Impact of KDM6B mosaic brain knockout on synaptic function and behavior.

Autism spectrum disorders (ASD) are complex neurodevelopmental conditions characterized by impairments in social communication, repetitive behaviors, and restricted interests. Epigenetic modifications serve as critical regulators of gene expression playing a crucial role in controlling brain function and behavior. Lysine (K)-specific demethylase 6B (KDM6B), a stress-inducible H3K27me3 demethylase, has emerged as one of the highest ASD risk genes, but the precise effects of KDM6B mutations on neuronal activity and behavioral function remain elusive. Here we show the impact of KDM6B mosaic brain knockout on the manifestation of different autistic-like phenotypes including repetitive behaviors, social interaction, and significant cognitive deficits. Moreover, KDM6B mosaic knockout display abnormalities in hippocampal excitatory synaptic transmission decreasing NMDA receptor mediated synaptic transmission and plasticity. Understanding the intricate interplay between epigenetic modifications and neuronal function may provide novel insights into the pathophysiology of ASD and potentially inform the development of targeted therapeutic interventions.

Endocytosis of Synaptic Vesicle in Motor Nerve Endings of FUS Transgenic Mice with a Model of Amyotrophic Lateral Sclerosis.

In experiments on the motor nerve endings of the diaphragm of transgenic FUS mice with a model of amyotrophic lateral sclerosis at the pre-symptomatic stage of the disease, the processes of transmitter release and endocytosis of synaptic vesicles were studied. In FUS mice, the intensity of transmitter release during high-frequency stimulation of the motor nerve (50 imp/sec) was lowered. At the same duration of stimulation, the loading of fluorescent dye FM1-43 was lower in FUS mice. However, at the time of stimulation, during which an equal number of quanta are released in wild-type and FUS mice, no differences in the intensity of dye loading were found. Thus, endocytosis is not the key factor in the mechanism of synaptic dysfunction in FUS mice at the pre-symptomatic stage.

The ALS-associated KIF5A P986L variant is not pathogenic for Drosophila motoneurons.

Amyotrophic lateral sclerosis (ALS) is a devastating paralytic disorder caused by the death of motoneurons. Several mutations in the KIF5A gene have been identified in patients with ALS. Some mutations affect the splicing sites of exon 27 leading to its deletion (Δ27 mutation). KIF5A Δ27 is aggregation-prone and pathogenic for motoneurons due to a toxic gain of function. Another mutation found to be enriched in ALS patients is a proline/leucine substitution at position 986 (P986L mutation). Bioinformatic analyses strongly suggest that this variant is benign. Our study aims to conduct functional studies in Drosophila to classify the KIF5A P986L variant. When expressed in motoneurons, KIF5A P986L does not modify the morphology of larval NMJ or the synaptic transmission. In addition, KIF5A P986L is uniformly distributed in axons and does not disturb mitochondria distribution. Locomotion at larval and adult stages is not affected by KIF5A P986L. Finally, both KIF5A WT and P986L expression in adult motoneurons extend median lifespan compared to control flies. Altogether, our data show that the KIF5A P986L variant is not pathogenic for motoneurons and may represent a hypomorphic allele, although it is not causative for ALS.

Mutations of Single Residues in the Complexin N-terminus Exhibit Distinct Phenotypes in Synaptic Vesicle Fusion.

