, especially attention-deficit/hyperactivity disorder (ADHD). Comparative studies of the phenomenology and psychosocial correlates of these conditions help to address this. Family functioning is an important topic for both BD and ADHD since both are associated with numerous family-related deficits. One previous study suggested that manic/hypomanic youths'family functioning differed from ADHD and typically developing control (TDC) groups. However, many family functioning studies with BD and ADHD youth have methodological limitations or fail to use comprehensive, validated measures. METHODS: This investigation used adolescent report on the Family Assessment Device (FAD), based on the McMaster Model of family functioning. Youth were recruited in BD (n=30), ADHD (n=36), and TDC (n=41) groups. RESULTS: Groups were similar on most demographic variables, but The TDC group scored somewhat higher than the others on IQ and socioeconomic status. FAD results indicated that BD and ADHD groups scored worse than TDC on the General Functioning and Roles scales of the FAD. In addition, the BD group showed impairment on the Problem Solving scale relative to TDC. LIMITATIONS: sample size, lack of parent report, ADHD comorbidity in BD group. CONCLUSIONS: Family functioning deficits distinguish both clinical groups from TDC, and problem-solving dysfunction may be specific to BD. These findings may apply to treatment models for both conditions.

Distribution of disease-associated copy number variants across distinct disorders of cognitive development.

OBJECTIVE: The purpose of the present study was to discover the extent to which distinct DSM disorders share large, highly recurrent copy number variants (CNVs) as susceptibility factors. We also sought to identify gene mechanisms common to groups of diagnoses and/or specific to a given diagnosis based on associations with CNVs. METHOD: Systematic review of 820 PubMed articles on autism spectrum disorder (ASD), intellectual disability (ID), schizophrenia, and epilepsy produced 54 CNVs associated with one or several disorders. Pathway analysis on genes implicated by CNVs in different groupings was conducted. RESULTS: The majority of CNVs were found in ID with the other disorders somewhat subsumed, yet certain CNVs were associated with isolated or groups of disorders. Based on genes implicated by CNVs, ID encompassed 96.8% of genes in ASD, 92.8% of genes in schizophrenia, and 100.0% of genes in epilepsy. Pathway analysis revealed that synapse processes were enriched in ASD, ID, and schizophrenia. Disease-specific processes were identified in ID (actin cytoskeleton processes), schizophrenia (ubiquitin-related processes), and ASD (synaptic vesicle transport and exocytosis). CONCLUSIONS: Intellectual disability may arise from the broadest range of genetic pathways, and specific subsets of these pathways appear to be relevant to other disorders or combinations of these disorders. It is clear that statistically significant CNVs across disorders of cognitive development are highly enriched for biological processes related to the synapse. There are also disorder-specific processes that may aid in understanding the distinct presentations and pathophysiology of these disorders.

Emotional face identification in youths with primary bipolar disorder or primary attention-deficit/hyperactivity disorder.

OBJECTIVE: Bipolar disorder (BD) and attention-deficit/hyperactivity disorder (ADHD) are often comorbid or confounded; therefore, we evaluated emotional face identification to better understand brain/behavior interactions in children and adolescents with either primary BD, primary ADHD, or typically developing controls (TDC). METHOD: Participants included individuals 7 to 17 years of age (overall sample mean age 12.40 ± 3.01 years), with "narrow-phenotype" pediatric BD (n = 30) or ADHD (n = 38), or typically developing controls (TDC) with no psychiatric disorders themselves or in their first-degree relatives (n = 41). In the BD group, comorbid diagnoses were allowed; however, youth in the ADHD group were excluded for comorbid mood or anxiety disorders. Patient groups were not excluded for psychotropic medication use. Emotional face identification was assessed using the computerized Diagnostic Analysis of Non-Verbal Accuracy (DANVA). RESULTS: Participants with BD made significantly more identification errors on child happy faces than either TDCs (p = .03) or participants with ADHD (p = .01). Furthermore, youth with BD (0.33 ± 0.55) were more likely than youth with ADHD (0.11 ± 0.31) to make errors on low-intensity child happy faces (p = .05) but not high-intensity happy faces (p = NS). Participants with BD and ADHD made significantly more total errors in child face labeling than did TDCs, although participants with BD and ADHD did not differ from one another. CONCLUSION: Our data suggest that youths with BD have specific alterations in emotional face identification of happy faces, an important finding that supports theories that response to positively valenced emotional stimuli may be especially salient in BD. Clinical trial registration information-Brain Imaging and Computer Games in Children With Either Bipolar Disorder, ADHD, Anxiety or Healthy Controls (BBPP); http://clinicaltrials.gov/; NCT01570426.

An unbalanced translocation involving loss of 10q26.2 and gain of 11q25 in a pedigree with autism spectrum disorder and cerebellar juvenile pilocytic astrocytoma.

