ITSN1: a novel candidate gene involved in autosomal dominant neurodevelopmental disorder spectrum.

ITSN1 plays an important role in brain development. Recent studies in large cohorts of subjects with neurodevelopmental disorders have identified de novo variants in ITSN1 gene thereby suggesting that this gene is involved in the development of such disorders. The aim of this study is to provide further proof of such a link. We performed trio exome sequencing in a patient presenting autism, intellectual disability, and severe behavioral difficulties. Additional affected patients with a neurodevelopmental disorder harboring a heterozygous variant in ITSN1 (NM_003024.2) were collected through a worldwide collaboration. All patients underwent detailed phenotypic and genetic assessment and data was collected and shared by healthcare givers. We identified ten novel patients from eight families with heterozygous truncating or missense variants in ITSN1 gene. In addition, four previously published patients from large meta-analysis studies were included. In total, 7/14 patients presented a de novo variant in ITSN1. All patients showed neurodevelopmental disorders from autism spectrum disorders (90%), intellectual disability (86%), and epilepsy (30%). We demonstrated that truncating variants are in the first half of ITSN1 whereas missense variants are clustered in C-terminal region. We suggest ITSN1 gene is involved in development of an autism spectrum disorder with variable additional neurodevelopmental deficiency, thus confirming the hypothesis that ITSN1 is important for brain development.

Caspase-dependent degradation of MDMx/MDM4 cell cycle regulatory protein in amyloid ß-induced neuronal damage.

MDMx/MDM4 is a negative regulator of the p53 tumor suppressor protein and is necessary for survival in dividing cells. MDMx is also expressed in postmitotic neurons, with prosurvival roles that are independent of its extensively described roles in carcinogenesis. We and others have shown a role for MDMx loss in neuronal death in vitro and in vivo in several neurodegenerative diseases. Further, we have recently shown that MDMx is targeted for proteolytic degradation by calcium-dependent proteases, calpains, in neurons in vitro, and that MDMx overexpression provided partial neuroprotection in a model of HIV-associated neurodegeneration. Here, we assessed whether amyloid ß (Aß)-induced MDMx degradation occurred in Alzheimer's Disease (AD) models. Our data shows an age-dependent reduction in MDMx levels in cholinergic neurons within the cortex of adult mice expressing the swedish mutant of the amyloid precursor protein, APP in the Tg2576 murine model of AD. In vitro, Aß treatment of primary cortical neurons led to the caspase-dependent MDMx degradation. Our findings suggest that MDMx degradation associated with neuronal death occurs via caspase activation in neurons, and that the progressive loss of MDMx protein represents a potential mechanism of Aß-induced neuronal death during disease progression in AD.

E2F1 in neurons is cleaved by calpain in an NMDA receptor-dependent manner in a model of HIV-induced neurotoxicity.

The transcription factor E2F1 activates gene targets required for G1 -S phase progression and for apoptosis, and exhibits increased expression levels in neurons in several CNS diseases including HIV encephalitis, Alzheimer disease, and Parkinson's Disease. While E2F1 is known to regulate cell viability through activation of caspases, here we present evidence supporting the involvement of E2F1 in N-methyl-d-aspartate (NMDA) receptor-dependent, HIV-induced neuronal death mediated by calpains. Using an in vitro model of HIV-induced neurotoxicity that is dependent on NMDA receptor and calpain activation, we have shown that cortical neurons lacking functional E2F1 are less susceptible to neuronal death. In addition, we report that neuronal E2F1 is cleaved by calpain to a stable 55-kiloDalton fragment following NR2B-dependent NMDA receptor stimulation. This cleavage of E2F1 is protein conformation-dependent and involves at least two cleavage events, one at each terminus of the protein. Intriguingly, the stabilized E2F1 cleavage product is produced in post-mitotic neurons of all ages, but fails to be stabilized in cycling cells. Finally, we show that a matching E2F1 cleavage product is produced in human fetal neurons, suggesting that calpain cleavage of E2F1 may be produced in human cortical tissue. These results suggest neuronal E2F1 is processed in a novel manner in response to NMDA receptor-mediated toxicity, a mechanism implicated in HIV-associated neurocognitive disorders pathogenesis as well as several other diseases of the CNS. After crossing the blood-brain barrier, HIV-infected monocytes differentiate into macrophages and release excitotoxins and inflammatory factors including glutamate into the brain parenchyma (1). These factors stimulate neuronal N-Methyl-d-aspartate (NMDA) receptors (2), causing calcium influx (3) and subsequent activation of the cysteine protease calpain (4). Activated calpain cleaves multiple substrates including E2F1, producing a stabilized protein fragment with truncations at the N- and C-terminus (5). Calpain-cleaved E2F1 may contribute to calpain-mediated neuronal damage observed in NMDA receptor-mediated neurotoxicity (6).