The release of neurotransmitters (NTs) at central synapses is dependent on a cascade of protein interactions, specific to the presynaptic compartment. Among those dedicated molecules, the cytosolic complexins play an incompletely defined role as synaptic transmission regulators. Complexins are multidomain proteins that bind soluble N-ethylmaleimide sensitive factor attachment protein receptor complexes, conferring both inhibitory and stimulatory functions. Using systematic mutagenesis and comparing reconstituted in vitro membrane fusion assays with electrophysiology in cultured neurons from mice of either sex, we deciphered the function of the N-terminus of complexin (Cpx) II. The N-terminus (amino acid 1-27) starts with a region enriched in hydrophobic amino acids (1-12), which binds lipids. Mutants maintaining this hydrophobic character retained the stimulatory function of Cpx, whereas exchanges introducing charged residues perturbed both spontaneous and evoked exocytosis. Mutants in the more distal region of the N-terminal domain (amino acid 11-18) showed a spectrum of effects. On the one hand, mutation of residue A12 increased spontaneous release without affecting evoked release. On the other hand, replacing D15 with amino acids of different shapes or hydrophobic properties (but not charge) not only increased spontaneous release but also impaired evoked release. Most surprising, this substitution reduced the size of the readily releasable pool, a novel function for Cpx at mammalian synapses. Thus, the exact amino acid composition of the Cpx N-terminus fine-tunes the degree of spontaneous and evoked NT release.

Congenital myasthenic syndromes: increasingly complex.

PURPOSE OF REVIEW: Congenital myasthenia syndromes (CMS) are treatable, inherited disorders affecting neuromuscular transmission. We highlight that the involvement of an increasing number of proteins is making the understanding of the disease mechanisms and potential treatments progressively more complex. RECENT FINDINGS: Although early studies identified mutations of proteins directly involved in synaptic transmission at the neuromuscular junction, recently, next-generation sequencing has facilitated the identification of many novel mutations in genes that encode proteins that have a far wider expression profile, some even ubiquitously expressed, but whose defective function leads to impaired neuromuscular transmission. Unsurprisingly, mutations in these genes often causes a wider phenotypic disease spectrum where defective neuromuscular transmission forms only one component. This has implications for the management of CMS patients. SUMMARY: Given the widening nonneuromuscular junction phenotypes in the newly identified forms of CMS, new therapies need to include disease-modifying approaches that address not only neuromuscular weakness but also the multisystem involvement. Whilst the current treatments for CMS are highly effective for many subtypes there remains, in a proportion of CMS patients, an unmet need for more efficacious therapies.

miR-7 controls glutamatergic transmission and neuronal connectivity in a Cdr1as-dependent manner.

The circular RNA (circRNA) Cdr1as is conserved across mammals and highly expressed in neurons, where it directly interacts with microRNA miR-7. However, the biological function of this interaction is unknown. Here, using primary cortical murine neurons, we demonstrate that stimulating neurons by sustained depolarization rapidly induces two-fold transcriptional upregulation of Cdr1as and strong post-transcriptional stabilization of miR-7. Cdr1as loss causes doubling of glutamate release from stimulated synapses and increased frequency and duration of local neuronal bursts. Moreover, the periodicity of neuronal networks increases, and synchronicity is impaired. Strikingly, these effects are reverted by sustained expression of miR-7, which also clears Cdr1as molecules from neuronal projections. Consistently, without Cdr1as, transcriptomic changes caused by miR-7 overexpression are stronger (including miR-7-targets downregulation) and enriched in secretion/synaptic plasticity pathways. Altogether, our results suggest that in cortical neurons Cdr1as buffers miR-7 activity to control glutamatergic excitatory transmission and neuronal connectivity important for long-lasting synaptic adaptations.

Expression profiling of cochlear genes uncovers sex-based cellular function in mouse cochleae.

Sex is a pivotal biological factor that significantly impacts tissue homeostasis and disease susceptibility. In the auditory system, sex differences have been observed in cochlear physiology and responses to pathological conditions. However, the underlying molecular mechanisms responsible for these differences remain elusive. The current research explores the differences in gene expression profiles in the cochlea between male and female mice, aiming to understand the functional implication of sex-biased gene expression in each sex. Using RNA-sequencing analysis on cochlear tissues obtained from male and female mice, we identified a significant number of genes exhibiting sex-biased expression differences. While some of these differentially expressed genes are located on sex chromosomes, most are found on autosomal chromosomes. Further bioinformatic analysis revealed that these genes are involved in several key cellular functions. In males, these genes are notably linked to oxidative phosphorylation and RNA synthesis and processing, suggesting their involvement in mitochondrial energy production and regulatory control of gene expression. In contrast, sex-biased genes are associated with mechano-transduction and synaptic transmission within female cochleae. Collectively, our study provides valuable insights into the molecular differences between the sexes and emphasizes the need for future research to uncover their functional implications and relevance to auditory health and disease development.

