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 and brain development.

Genetic studies are refining our understanding of neurodevelopmental mechanisms in autism. Some autism-related mutations appear to disrupt genes regulated by neuronal activity, which are especially important in development of the postnatal nervous system. Gene replacement studies in mice indicate that the developmental window to ameliorate symptoms may be wider than previously anticipated.

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.

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.

GPT2 mutations in autosomal recessive developmental disability: extending the clinical phenotype and population prevalence estimates.

The glutamate pyruvate transaminase 2 (GPT2) gene produces a nuclear-encoded mitochondrial enzyme that catalyzes the reversible transfer of an amino group from glutamate to pyruvate, generating alanine and alpha-ketoglutarate. Recessive mutations in GPT2 have been recently identified in a new syndrome involving intellectual and developmental disability (IDD), postnatal microcephaly, and spastic paraplegia. We have identified additional families with recessive GPT2 mutations and expanded the phenotype to include small stature. GPT2 loss-of-function mutations were identified in four families, nine patients total, including: a homozygous mutation in one child [c.775T>C (p.C259R)]; compound heterozygous mutations in two siblings [c.812A>C (p.N271T)/c.1432_1433delGT (p.V478Rfs*73)]; a novel homozygous, putative splicing mutation [c.1035C>T (p.G345=)]; and finally, a recurrent mutation, previously identified in a distinct family [c.1210C>T (p.R404*)]. All patients were diagnosed with IDD. A majority of patients had remarkably small stature throughout development, many < 1st percentile for height and weight. Given the potential biological function of GPT2 in cellular growth, this phenotype is strongly suggestive of a newly identified clinical susceptibility. Further, homozygous GPT2 mutations manifested in at least 2 of 176 families with IDD (approximately 1.1%) in a Pakistani cohort, thereby representing a relatively common cause of recessive IDD in this population, with recurrence of the p.R404* mutation in this population. Based on variants in the ExAC database, we estimated that approximately 1 in 248 individuals are carriers of moderately or severely deleterious variants in GPT2.

Longitudinal MRI findings in patient with SLC25A12 pathogenic variants inform disease progression and classification.

Aspartate-glutamate carrier 1 (AGC1) is one of two exchangers within the malate-aspartate shuttle. AGC1 is encoded by the SLC25A12 gene. Three patients with pathogenic variants in SLC25A12 have been reported in the literature. These patients were clinically characterized by neurodevelopmental delay, epilepsy, hypotonia, cerebral atrophy, and hypomyelination; however, there has been discussion in the literature as to whether this hypomyelination is primary or secondary to a neuronal defect. Here we report a 12-year-old patient with variants in SLC25A12 and magnetic resonance imaging (MRI) at multiple ages. Novel compound heterozygous, recessive variants in SLC25A12 were identified: c.1295C>T (p.A432V) and c.1447-2_1447-1delAG. Clinical presentation is characterized by severe intellectual disability, nonambulatory, nonverbal status, hypotonia, epilepsy, spastic quadriplegia, and a happy disposition. The serial neuroimaging findings are notable for cerebral atrophy with white matter involvement, namely, early hypomyelination yet subsequent progression of myelination. The longitudinal MRI findings are most consistent with a leukodystrophy of the leuko-axonopathy category, that is, white matter abnormalities that are most suggestive of mechanisms that result from primary neuronal defects. We present here the first case of a patient with compound heterozygous variants in SLC25A12, including brain MRI findings, in the oldest individual reported to date with this neurogenetic condition.

Mutations in mitochondrial enzyme GPT2 cause metabolic dysfunction and neurological disease with developmental and progressive features.

Mutations that cause neurological phenotypes are highly informative with regard to mechanisms governing human brain function and disease. We report autosomal recessive mutations in the enzyme glutamate pyruvate transaminase 2 (GPT2) in large kindreds initially ascertained for intellectual and developmental disability (IDD). GPT2 [also known as alanine transaminase 2 (ALT2)] is one of two related transaminases that catalyze the reversible addition of an amino group from glutamate to pyruvate, yielding alanine and α-ketoglutarate. In addition to IDD, all affected individuals show postnatal microcephaly and ∼80% of those followed over time show progressive motor symptoms, a spastic paraplegia. Homozygous nonsense p.Arg404* and missense p.Pro272Leu mutations are shown biochemically to be loss of function. The GPT2 gene demonstrates increasing expression in brain in the early postnatal period, and GPT2 protein localizes to mitochondria. Akin to the human phenotype, Gpt2-null mice exhibit reduced brain growth. Through metabolomics and direct isotope tracing experiments, we find a number of metabolic abnormalities associated with loss of Gpt2. These include defects in amino acid metabolism such as low alanine levels and elevated essential amino acids. Also, we find defects in anaplerosis, the metabolic process involved in replenishing TCA cycle intermediates. Finally, mutant brains demonstrate misregulated metabolites in pathways implicated in neuroprotective mechanisms previously associated with neurodegenerative disorders. Overall, our data reveal an important role for the GPT2 enzyme in mitochondrial metabolism with relevance to developmental as well as potentially to neurodegenerative mechanisms.