Targeted gene mutation of E2F1 evokes age-dependent synaptic disruption and behavioral deficits.

Aberrant expression and activation of the cell cycle protein E2F1 in neurons has been implicated in many neurodegenerative diseases. As a transcription factor regulating G1 to S phase progression in proliferative cells, E2F1 is often up-regulated and activated in models of neuronal death. However, despite its well-studied functions in neuronal death, little is known regarding the role of E2F1 in the mature brain. In this study, we used a combined approach to study the effect of E2F1 gene disruption on mouse behavior and brain biochemistry. We identified significant age-dependent olfactory and memory-related deficits in E2f1 mutant mice. In addition, we found that E2F1 exhibits punctated staining and localizes closely to the synapse. Furthermore, we found a mirroring age-dependent loss of post-synaptic protein-95 in the hippocampus and olfactory bulb as well as a global loss of several other synaptic proteins. Coincidently, E2F1 expression is significantly elevated at the ages, in which behavioral and synaptic perturbations were observed. Finally, we show that deficits in adult neurogenesis persist late in aged E2f1 mutant mice which may partially contribute to the behavior phenotypes. Taken together, our data suggest that the disruption of E2F1 function leads to specific age-dependent behavioral deficits and synaptic perturbations. E2F1 is a transcription factor regulating cell cycle progression and apoptosis. Although E2F1 dysregulation under toxic conditions can lead to neuronal death, little is known about its physiologic activity in the healthy brain. Here, we report significant age-dependent olfactory and memory deficits in mice with dysfunctional E2F1. Coincident with these behavioral changes, we also found age-matched synaptic disruption and persisting reduction in adult neurogenesis. Our study demonstrates that E2F1 contributes to physiologic brain structure and function.

Differential roles for caspase-mediated and calpain-mediated cell death in 1- and 3-week-old rat cortical cultures.

Necrosis and apoptosis are well established as two primary cell death pathways. Mixed neuroglial cultures are commonly used to study cell death mechanisms in neural cells. However, the ages of these cultures vary across studies and little attention has been paid to how cell death processes may change as the cultures mature. To clarify whether neuroglial culture age affects cell death mechanisms, we treated 1- and 3-week-old neuroglial cultures with either the excitotoxic stimulus, N-methyl-D-aspartate (NMDA), or with the oxidative stressor, hydrogen peroxide (H2O2). Although NMDA is known to be toxic only in cultures that are at least 2 weeks old, H2O2 is toxic in cultures of all ages. Here, we confirm that, in 1-week-old neuroglial cultures, NMDA does not induce toxicity, whereas H2O2 induces both calpain-mediated and caspase-mediated neuronal death. In 3-week-old cultures, both NMDA and H2O2 trigger calpain-mediated, but not caspase-mediated, neuronal death. Further, we observed a decrease in caspase-3 levels and an increase in calpain levels in untreated neuroglial cultures as they aged. The findings presented here show that neuronal cell death mechanisms vary with culture age and highlight the necessity of considering culture age when interpreting neural cell culture data.