We report on a pedigree with a pair of brothers each with minor anomalies, developmental delay, and autistic-symptoms who share an unbalanced translocation (not detectable by karyotype). The unbalanced translocation involves a 7.1 Mb loss of the terminal portion of 10q, and a 4.2 Mb gain of 11q. One of the brothers also developed a cerebellar juvenile pilocytic astrocytoma. The father was found to be a balanced carrier and the couple had a previous miscarriage. We demonstrate that the breakpoint for the triplicated region from chromosome 11 is adjacent to two IgLON genes, namely Neurotrimin (NTM) and Opioid Binding Protein/Cell Adhesion Molecule-like (OPCML). These genes are highly similar neural cell adhesion molecules that have been implicated in synaptogenesis and oncogenesis, respectively. The children also have a 10q deletion and are compared to other children with the 10q deletion syndrome which generally does not involve autism spectrum disorders (ASDs) or cancer. Together these data support a role for NTM and OPCML in developmental delay and potentially in cancer susceptibility.

Developmental meta-analysis of the functional neural correlates of autism spectrum disorders.

OBJECTIVE: There is a pressing need to elucidate the brain-behavior interactions underlying autism spectrum disorders (ASD) given the marked rise in ASD diagnosis over the past decade. Functional magnetic resonance imaging (fMRI) has begun to address this need, but few fMRI studies have evaluated age-related changes in ASD. Therefore, we conducted a developmental analysis of activation likelihood estimation (ALE) meta-analysis to compare child versus adult ASD fMRI studies. We hypothesized that children and adolescents with ASD (<18 years old) would rely less on prefrontal cortex structures than adults (≥18 years old). METHOD: PubMed and PsycInfo literature searches were conducted to identify task-dependent fMRI studies of children or adults with ASD. Then recent GingerALE software improvements were leveraged to perform direct comparisons of child (n = 18) versus adult (n = 24) studies. RESULTS: ALE meta-analyses of social tasks showed that children and adolescents with ASD versus adults had significantly greater hyperactivation in the left post-central gyrus, and greater hypoactivation in the right hippocampus and right superior temporal gyrus. ALE meta-analyses of nonsocial tasks showed that children with ASD versus adults had significantly greater hyperactivation in the right insula and left cingulate gyrus, and hypoactivation in the right middle frontal gyrus. CONCLUSION: Our data suggest that the neural alterations associated with ASD are not static, occurring only in early childhood. Instead, children with ASD have altered neural activity compared to adults during both social and nonsocial tasks, especially in fronto-temporal structures. Longitudinal neuroimaging studies are required to examine these changes prospectively, as potential targets for brain-based treatments for ASD.

Lighting a path: genetic studies pinpoint neurodevelopmental mechanisms in autism and related disorders.

In this review, we outline critical molecular processes that have been implicated by discovery of genetic mutations in autism. These mechanisms need to be mapped onto the neurodevelopment step(s) gone awry that may be associated with cause in autism. Molecular mechanisms include: (i) regulation of gene expression; (ii) pre-mRNA splicing; (iii) protein localization, translation, and turnover; (iv) synaptic transmission; (v) cell signaling; (vi) the functions of cytoskeletal and scaffolding proteins; and (vii) the function of neuronal cell adhesion molecules. While the molecular mechanisms appear broad, they may converge on only one of a few steps during neurodevelopment that perturbs the structure, function, and/or plasticity of neuronal circuitry. While there are many genetic mutations involved, novel treatments may need to target only one of few developmental mechanisms.

A novel familial 11p15.4 microduplication associated with intellectual disability, dysmorphic features, and obesity with involvement of the ZNF214 gene.

We evaluated a patient with mild intellectual disability, obesity, overgrowth, and dysmorphic features. Array comparative genomic hybridization (aCGH) analysis showed a single copy number increase of a BAC clone in the 11p15.4 region. Oligonucleotide aCGH refined the duplication to approximately 2.29 megabases (Mb) in size. Testing the parents revealed that the father, who had learning disabilities and overgrowth, also had the 11p15.4 duplication, and the mother had a normal microarray. In addition, the patient's brother and grandmother all share clinical features with the proband and tested positive for the duplication. The duplicated region (Chr11:6,934,067-9,220,605) encompasses 29 genes, including the ZNF214 gene, which has been postulated to play a role in Beckwith-Wiedemann syndrome [Alders et al., 2000]. This three-generation pedigree outlines features of a novel microduplication syndrome.

Translational neuroscience in pediatric bipolar disorder.

While controversial and often confounded with other forms of psychopathology, recent studies have shown that bipolar disorder (BD) is on the rise in children and adolescents. Research has made important strides in advancing our understanding of the phenomenology, neural underpinnings and treatment outcomes for BD youths. However, there is an increasing need to unite these domains to identify potential neural effects and predictors of treatment outcome. Pavuluri et al. have conducted such a study, evaluating the neural effects of divalproex or risperidone for pediatric BD. The future is likely to bring more of such studies, potentially resulting in a biomarker augmented approach to the diagnosis and treatment of pediatric BD.