No association between peripheral serotonin-gene-related DNA methylation and brain serotonin neurotransmission in the healthy and depressed state.

BACKGROUND: Methylation of serotonin-related genes has been proposed as a plausible gene-by-environment link which may mediate environmental stress, depressive and anxiety symptoms. DNA methylation is often measured in blood cells, but little is known about the association between this peripheral epigenetic modification and brain serotonergic architecture. Here, we evaluated the association between whole-blood-derived methylation of four CpG sites in the serotonin transporter (SLC6A4) and six CpG sites of the tryptophan hydroxylase 2 (TPH2) gene and in-vivo brain levels of serotonin transporter (5-HTT) and serotonin 4 receptor (5-HT4) in a cohort of healthy individuals (N = 254) and, for 5-HT4, in a cohort of unmedicated patients with depression (N = 90). To do so, we quantified SLC6A4/TPH2 methylation using bisulfite pyrosequencing and estimated brain 5-HT4 and 5-HTT levels using positron emission tomography. In addition, we explored the association between SLC6A4 and TPH2 methylation and measures of early life and recent stress, depressive and anxiety symptoms on 297 healthy individuals. RESULTS: We found no statistically significant association between peripheral DNA methylation and brain markers of serotonergic neurotransmission in patients with depression or in healthy individuals. In addition, although SLC6A4 CpG2 (chr17:30,236,083) methylation was marginally associated with the parental bonding inventory overprotection score in the healthy cohort, statistical significance did not remain after accounting for blood cell heterogeneity. CONCLUSIONS: We suggest that findings on peripheral DNA methylation in the context of brain serotonin-related features should be interpreted with caution. More studies are needed to rule out a role of SLC6A4 and TPH2 methylation as biomarkers for environmental stress, depressive or anxiety symptoms.

Gene replacement therapies for inherited disorders of neurotransmission: Current progress in succinic semialdehyde dehydrogenase deficiency.

Neurodevelopment is a highly organized and complex process involving lasting and often irreversible changes in the central nervous system. Inherited disorders of neurotransmission (IDNT) are a group of genetic disorders where neurotransmission is primarily affected, resulting in abnormal brain development from early life, manifest as neurodevelopmental disorders and other chronic conditions. In principle, IDNT (particularly those of monogenic causes) are amenable to gene replacement therapy via precise genetic correction. However, practical challenges for gene replacement therapy remain major hurdles for its translation from bench to bedside. We discuss key considerations for the development of gene replacement therapies for IDNT. As an example, we describe our ongoing work on gene replacement therapy for succinic semialdehyde dehydrogenase deficiency, a GABA catabolic disorder.

Reduced synaptic depression in human neurons carrying homozygous disease-causing STXBP1 variant L446F.

MUNC18-1 is an essential protein of the regulated secretion machinery. De novo, heterozygous mutations in STXBP1, the human gene encoding this protein, lead to a severe neurodevelopmental disorder. Here, we describe the electrophysiological characteristics of a unique case of STXBP1-related disorder caused by a homozygous mutation (L446F). We engineered this mutation in induced pluripotent stem cells from a healthy donor (STXBP1LF/LF) to establish isogenic cell models. We performed morphological and electrophysiological analyses on single neurons grown on glial micro-islands. Human STXBP1LF/LF neurons displayed normal morphology and normal basal synaptic transmission but increased paired-pulse ratios and charge released, and reduced synaptic depression compared to control neurons. Immunostainings revealed normal expression levels but impaired recognition by a mutation-specific MUNC18-1 antibody. The electrophysiological gain-of-function phenotype is in line with earlier overexpression studies in Stxbp1 null mouse neurons, with some potentially human-specific features. Therefore, the present study highlights important differences between mouse and human neurons critical for the translatability of pre-clinical studies.

