Biology subjects

Rotenberg, A.

Publications and source records attributed to Rotenberg, A..

7 recordsLinked to original sources

bioRxiv2026

Postnatal gene restoration in succinic semialdehyde dehydrogenase deficiency (SSADHD) reveals phenotype reversibility

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare autosomal recessive metabolic disorder due to loss-of-function ALDH5A1 mutations impairing the catabolism of {gamma}-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the brain. In SSADHD, pathologic accumulation of GABA and its metabolic by-product {gamma}-hydroxybutyrate (GHB) corresponds to a clinical syndrome dominated by developmental delay and epilepsy in half of patients with risk of sudden death in adolescence and adulthood. Brain-wide ALDH5A1 gene replacement for SSADHD is unavailable, and whether such treatment will reverse the SSADHD phenotype is unknown. We developed an inducible mouse SSADHD model, Aldh5a1lox-STOP, enabling Cre-dependent Aldh5a1 restoration to evaluate gene therapy feasibility. In the absence of SSADH, Aldh5a1lox-STOP mice exhibit hyperactivity and excessive serum GHB levels, culminating in death by [~]postnatal day 22, recapitulating the severe SSADHD condition. Systemic delivery of a blood-brain barrier (BBB)-penetrating adeno-associated virus (AAV) carrying a Cre gene to Aldh5a1lox-STOP mice leads to brain-wide SSADH restoration, serum GHB level reduction, normalization of hyperactivity, and substantial increase in survival. As a step toward clinical translation, we further assessed an AAV encompassing a functional native promoter (FLnP) of ALDH5A1 tethered to its human coding sequence, namely AAV-FLnP-hALDH5A1. Aldh5a1lox-STOP mice were effectively rescued when treated with AAV-FLnP-hALDH5A1 packaged in the blood-brain barrier (BBB)-penetrating capsid PHP.eB. These findings provide preclinical proof that SSADH gene replacement therapy is feasible and potentially effective.

neuroscience↗

bioRxiv2026

FMR1 reduction alters cellular and circuit properties in human cortex

Transcriptional silencing of FMR1 results in Fragile X syndrome (FXS), the leading inherited cause of intellectual disability (ID) and autism. The Fmr1-/y mouse model has been used to identify FXS disease mechanisms, whereas mechanistic insights from human brain are lacking. By leveraging organotypic human cortical slices and viral tools to reduce FMR1 expression, we create a new model that captures cell type-specific transcriptomic changes similar to FXS patient cortex that are not seen in the Fmr1-/y mouse. Among these are ion channel subunit changes in deep layer pyramidal neurons, which are consistent with a robust hyperexcitability seen by whole-cell patch-clamp recordings, and increased synchronized activity revealed by 2-photon calcium imaging. Together, this work defines the impact of FMR1 reduction in human cortex and provides a new model for testing therapeutic interventions in FXS.

neuroscience↗

bioRxiv2025

Injured SSTR2+ nociceptor axons in neuromas drive chronic spontaneous neuropathic pain

Spontaneous pain is a common but poorly understood consequence of peripheral nerve injury1-3, including injuries that lead to the formation of neuromas4,5. We developed a chronic neuroma model for measuring spontaneous pain-related behaviours in mice, which revealed that limb flicks - emerging predominantly 2 months post-injury - reflect spontaneous paroxysmal pain. Ectopic activity of injured dorsal root ganglia (DRG) sensory neurons whose axonal endings terminate within the neuroma drives this spontaneous pain. In vivo imaging showed that a subset of small-diameter DRG sensory neurons are the source of spontaneous neural signals emanating from the neuroma, and these spontaneously active neurons are distinct from the intact larger diameter sensory neurons that mediate stimulus-evoked mechanical allodynia from spared nerves. Cell-type-specific gain- and loss-of-function studies identified a genetically- and functionally-defined subtype of small-diameter C-fibre nociceptors whose injured axons in neuromas drive spontaneous limb flicks/neuropathic pain. These findings establish the neurobiological basis of spontaneous pain enabling targeted pain management strategies and define a cellular and mechanistic separation between spontaneous and evoked neuropathic pain.

neuroscience↗

bioRxiv2024

Zebrafish models of candidate human epilepsy-associated genes provide evidence of hyperexcitability

Hundreds of novel candidate human epilepsy-associated genes have been identified thanks to advancements in next-generation sequencing and large genome-wide association studies, but establishing genetic etiology requires functional validation. We generated a list of >2200 candidate epilepsy-associated genes, of which 81 were determined suitable for the generation of loss-of-function zebrafish models via CRISPR/Cas9 gene editing. Of those 81 crispants, 48 were successfully established as stable mutant lines and assessed for seizure-like swim patterns in a primary F2 screen. Evidence of seizure-like behavior was present in 5 (arfgef1, kcnd2, kcnv1, ubr5, wnt8b) of the 48 mutant lines assessed. Further characterization of those 5 lines provided evidence for epileptiform activity via electrophysiology in kcnd2 and wnt8b mutants. Additionally, arfgef1 and wnt8b mutants showed a decrease in the number of inhibitory interneurons in the optic tectum of larval animals. Furthermore, RNAseq revealed convergent transcriptional abnormalities between mutant lines, consistent with their developmental defects and hyperexcitable phenotypes. These zebrafish models provide strongest experimental evidence supporting the role of ARFGEF1, KCND2, and WNT8B in human epilepsy and further demonstrate the utility of this model system for evaluating candidate human epilepsy genes. HighlightsO_LIZebrafish models generated by CRISPR/Cas9 gene editing display seizure-like swim patterns in five candidate human epilepsy genes: arfgef1, kcnd2, kcnv1, ubr5, wnt8b. C_LIO_LILocal field potential abnormalities recorded from kcnd2 and wnt8b crispants provide additional evidence of hyperexcitability. C_LIO_LIArfgef1 and wnt8b mutant larvae have fewer inhibitory interneurons than wild type in the optic tectum. C_LIO_LICRISPR-generated mutants of epilepsy genes displayed convergent transcriptional dysregulation, consistent with developmental abnormalities and their hyperexcitability phenotype. C_LI

