Biology subjects

Marsh, E. D.

Publications and source records attributed to Marsh, E. D..

8 recordsLinked to original sources

bioRxiv2025

Chromatin Disruption After Prenatal Hypoxia Predicts Lasting Neuron Deficits

Prenatal exposure to transient hypoxia can result in various developmental disabilities presumably by disrupting normal neurodevelopmental processes, even in milder injuries without detectable cell death or structural damage1,2. Emerging evidence suggests that disruption of the epigenome may be one of the most critical consequences of prenatal brain exposure to hypoxia3-8. However, the cell-type-selective effects of hypoxia on the developing brains transcriptome and epigenome remain unknown. Here, we demonstrate that immediately after hypoxia, transcriptional and chromatin disruptions occur in all cell types, consistent with the energy deficit dynamics caused by prenatal hypoxia. Unexpectedly, we identify a selective dissociation of transcriptional and chromatin regulation in glutamatergic neurons after hypoxia. Genes neighboring sites of differential chromatin accessibility, but not differential gene expression, correlate with structural and functional deficits in glutamatergic neurons one month after the injury. Our approach reveals novel, shared, and cell-type-specific changes that reflect potential short- and long-term consequences of the hypoxic response. Together, these data provide insight into the mechanisms that lead to lasting changes in the epigenome following an injury to the developing brain, resulting in long-term functional deficits.

neuroscience↗

bioRxiv2024

Impaired excitability of fast-spiking neurons in a novel mouse model of KCNC1 epileptic encephalopathy

The recurrent pathogenic variant KCNC1-p.Ala421Val (A421V) is a cause of developmental and epileptic encephalopathy characterized by moderate-to-severe developmental delay/intellectual disability, and infantile-onset treatment-resistant epilepsy with multiple seizure types including myoclonic seizures. Yet, the mechanistic basis of this disease, and of the KCNC1 disease spectrum, is unclear. KCNC1 encodes Kv3.1, a voltage-gated potassium channel subunit that is highly and selectively expressed in neurons capable of generating action potentials at high frequency, including parvalbumin-positive fast-spiking GABAergic inhibitory interneurons in cerebral cortex (PV-INs) known to be important for cognitive function and plasticity as well as control of network excitation to prevent seizures. In this study, we generate a novel transgenic mouse model with conditional expression of the Ala421Val pathogenic missense variant (Kcnc1-A421V/+ mice) to explore the specific physiological mechanisms of KCNC1 developmental and epileptic encephalopathy. Our results indicate that global heterozygous expression of the A421V variant leads to cognitive impairment, epilepsy, and premature lethality. We observe decreased PV-IN cell surface expression of Kv3.1 via immunohistochemistry, decreased voltage-gated potassium current density in PV-INs using outside-out nucleated macropatch recordings in brain slice, and profound impairments in the intrinsic excitability of cerebral cortex PV-INs (but not excitatory neurons) in current-clamp electrophysiology. In vivo two-photon calcium imaging revealed altered basal activity in Kcnc1-A421V/+ PV-INs and excitatory cells, as well as hypersynchronous discharges correlated with brief paroxysmal movements that were subsequently shown to be myoclonic seizures on electroencephalography. We found alterations in PV-IN-mediated inhibitory neurotransmission in young adult but not juvenile Kcnc1-A421V/+ mice relative to wild-type controls. Together, these results establish the specific impact of the recurrent Kv3.1-A421V variant on neuronal excitability and synaptic physiology across development to drive network dysfunction underlying KCNC1 epileptic encephalopathy.