Sorbs2 regulates seizure activity by influencing AMPAR-mediated excitatory synaptic transmission in temporal lobe epilepsy.

Temporal lobe epilepsy (TLE), the most common type of drug-resistant epilepsy, severely affects quality of life. However, the underlying mechanism of TLE remains unclear and deserves further exploration. Sorbs2, a key synaptic regulatory protein, plays an important role in the regulation of synaptic transmission in the mammalian brain. In this study, we aimed to investigate the expression pattern of Sorbs2 in a kainic acid (KA)-induced TLE mouse model and in patients with TLE to further determine whether Sorbs2 is involved in seizure activity and to explore the potential mechanism by which Sorbs2 affects seizures in this TLE mouse model. First, we found that the expression of Sorbs2 was obviously increased in the hippocampus and cortex of a TLE mouse model and in the temporal cortex of TLE patients, indicating an abnormal expression pattern of Sorbs2 in TLE. Importantly, subsequent behavioral analyses and local field potential (LFP) analyses of a TLE mouse model demonstrated that the downregulation of hippocampal Sorbs2 could prolong the latency to spontaneous recurrent seizures (SRSs) and protect against SRSs. We also found that the knockdown of Sorbs2 in the hippocampus could decrease excitatory synaptic transmission in pyramidal neurons (PNs) in the hippocampal CA1 region and reduce the expression levels of the AMPAR subunits GluA1 and GluA2. Thus, we speculated that Sorbs2 may promote epileptogenesis and the development of TLE by affecting AMPAR-mediated excitatory synaptic transmission in PNs in the CA1 region. Therefore, reducing the expression of hippocampal Sorbs2 could restrain epileptogenesis and the development of TLE.

Impaired synaptic function and hyperexcitability of the pyramidal neurons in the prefrontal cortex of autism-associated Shank3 mutant dogs.

BACKGROUND: SHANK3 gene is a highly replicated causative gene for autism spectrum disorder and has been well characterized in multiple Shank3 mutant rodent models. When compared to rodents, domestic dogs are excellent animal models in which to study social cognition as they closely interact with humans and exhibit similar social behaviors. Using CRISPR/Cas9 editing, we recently generated a dog model carrying Shank3 mutations, which displayed a spectrum of autism-like behaviors, such as social impairment and heightened anxiety. However, the neural mechanism underlying these abnormal behaviors remains to be identified. METHODS: We used Shank3 mutant dog models to examine possible relationships between Shank3 mutations and neuronal dysfunction. We studied electrophysiological properties and the synaptic transmission of pyramidal neurons from acute brain slices of the prefrontal cortex (PFC). We also examined dendrite elaboration and dendritic spine morphology in the PFC using biocytin staining and Golgi staining. We analyzed the postsynaptic density using electron microscopy. RESULTS: We established a protocol for the electrophysiological recording of canine brain slices and revealed that excitatory synaptic transmission onto PFC layer 2/3 pyramidal neurons in Shank3 heterozygote dogs was impaired, and this was accompanied by reduced dendrite complexity and spine density when compared to wild-type dogs. Postsynaptic density structures were also impaired in Shank3 mutants; however, pyramidal neurons exhibited hyperexcitability. LIMITATIONS: Causal links between impaired PFC pyramidal neuron function and behavioral alterations remain unclear. Further experiments such as manipulating PFC neuronal activity or restoring synaptic transmission in Shank3 mutant dogs are required to assess PFC roles in altered social behaviors. CONCLUSIONS: Our study demonstrated the feasibility of using canine brain slices as a model system to study neuronal circuitry and disease. Shank3 haploinsufficiency causes morphological and functional abnormalities in PFC pyramidal neurons, supporting the notion that Shank3 mutant dogs are new and valid animal models for autism research.