Genetic control of postnatal human brain growth.

PURPOSE OF REVIEW: Studies investigating postnatal brain growth disorders inform the biology underlying the development of human brain circuitry. This research is becoming increasingly important for the diagnosis and treatment of childhood neurodevelopmental disorders, including autism and related disorders. Here, we review recent research on typical and abnormal postnatal brain growth and examine potential biological mechanisms. RECENT FINDINGS: Clinically, brain growth disorders are heralded by diverging head size for a given age and sex, but are more precisely characterized by brain imaging, post-mortem analysis, and animal model studies. Recent neuroimaging and molecular biological studies on postnatal brain growth disorders have broadened our view of both typical and pathological postnatal neurodevelopment. Correlating gene and protein function with brain growth trajectories uncovers postnatal biological mechanisms, including neuronal arborization, synaptogenesis and pruning, and gliogenesis and myelination. Recent investigations of childhood neurodevelopmental and neurodegenerative disorders highlight the underlying genetic programming and experience-dependent remodeling of neural circuitry. SUMMARY: To understand typical and abnormal postnatal brain development, clinicians and researchers should characterize brain growth trajectories in the context of neurogenetic syndromes. Understanding mechanisms and trajectories of postnatal brain growth will aid in differentiating, diagnosing, and potentially treating neurodevelopmental disorders.

Intellectual disability is associated with increased runs of homozygosity in simplex autism.

Intellectual disability (ID), often attributed to autosomal-recessive mutations, occurs in 40% of autism spectrum disorders (ASDs). For this reason, we conducted a genome-wide analysis of runs of homozygosity (ROH) in simplex ASD-affected families consisting of a proband diagnosed with ASD and at least one unaffected sibling. In these families, probands with an IQ ≤ 70 show more ROH than their unaffected siblings, whereas probands with an IQ > 70 do not show this excess. Although ASD is far more common in males than in females, the proportion of females increases with decreasing IQ. Our data do support an association between ROH burden and autism diagnosis in girls; however, we are not able to show that this effect is independent of low IQ. We have also discovered several autism candidate genes on the basis of finding (1) a single gene that is within an ROH interval and that is recurrent in autism or (2) a gene that is within an autism ROH block and that harbors a homozygous, rare deleterious variant upon analysis of exome-sequencing data. In summary, our data suggest a distinct genetic architecture for participants with autism and co-occurring intellectual disability and that this architecture could involve a role for recessively inherited loci for this autism subgroup.

Graph Metrics of Structural Brain Networks in Individuals with Schizophrenia and Healthy Controls: Group Differences, Relationships with Intelligence, and Genetics.

OBJECTIVES: One of the most prominent features of schizophrenia is relatively lower general cognitive ability (GCA). An emerging approach to understanding the roots of variation in GCA relies on network properties of the brain. In this multi-center study, we determined global characteristics of brain networks using graph theory and related these to GCA in healthy controls and individuals with schizophrenia. METHODS: Participants (N=116 controls, 80 patients with schizophrenia) were recruited from four sites. GCA was represented by the first principal component of a large battery of neurocognitive tests. Graph metrics were derived from diffusion-weighted imaging. RESULTS: The global metrics of longer characteristic path length and reduced overall connectivity predicted lower GCA across groups, and group differences were noted for both variables. Measures of clustering, efficiency, and modularity did not differ across groups or predict GCA. Follow-up analyses investigated three topological types of connectivity--connections among high degree "rich club" nodes, "feeder" connections to these rich club nodes, and "local" connections not involving the rich club. Rich club and local connectivity predicted performance across groups. In a subsample (N=101 controls, 56 patients), a genetic measure reflecting mutation load, based on rare copy number deletions, was associated with longer characteristic path length. CONCLUSIONS: Results highlight the importance of characteristic path lengths and rich club connectivity for GCA and provide no evidence for group differences in the relationships between graph metrics and GCA.

