Impaired Neurite Contact Guidance in Ubiquitin Ligase E3a (Ube3a)‐Deficient Hippocampal Neurons on Nanostructured Substrates

Abstract

Recent discoveries indicate that during neuronal development the signaling processes that regulate extracellular sensing (e.g., adhesion, cytoskeletal dynamics) are important targets for ubiquitination-dependent regulation, in particular through E3 ubiquitin ligases. Among these, Ubiquitin E3a ligase (UBE3A) has a key role in brain functioning, but its function and how its deficiency results in the neurodevelopmental disorder Angelman syndrome is still unclear. Here, the role of UBE3A is investigated in neurite contact guidance during neuronal development, in vitro. The microtopography sensing of wild-type and Ube3a-deficient hippocampal neurons is studied by exploiting gratings with different topographical characteristics, with the aim to compare their capabilities to read and follow physical directional stimuli. It is shown that neuronal contact guidance is defective in Ube3a-deficient neurons, and this behavior is linked to an impaired activation of the focal adhesion signaling pathway. Taken together, the results suggest that the neuronal contact sensing machinery might be affected in Angelman syndrome.

Dysfunctional mTORC1 Signaling: A Convergent Mechanism between Syndromic and Nonsyndromic Forms of Autism Spectrum Disorder?

“…Moreover, an overall excitatory network, due to a more severe decrease in inhibitory than excitatory inputs [197], possibly contributes to the increased seizure susceptibility observed in AS patients. In vitro studies using Ube3a knockdown or Ube3a KO neurons from mice have also shown that Ube3a loss of function decreased dendrite arborization, disrupted dendrite polarity and reduced apical dendrite length [198,199]. In addition, disrupted synaptic plasticity in the form of impaired LTP in different brain areas [45,193,200] and enhanced hippocampal mGluR-LTD [201] were also observed in AS mice (Table 1).…”

Neuronal contact guidance and YAP signaling on ultra-small nanogratings

“…Because we know that neurite growth is usually favored along NGs (i.e. aligned neurites are longer than misaligned ones) 55 , we quantitatively examined the spatial development of neurite arborization and we calculated the total length of the neurites aligned (alignment angle 0-15°) or perpendicular (alignment angle 75°-90°) to NG pattern on each substrate (reported in % over the total network length analyzed) (Fig. 8b).…”

“…Cell lysates (10 µg/line) were processed by immunoblot, as in 55 . Briefly, samples were resolved by gel electrophoresis (SDS-PAGE) using Gel Criterion XT-Precasted polyacrylamide gel 4-12% Bis-Tris (Biorad), transferred to nitrocellulose membranes by Trans-Blot Turbo transfer system (Biorad) and probed overnight at 4 °C with primary antibodies.…”

“…YAP immunocytochemistry and fluorescence confocal microscopy. PC12 cells were cultured, fixed for 15 min with 4% formaldehyde with 4% sucrose in PBS at room temperature and processed as previously reported 55 . Here cells were incubated with primary anti-YAP1 antibody (1:500, ab39361) and phalloidin-Alexa fluor 647 (1:40, Thermo Fisher A22287) in GDB buffer (0.2% BSA, 0.8 M NaCl, 0.5% Triton X-100, 30 mM phosphate buffer, pH 7.4) overnight at 4 °C and then with appropriate secondary antibody conjugated to AlexaFluor 488; in the end samples were mounted using Fluorashield mounting medium with 4′,6-diamidin-2-fenilindolo (DAPI) to stain nuclei (Sigma, F6057).…”

“…primary hippocampal neurons. Hippocampal neuronal cultures (HNs) were prepared from brains of single E17-18 mouse embryos (C57 background), as previously described 55 . Mice were maintained under standard housing conditions and used according to the protocols and ethical guidelines approved by the Italian Ministry of Health (Permit Number: CBS-not.…”