neuroscience↗

bioRxiv2024

Therapeutic suppression of Tubb4a rescues H-ABC leukodystrophy

Hypomyelination and atrophy of basal ganglia and cerebellum (H-ABC) is a rare leukodystrophy associated with causal variants in {beta}-tubulin 4A (TUBB4A). The recurring variant p.Asp249Asn (D249N) presents in infancy with dystonia, communication deficits, and loss of ambulation during the first decade of life. In this study, we characterized a genetic murine series (Tubb4aKO/KO, Tubb4aD249N/+, Tubb4aD249N/KO, and Tubb4aD249N/D249N) to demonstrate that disease severity correlates with the expression of mutant Tubb4a and relative preservation of WT tubulin. To further evaluate the translational potential of Tubb4a suppression as a therapy in H-ABC, we identified a well-tolerated Tubb4a-targeted antisense oligonucleotide (ASO) candidate that selectively reduces Tubb4a. Notably, single intracerebroventricular (i.c.v.) administration of ASO in postnatal Tubb4aD249N/KO mice drastically extends its lifespan, improves motor phenotypes, and reduces seizures. Neuropathologically, treating ASO Tubb4aD249N/KO mice prevents myelin and oligodendrocyte loss and recovers visual evoked potential latencies in vivo. Furthermore, the microtubule function of Mbp mRNA transport from oligodendrocyte (OL) soma to myelin sheath is retained. A major limitation we noted is that ASOs fail to target cerebellar granule neurons even with multiple routes of administration in the brain. This is the first preclinical proof-of-concept for Tubb4a suppression via ASO as a disease-modifying therapy for H-ABC.

neuroscience↗

bioRxiv2022

Mechanisms of vascular maturation and maintenance captured by longitudinal imaging of live mouse skin

A functional network of blood vessels is essential for organ growth and homeostasis. Yet, how the vasculature matures and maintains adult homeostasis remains elusive in live mice. By longitudinally tracking the same neonatal endothelial cells (ECs) over days to weeks, we found that capillary plexus expansion is driven by network-wide vessel regression and transient angiogenesis. A fixed number of neonatal ECs rearrange their positions to evenly distribute throughout the developing plexus and become positionally stable in adulthood. Upon injury, while neonatal ECs are predisposed to die, adult ECs survive through a plasmalemmal self-repair response. Furthermore, adult neighboring ECs reactivate migration to assist vessel repair. Lastly, neonatal vessel regression and adult vascular maintenance are orchestrated by temporally restricted VEGFR2 dependent signaling. Our work sheds light on fundamental cellular mechanisms that underlie both vascular maturation and adult homeostasis in vivo.

developmental biology↗

bioRxiv2021

Transient late gestation prenatal hypoxic insult results in functional deficits but not gross neuroanatomic deficits in mice

Intrauterine hypoxia is a common cause of brain injury in children resulting in a broad spectrum of long-term neurodevelopmental sequela, including life-long disabilities that can occur even in the absence of severe neuroanatomic damage. Postnatal hypoxia-ischemia rodent models are commonly used to understand the effects of ischemia and transient hypoxia on the developing brain. Postnatal models, however, have some limitations. First, they do not test the impact of placental pathologies on outcomes from hypoxia. Second, they primarily recapitulate severe injury because they provoke substantial cell death, which is not seen in children with mild hypoxic injury. Lastly, they do not model preterm hypoxic injury. Prenatal models of hypoxia in mice may allow us to address some of these limitations to expand our understanding of developmental brain injury. The published rodent models of prenatal hypoxia employ multiple days of hypoxic exposure or complicated surgical procedures, making these models challenging to perform consistently in mice. Furthermore, large animal models suggest that transient prenatal hypoxia without ischemia is sufficient to lead to significant functional impairment to the developing brain. However, these large animal studies are resource-intensive and not readily amenable to mechanistic molecular studies. Therefore, here we characterized the effect of late gestation (embryonic day 17.5) transient prenatal hypoxia (5% inspired oxygen) on long-term anatomical and neurodevelopmental outcomes in mice. Late gestation transient prenatal hypoxia increased hypoxia-inducible factor 1 alpha protein levels (a marker of hypoxic exposure) in the fetal brain. Hypoxia exposure predisposed animals to decreased weight at postnatal day 2, which normalized by day 8. However, hypoxia did not affect gestational age at birth, litter size at birth, or pup survival. No differences in fetal brain cell death or long-term gray or white matter changes resulted from hypoxia. Animals exposed to prenatal hypoxia did have several long-term functional consequences, including sex-dichotomous changes. Hypoxia exposure was associated with a decreased seizure threshold and abnormalities in hindlimb strength and repetitive behaviors in males and females. Males exposed to hypoxia had increased anxiety-related deficits, whereas females had deficits in social interaction. Neither sex developed any motor or visual learning deficits. This study demonstrates that late gestation transient prenatal hypoxia in mice is a simple, clinically relevant paradigm for studying putative environmental and genetic modulators of the long-term effects of hypoxia on the developing brain.