Loss of SLC30A10 manganese transporter alters expression of neurotransmission genes and activates hypoxia-inducible factor signaling in mice.

The essential metal manganese (Mn) induces neuromotor disease at elevated levels. The manganese efflux transporter SLC30A10 regulates brain Mn levels. Homozygous loss-of-function mutations in SLC30A10 induce hereditary Mn neurotoxicity in humans. Our prior characterization of Slc30a10 knockout mice recapitulated the high brain Mn levels and neuromotor deficits reported in humans. But, mechanisms of Mn-induced motor deficits due to SLC30A10 mutations or elevated Mn exposure are unclear. To gain insights into this issue, we characterized changes in gene expression in the basal ganglia, the main brain region targeted by Mn, of Slc30a10 knockout mice using unbiased transcriptomics. Compared with littermates, >1000 genes were upregulated or downregulated in the basal ganglia sub-regions (i.e. caudate putamen, globus pallidus, and substantia nigra) of the knockouts. Pathway analyses revealed notable changes in genes regulating synaptic transmission and neurotransmitter function in the knockouts that may contribute to the motor phenotype. Expression changes in the knockouts were essentially normalized by a reduced Mn chow, establishing that changes were Mn dependent. Upstream regulator analyses identified hypoxia-inducible factor (HIF) signaling, which we recently characterized to be a primary cellular response to elevated Mn, as a critical mediator of the transcriptomic changes in the basal ganglia of the knockout mice. HIF activation was also evident in the liver of the knockout mice. These results: (i) enhance understanding of the pathobiology of Mn-induced motor disease; (ii) identify specific target genes/pathways for future mechanistic analyses; and (iii) independently corroborate the importance of the HIF pathway in Mn homeostasis and toxicity.

Role of the Glycine Receptor ß Subunit in Synaptic Localization and Pathogenicity in Severe Startle Disease.

Startle disease is due to the disruption of recurrent inhibition in the spinal cord. Most common causes are genetic variants in genes (GLRA1, GLRB) encoding inhibitory glycine receptor (GlyR) subunits. The adult GlyR is a heteropentameric complex composed of α1 and ß subunits that localizes at postsynaptic sites and replaces embryonically expressed GlyRα2 homomers. The human GlyR variants of GLRA1 and GLRB, dominant and recessive, have been intensively studied in vitro. However, the role of unaffected GlyRß, essential for synaptic GlyR localization, in the presence of mutated GlyRα1 in vivo is not fully understood. Here, we used knock-in mice expressing endogenous mEos4b-tagged GlyRß that were crossed with mouse Glra1 startle disease mutants. We explored the role of GlyRß under disease conditions in mice carrying a missense mutation (shaky) or resulting from the loss of GlyRα1 (oscillator). Interestingly, synaptic targeting of GlyRß was largely unaffected in both mouse mutants. While synaptic morphology appears unaltered in shaky animals, synapses were notably smaller in homozygous oscillator animals. Hence, GlyRß enables transport of functionally impaired GlyRα1 missense variants to synaptic sites in shaky animals, which has an impact on the efficacy of possible compensatory mechanisms. The observed enhanced GlyRα2 expression in oscillator animals points to a compensation by other GlyRα subunits. However, trafficking of GlyRα2ß complexes to synaptic sites remains functionally insufficient, and homozygous oscillator mice still die at 3 weeks after birth. Thus, both functional and structural deficits can affect glycinergic neurotransmission in severe startle disease, eliciting different compensatory mechanisms in vivo.

Lethal variant in the C2A domain may cause severe SYT1-associated neurodevelopmental disorder in the newborns.