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.

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.

DELISHUS: an efficient and exact algorithm for genome-wide detection of deletion polymorphism in autism.

MOTIVATION: The understanding of the genetic determinants of complex disease is undergoing a paradigm shift. Genetic heterogeneity of rare mutations with deleterious effects is more commonly being viewed as a major component of disease. Autism is an excellent example where research is active in identifying matches between the phenotypic and genomic heterogeneities. A considerable portion of autism appears to be correlated with copy number variation, which is not directly probed by single nucleotide polymorphism (SNP) array or sequencing technologies. Identifying the genetic heterogeneity of small deletions remains a major unresolved computational problem partly due to the inability of algorithms to detect them. RESULTS: In this article, we present an algorithmic framework, which we term DELISHUS, that implements three exact algorithms for inferring regions of hemizygosity containing genomic deletions of all sizes and frequencies in SNP genotype data. We implement an efficient backtracking algorithm-that processes a 1 billion entry genome-wide association study SNP matrix in a few minutes-to compute all inherited deletions in a dataset. We further extend our model to give an efficient algorithm for detecting de novo deletions. Finally, given a set of called deletions, we also give a polynomial time algorithm for computing the critical regions of recurrent deletions. DELISHUS achieves significantly lower false-positive rates and higher power than previously published algorithms partly because it considers all individuals in the sample simultaneously. DELISHUS may be applied to SNP array or sequencing data to identify the deletion spectrum for family-based association studies. AVAILABILITY: DELISHUS is available at http://www.brown.edu/Research/Istrail_Lab/.

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.

Further clinical and molecular delineation of the 15q24 microdeletion syndrome.

BACKGROUND: Chromosome 15q24 microdeletion syndrome is a rare genomic disorder characterised by intellectual disability, growth retardation, unusual facial morphology and other anomalies. To date, 20 patients have been reported; 18 have had detailed breakpoint analysis. AIM: To further delineate the features of the 15q24 microdeletion syndrome, the clinical and molecular characterisation of fifteen patients with deletions in the 15q24 region was performed, nearly doubling the number of reported patients. METHODS: Breakpoints were characterised using a custom, high-density array comparative hybridisation platform, and detailed phenotype information was collected for each patient. RESULTS: Nine distinct deletions with different breakpoints ranging in size from 266 kb to 3.75 Mb were identified. The majority of breakpoints lie within segmental duplication (SD) blocks. Low sequence identity and large intervals of unique sequence between SD blocks likely contribute to the rarity of 15q24 deletions, which occur 8-10 times less frequently than 1q21 or 15q13 microdeletions in our series. Two small, atypical deletions were identified within the region that help delineate the critical region for the core phenotype in the 15q24 microdeletion syndrome. CONCLUSION: The molecular characterisation of these patients suggests that the core cognitive features of the 15q24 microdeletion syndrome, including developmental delays and severe speech problems, are largely due to deletion of genes in a 1.1-Mb critical region. However, genes just distal to the critical region also play an important role in cognition and in the development of characteristic facial features associated with 15q24 deletions. Clearly, deletions in the 15q24 region are variable in size and extent. Knowledge of the breakpoints and size of deletion combined with the natural history and medical problems of our patients provide insights that will inform management guidelines. Based on common phenotypic features, all patients with 15q24 microdeletions should receive a thorough neurodevelopmental evaluation, physical, occupational and speech therapies, and regular audiologic and ophthalmologic screening.

Genome-wide transcriptome analysis in murine neural retina using high-throughput RNA sequencing.

Genome-wide characterization of the retinal transcriptome is central to understanding development, physiology and disorders of the visual system. Massively parallel, short-read sequencing of mRNA libraries was used to generate an extensive map of the transcriptome of the adult, murine neural retina. RNA-seq data strongly corroborates prior transcriptome studies by microarray and SAGE. However, several novel features of the retinal transcriptome were discovered. For example, retinal disease genes were discovered to be among the most highly expressed in the transcriptome. We also demonstrate other interesting features of the retinal transcriptome, for example, that the retina appears to employ a very specific and restricted set of synaptic vesicle genes, and also that there is persistence of expression of a majority of "neurodevelopmental" genes into adulthood. Retina transcriptome studies utilizing novel sequencing methods have been highly informative and these data may also serve as a resource for the community of researchers.

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.

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.