genetics↗

bioRxiv2023

Sex differences in seizure presentation in Dravet syndrome model mice, Scn1a+/-

Dravet syndrome (DS) is an epileptic encephalopathy mostly due to haploinsufficiency of the SCN1A voltagegated sodium channel subunit. Disease presentation (i.e., severe seizures and early life mortality) is highly recapitulated in mice haploinsufficient in Scn1a (Scn1a+/-). However, phenotypic characterization in Scn1a+/- mice in a sex and temporal manner is limited. Given the reliance of mouse models for studying disease pathophysiology and for the development of novel treatments, we tested whether mortality and seizure morbidity differed among young and adult male and female Scn1a+/- animals in the F1 hybrid C57x129S6 background. We found increased mortality in female Scn1a+/- mice regardless of age compared to their male counterparts (n = 120-125 mice/sex; p < 0.05). Interestingly, long-term video EEG recordings revealed the opposite for morbidity as seizure frequency and severity were escalated in adult male Scn1a+/- animals (n = 21-30 mice/sex; p < 0.05 or p < 0.01). Adult female Scn1a+/- mice, however, are more hyperactive (p < 0.05), which could be related to sleep impairment and contribute to the increased mortality despite decreased seizure morbidity. Overall, the phenotypic presentation of Scn1a+/- mice is sex-dependent and may have translational implications for therapeutic drug discovery and basic biology understanding in DS. Short SummarySex differences in mortality and seizure morbidity are discovered in Scn1a haploinsufficient mice, Scn1a+/-, which faithfully model the epileptic encephalopathy disorder, Dravet syndrome (DS). Female Scn1a+/- mice die more across all ages, whereas adult male Scn1a+/- mice have more seizures that are of greater severity. Hyperactivity, as a proxy for sleep disruption, may contribute to the increased mortality in female Scn1a+/- mice despite decreased seizure incidence and severity. These sex-specific findings may have considerable impact in therapeutic discovery and development for DS and other SCN1A-related disorders.

neuroscience↗

bioRxiv2021

Mosaic and non-mosaic pcdh19 mutation leads to neuronal hyperexcitability in zebrafish

Epilepsy is one of the most common neurological disorders. The X-linked gene PCDH19 is associated with sporadic and familial epilepsy in humans, typically with early-onset clustering seizures and intellectual disability in females but not in so-called carrier males, suggesting that mosaic PCDH19 expression is required to produce epilepsy. To characterize the role of loss of PCDH19 function in epilepsy, we generated zebrafish with truncating pcdh19 variants. Evaluating zebrafish larvae for electrophysiological abnormalities, we observed hyperexcitability phenotypes in both mosaic and non-mosaic pcdh19+/- and pcdh19-/- mutant larvae. Thus, we demonstrate that the key feature of epilepsy--network hyperexcitability--can be modeled effectively in zebrafish, even though overt spontaneous seizure-like swim patterns were not observed. Further, zebrafish with non-mosaic pcdh19 mutation displayed reduced numbers of inhibitory interneurons suggesting a potential cellular basis for the observed hyperexcitability. Our findings in both mosaic and non-mosaic pcdh19 mutant zebrafish challenge the prevailing theory that mosaicism governs all PCDH19-related phenotypes and point to interneuron-mediated mechanisms underlying these phenotypes.

neuroscience↗

bioRxiv2020

Novel genetic tools to model functional enzyme restoration in succinic semialdehyde dehydrogenase deficiency (SSADHD)

SSADHD is a rare inborn metabolic disorder caused by the functional impairment of SSADH (encoded by the aldh5a1 gene), an enzyme essential for breaking down the inhibitory neurotransmitter{gamma} -aminobutyric acid (GABA). In SSADHD, pathologic accumulation of GABA results in broad spectrum encephalopathy including developmental delay, ataxia, seizures and a risk of sudden unexpected death in epilepsy (SUDEP). Proof-of-concept systemic SSADH restoration via enzyme replacement therapy (ERT) or aldh5a1 gene transfer increased survival of SSADH knockout mice, suggesting that SSADH restoration might be a viable cure for SSADHD. However, before testing SSADH restoration therapy in patients, we must consider its safety and feasibility in context of the unique SSADHD pathophysiology. Specifically, a profound use-dependent down-regulation of GABAA receptors in SSADHD indicates a risk that sudden SSADH restoration might diminish GABAergic tone and provoke seizures. Such risk may be mitigated by gradual, rather than abrupt, SSADH restoration, or by restoration that is confined to critical cell types and brain regions. We therefore describe early work to construct a novel SSADHD mouse model that allows on-demand SSADH restoration for the systematic investigation of the rate, timing and cell-specific parameters of SSADH-restoring therapies. We aim to understand the clinical readiness of specific SSADH restoration protocols on brain physiology for purposes of accelerating the bench to bed-side development of ERT or gene therapy for SSADHD patients.

neuroscience↗