Synaptotagmin-1 (SYT1) plays a pivotal role in regulating presynaptic processes, including neurotransmitter release. SYT1 variants perturb synaptic vesicle endocytosis and exocytosis, resulting in a series of neurodevelopmental disorders defined as Baker-Gordon syndrome. Herein, we report the case of a newborn with dysmorphic facial appearance, severe hypotonia, poor feeding, gastroesophageal reflux, and an inability to eat and breathe, diagnosed with Baker-Gordon syndrome. A retrospective search was performed on a newborn with Baker-Gordon syndrome. Medical charts were reviewed, with focus on the clinical presentation, diagnostic process, and treatment outcomes. Whole-genome high-throughput DNA sequencing was performed to identify genetic variants. Whole-exome sequencing identified the likely pathogenic variant as SYT1 C.551 T > C(p.V184A). Sanger sequencing results indicated that this variant was a de novo mutation in a conservative site located in the C2A domain of the protein. The patient died at 57 days old because of severe feeding and breathing problems. Our findings of a novel lethal variant in the C2A domain of SYT1 in the youngest patient diagnosed infantile Baker-Gordon syndrome who presented with the most severe hypotonia reported to date expands the spectrum of SYT1- associated neurodevelopmental disorders.

Microcircuit failure in STXBP1 encephalopathy leads to hyperexcitability.

De novo mutations in STXBP1 are among the most prevalent causes of neurodevelopmental disorders and lead to haploinsufficiency, cortical hyperexcitability, epilepsy, and other symptoms in people with mutations. Given that Munc18-1, the protein encoded by STXBP1, is essential for excitatory and inhibitory synaptic transmission, it is currently not understood why mutations cause hyperexcitability. We find that overall inhibition in canonical feedforward microcircuits is defective in a P15-22 mouse model for Stxbp1 haploinsufficiency. Unexpectedly, we find that inhibitory synapses formed by parvalbumin-positive interneurons were largely unaffected. Instead, excitatory synapses fail to recruit inhibitory interneurons. Modeling confirms that defects in the recruitment of inhibitory neurons cause hyperexcitation. CX516, an ampakine that enhances excitatory synapses, restores interneuron recruitment and prevents hyperexcitability. These findings establish deficits in excitatory synapses in microcircuits as a key underlying mechanism for cortical hyperexcitability in a mouse model of Stxbp1 disorder and identify compounds enhancing excitation as a direction for therapy.

Removal of a partial genomic duplication restores synaptic transmission and behavior in the MyosinVA mutant mouse Flailer.

BACKGROUND: Copy number variations, and particularly duplications of genomic regions, have been strongly associated with various neurodegenerative conditions including autism spectrum disorder (ASD). These genetic variations have been found to have a significant impact on brain development and function, which can lead to the emergence of neurological and behavioral symptoms. Developing strategies to target these genomic duplications has been challenging, as the presence of endogenous copies of the duplicate genes often complicates the editing strategies. RESULTS: Using the ASD and anxiety mouse model Flailer, which contains a partial genomic duplication working as a dominant negative for MyoVa, we demonstrate the use of DN-CRISPRs to remove a 700 bp genomic region in vitro and in vivo. Importantly, DN-CRISPRs have not been used to remove genomic regions using sgRNA with an offset greater than 300 bp. We found that editing the flailer gene in primary cortical neurons reverts synaptic transport and transmission defects. Moreover, long-term depression (LTD), disrupted in Flailer animals, is recovered after gene editing. Delivery of DN-CRISPRs in vivo shows that local delivery to the ventral hippocampus can rescue some of the mutant behaviors, while intracerebroventricular delivery, completely recovers the Flailer animal phenotype associated to anxiety and ASD. CONCLUSIONS: Our results demonstrate the potential of DN-CRISPR to efficiently remove larger genomic duplications, working as a new gene therapy approach for treating neurodegenerative diseases.

Regulation of Syntaxin3B-Mediated Membrane Fusion by T14, Munc18, and Complexin.