animal behavior and cognition↗

bioRxiv2021

Neuronal genetic rescue normalizes brain network dynamics in a lysosomal storage disorder despite persistent storage accumulation

Although neurologic symptoms occur in two-thirds of lysosomal storage disorders (LSDs), for most we do not understand the mechanisms underlying brain dysfunction. A major unanswered question is if the pathogenic hallmark of LSDs, storage accumulation, induces functional defects directly or is a disease bystander. Also, for most LSDs we do not know the impact of loss-of-function in individual cell types. Understanding these critical questions are essential to therapy development. Here, we determined the impact of genetic rescue in distinct cell types on neural circuit dysfunction in CLN3 disease, the most common pediatric dementia and a representative LSD. We restored Cln3 expression via AAV-mediated gene delivery and conditional genetic rescue in a CLN3 mouse model. Surprisingly, we found that low-level rescue of Cln3 expression in neurons alone normalized clinically-relevant electrophysiologic markers of network dysfunction, despite the presence of substantial residual histopathology, in contrast to restoring expression in astrocytes. Thus, loss of Cln3 function in neurons, not storage accumulation, underlies neurologic dysfunction in CLN3 disease, implying that storage clearance may be an inappropriate target for therapy development and an ineffectual biomarker.

neuroscience↗

bioRxiv2021

Skin-resident immune cells actively coordinate their distribution with epidermal cells during homeostasis

Our organs consist of multiple cell types that ensure proper architecture and function. How different cell types coexist and interact to maintain their homeostasis in vivo remain elusive. The skin epidermis comprises mostly epithelial cells, but also harbors Langerhans cells (LCs) and Dendritic Epidermal T cells (DETCs). In response to injury or infection, LCs and DETCs become activated and play critical immunological roles. During homeostasis, they coexist with epithelial cells in the basal layer of the epidermis. Whether, and how, distributions of LCs and DETCs are regulated during homeostasis is unclear. Here, we addressed this question by tracking LCs, DETCs and epithelial basal cells over time within the skin of live adult mice. We show that LCs and DETCs maintain their overall position despite continuous turnover of neighboring basal epithelial stem cells. Moreover, LCs and DETCs rapidly and maximally explore basal epithelial cell junctions through their dendritic extensions. Altering the epithelial cell density triggers corresponding changes in the immune cell density, but not vice versa, suggesting that epithelial cells determine immune tissue composition in the epidermis. Moreover, LCs and DETCs are organized in a tiling pattern that is actively maintained. When LCs or DETCs are ectopically removed, neighboring epidermal LCs or DETCs, respectively, move into the emptied spaces and re-establish the tiling pattern. Finally, LCs require the GTPase Rac1 to maintain their positional stability, density and tiling pattern. Overall, we discovered that epidermal cells regulate the density of immune cells during homeostasis, and that immune cells actively maintain a non-random spatial distribution, reminiscent of neuronal self-avoidance. We propose that these cellular mechanisms provide the epidermis with an optimal response to environmental insults.

cell biology↗

bioRxiv2019

High throughput Characterization of KCNB1 variants Associated with Developmental and Epileptic Encephalopathy

Pathogenic variants in KCNB1, encoding the voltage-gated potassium channel Kv2.1, are associated with developmental and epileptic encephalopathies (DEE). Previous functional studies on a limited number of KCNB1 variants indicated a range of molecular mechanisms by which variants affect channel function, including loss of voltage sensitivity, loss of ion selectivity, and reduced cell-surface expression. We evaluated a series of 17 KCNB1 variants associated with DEE or neurodevelopmental disorder (NDD) to rapidly ascertain channel dysfunction using high-throughput functional assays. Specifically, we investigated the biophysical properties and cell-surface expression of variant Kv2.1 channels expressed in heterologous cells using high-throughput automated electrophysiology and immunocytochemistry-flow cytometry. Pathogenic variants exhibited diverse functional defects, including altered current density and shifts in the voltage-dependence of activation and/or inactivation, as homotetramers or when co-expressed with wild-type Kv2.1. Quantification of protein expression also identified variants with reduced total Kv2.1 expression or deficient cell-surface expression.\n\nOur study establishes a platform for rapid screening of functional defects of KCNB1 variants associated with DEE and other NDDs, which will aid in establishing KCNB1 variant pathogenicity and may enable discovery of targeted strategies for therapeutic intervention based on molecular phenotype.

genetics↗