The role of ubiquitin ligase E3A in polarized contact guidance and rescue strategies in UBE3A-deficient hippocampal neurons

Abstract

BackgroundAlthough neuronal extracellular sensing is emerging as crucial for brain wiring and therefore plasticity, little is known about these processes in neurodevelopmental disorders. Ubiquitin protein ligase E3A (UBE3A) plays a key role in neurodevelopment. Lack of UBE3A leads to Angelman syndrome (AS), while its increase is among the most prevalent genetic causes of autism (e.g., Dup15q syndrome). By using microstructured substrates that can induce specific directional stimuli in cells, we previously found deficient topographical contact guidance in AS neurons, which was linked to a dysregulated activation of the focal adhesion pathway.MethodsHere, we study axon and dendrite contact guidance and neuronal morphological features of wild-type, AS, and UBE3A-overexpressing neurons (Dup15q autism model) on micrograting substrates, with the aim to clarify the role of UBE3A in neuronal guidance.ResultsWe found that loss of axonal contact guidance is specific for AS neurons while UBE3A overexpression does not affect neuronal directional polarization along microgratings. Deficits at the level of axonal branching, growth cone orientation and actin fiber content, focal adhesion (FA) effectors, and actin fiber–binding proteins were observed in AS neurons. We tested different rescue strategies for restoring correct topographical guidance in AS neurons on microgratings, by either UBE3A protein re-expression or by pharmacological treatments acting on cytoskeleton contractility. Nocodazole, a drug that depolymerizes microtubules and increases cell contractility, rescued AS axonal alignment to the gratings by partially restoring focal adhesion pathway activation. Surprisingly, UBE3A re-expression only resulted in partial rescue of the phenotype.ConclusionsWe identified a specific in vitro deficit in axonal topographical guidance due selectively to the loss of UBE3A, and we further demonstrate that this defective guidance can be rescued to a certain extent by pharmacological or genetic treatment strategies. Overall, cytoskeleton dynamics emerge as important partners in UBE3A-mediated contact guidance responses. These results support the view that UBE3A-related deficits in early neuronal morphogenesis may lead to defective neuronal connectivity and plasticity.

Angelman syndrome: a journey through the brain

“…Recently, another study on axonal dynamics was conducted. Axon and dendrite contact guidance of AS hippocampal neurons was assessed on micrograting substrates that can provide a directional stimulus in vitro . Axonal guidance deficit and increased axonal branching with frequent secondary axonal segments were found to be related to the UBE3A loss.…”

Neuronal contact guidance and YAP signaling on ultra-small nanogratings

“…Only neurons fully visible and with soma cleanly abutting neighbouring cells were traced and analysed. Neurites were semi-automatically segmented by NeuronJ, according to their branching, as in 69,70 . Briefly, neurite segments were divided in categories: 1° order for neurites directly emerging from the cell soma (from soma to the tip); 2° order for neurites emerging from 1° order neurites.…”

“…Briefly, neurite segments were divided in categories: 1° order for neurites directly emerging from the cell soma (from soma to the tip); 2° order for neurites emerging from 1° order neurites. We focused our analysis on 1° order neurites, being them the vast majority of total neuritic segments at early stages 55,69 . Neurite analysis was then performed such as for PC12 cells, as reported above.…”

The Research Advance of Cell Bridges in vitro

“…Notably, some novel cell morphologies which in part dictates the cell such as contact guidance (Nguyen et al, 2016;Ray et al, 2017) and cell bridges (Goldner et al, 2006;Zhang et al, 2015b), have been observed on the patterned substrates at micro and nano scale. While contact guidance has been extensively investigated in regeneration medicine and tissue engineering (Kamyar et al, 2018;Tonazzini et al, 2019), studies on cell bridges observed on chemical pattern substrates and some nano/micro topographical substrates remain scarce.…”

“…With the recent advancement of tissue engineering, cell biology, and biomaterials science, various substrates or scaffolds with more defined topographic (Zhao et al, 2018) and chemical (Na et al, 2017) pattern are prepared to mimic the natural cell niche in tissue in order to guide the cells behavior. Contact guidance (Zhang et al, 2015a;Tonazzini et al, 2019), cell adhesion (Vasiliki and Triantafyllos, 2018), differentiation (Yao et al, 2016;Zhang et al, 2019) and gene expression (Qiongfang et al, 2018) have been paid much attention and investigated extensively; however, cell bridges, a phenomenon as consistent as contact guidance remain to be completely elucidated. As an essential behavior of cells, cell bridges may play crucial role in organization of cells, migration and development.…”