Retinal neurons that form ribbon-style synapses operate over a wide dynamic range, continuously relaying visual information to their downstream targets. The remarkable signaling abilities of these neurons are supported by specialized presynaptic machinery, one component of which is syntaxin3B. Syntaxin3B is an essential t-SNARE protein of photoreceptors and bipolar cells that is required for neurotransmitter release. It has a light-regulated phosphorylation site in its N-terminal domain at T14 that has been proposed to modulate membrane fusion. However, a direct test of the latter has been lacking. Using a well-controlled in vitro fusion assay, we found that a phosphomimetic T14 syntaxin3B mutation leads to a small but significant enhancement of SNARE-mediated membrane fusion following the formation of the t-SNARE complex. While the addition of Munc18a had only a minimal effect on membrane fusion mediated by SNARE complexes containing wild-type syntaxin3B, a more significant enhancement was observed in the presence of Munc18a when the SNARE complexes contained a syntaxin3B T14 phosphomimetic mutant. Finally, we showed that the retinal-specific complexins (Cpx III and Cpx IV) inhibited membrane fusion mediated by syntaxin3B-containing SNARE complexes in a dose-dependent manner. Collectively, our results establish that membrane fusion mediated by syntaxin3B-containing SNARE complexes is regulated by the T14 residue of syntaxin3B, Munc18a, and Cpxs III and IV.

In Cerebellar Atrophy of 12-Month-Old ATM-Null Mice, Transcriptome Upregulations Concern Most Neurotransmission and Neuropeptide Pathways, While Downregulations Affect Prominently Itpr1, Usp2 and Non-Coding RNA.

The autosomal recessive disorder Ataxia-Telangiectasia is caused by a dysfunction of the stress response protein, ATM. In the nucleus of proliferating cells, ATM senses DNA double-strand breaks and coordinates their repair. This role explains T-cell dysfunction and tumour risk. However, it remains unclear whether this function is relevant for postmitotic neurons and underlies cerebellar atrophy, since ATM is cytoplasmic in postmitotic neurons. Here, we used ATM-null mice that survived early immune deficits via bone-marrow transplantation, and that reached initial neurodegeneration stages at 12 months of age. Global cerebellar transcriptomics demonstrated that ATM depletion triggered upregulations in most neurotransmission and neuropeptide systems. Downregulated transcripts were found for the ATM interactome component Usp2, many non-coding RNAs, ataxia genes Itpr1, Grid2, immediate early genes and immunity factors. Allelic splice changes affected prominently the neuropeptide machinery, e.g., Oprm1. Validation experiments with stressors were performed in human neuroblastoma cells, where ATM was localised only to cytoplasm, similar to the brain. Effect confirmation in SH-SY5Y cells occurred after ATM depletion and osmotic stress better than nutrient/oxidative stress, but not after ATM kinase inhibition or DNA stressor bleomycin. Overall, we provide pioneer observations from a faithful A-T mouse model, which suggest general changes in synaptic and dense-core vesicle stress adaptation.

Expression and function of Caenorhabditis elegans UNCP-18, a paralog of the SM protein UNC-18.

Sec1/Munc18 (SM) proteins are important regulators of SNARE complex assembly during exocytosis throughout all major animal tissue types. However, expression of a founding member of the SM family, UNC-18, is mostly restricted to the nervous system of the nematode Caenorhabditis elegans, where it is important for synaptic transmission. Moreover, unc-18 null mutants do not display the lethality phenotype associated with (a) loss of all Drosophila and mouse orthologs of unc-18 and (b) with complete elimination of synaptic transmission in C. elegans. We investigated whether a previously uncharacterized unc-18 paralog, which we named uncp-18, may be able to explain the restricted expression and limited phenotypes of unc-18 null mutants. A reporter allele shows ubiquitous expression of uncp-18. Analysis of uncp-18 null mutants, unc-18 and uncp-18 double null mutants, as well as overexpression of uncp-18 in an unc-18 null mutant background, shows that these 2 genes can functionally compensate for one another and are redundantly required for embryonic viability. Our results indicate that the synaptic transmission defects of unc-18 null mutants cannot necessarily be interpreted as constituting a null phenotype for SM protein function at the synapse.