Alessandra Folci

Genetic defects of the gene encoding the ubiquitin ligase UBE3A cause a severe neurodevelopmental disorder, the Angelman syndrome (AS). The pathophysiology of AS remains unclear, hindering the development of effective therapies. Using AS animal models, we show here that UBE3A controls the development of excitatory and distinct subtypes of inhibitory synapses in cortical pyramidal neurons through cell-autonomous mechanisms, ultimately leading to alteration of synaptic transmission and hyperexcitability. Replacing endogenous Ube3a with individual isoforms, we demonstrate that their uneven nuclear (hUBE3A isoform 1) and cytosolic (hUBE3A isoforms 2/3) distribution is critical for regulating distinct aspects of synaptic development. We also define the molecular requirements underpinning this regulation, showing that: (i) both nuclear and cytosolic UBE3A rely on their ubiquitin ligase activity to ensure proper assembly of synapses; (ii) in addition to the nucleus, UBE3A isoform 1 is also localized in the cytosol, where it is functionally interchangeable with UBE3A isoform 3. Our findings identify a subcellular distribution-dependent mechanism by which UBE3A coordinates cortical circuit development and suggest pathogenic mechanisms of AS.

The CACNA1C gene encodes the CaV1.2 L-type voltage-gated calcium channel, which plays a crucial role in neuronal signaling. CACNA1C is a risk gene for psychiatric conditions involving disruption of neuronal connectivity such as schizophrenia, autism, and bipolar disorders. While genomic studies are consistently reinforcing the notion of CACNA1C as an important locus related to these diseases, the role of CaV1.2 channels in determining neuronal architecture is incompletely understood. Several studies pinpoint the L-type current (ICaL) as regulator of dendritic arborization development. ICaL lays upstream of competing cellular mechanisms leading to both inhibition and promotion of dendritic growth. How signal selectivity is achieved remains an open question. Here, we report that ICaL -dependent dendritic development of murine cultured hippocampal neurons relies on CaV1.2 and is determined by an equilibrium between the level of CaV1.2 protein expression and ICaL activity. Indeed, increasing ICaL enhances dendritic complexity only when CaV1.2 expression level is reduced. In contrast, when channel levels are at baseline, the CaV1.2-dependent growing signal is overcome by the elevation of the dendritic growth inhibiting CaMKIIα signaling beyond basal conditions. These findings suggest that CaV1.2 expression level acts as a molecular switch between dendritic growing and inhibiting signals. Consequently, altered CaV1.2 expression during early development may alter neuronal structure, potentially impairing neural network formation and increasing susceptibility to psychiatric disorders.

The Fragile X Mental Retardation Protein (FMRP) is an RNA-binding protein essential to the regulation of local translation at synapses. In the mammalian brain, synapses are constantly formed and eliminated throughout development to achieve functional neuronal networks. At the molecular level, thousands of proteins cooperate to accomplish efficient neuronal communication. Therefore, synaptic protein levels and their functional interactions need to be tightly regulated. FMRP generally acts as a translational repressor of its mRNA targets. FMRP is the target of several post-translational modifications (PTMs) that dynamically regulate its function. Here we provide an overview of the PTMs controlling the FMRP function and discuss how their spatiotemporal interplay contributes to the physiological regulation of FMRP. Importantly, FMRP loss-of-function leads to Fragile X syndrome (FXS), a rare genetic developmental condition causing a range of neurological alterations including intellectual disability (ID), learning and memory impairments, autistic-like features and seizures. Here, we also explore the possibility that recently reported missense mutations in the FMR1 gene disrupt the PTM homoeostasis of FMRP, thus participating in the aetiology of FXS. This suggests that the pharmacological targeting of PTMs may be a promising strategy to develop innovative therapies for patients carrying such missense mutations.

Sumoylation is a reversible post-translational modification essential to the modulation of neuronal function, including neurotransmitter release and synaptic plasticity. A tightly regulated equilibrium between the sumoylation and desumoylation processes is critical to the brain function and its disruption has been associated with several neurological disorders. This sumoylation/desumoylation balance is governed by the activity of the sole SUMO-conjugating enzyme Ubc9 and a group of desumoylases called SENPs, respectively. We previously demonstrated that the activation of type 5 metabotropic glutamate receptors (mGlu5R) triggers the transient trapping of Ubc9 in dendritic spines, leading to a rapid increase in the overall synaptic sumoylation. However, the mechanisms balancing this increased synaptic sumoylation are still not known. Here, we examined the diffusion properties of the SENP1 enzyme using a combination of advanced biochemical approaches and restricted photobleaching/photoconversion of individual hippocampal spines. We demonstrated that the activation of mGlu5R leads to a time-dependent decrease in the exit rate of SENP1 from dendritic spines. The resulting post-synaptic accumulation of SENP1 restores synaptic sumoylation to initial levels. Altogether, our findings reveal the mGlu5R system as a central activity-dependent mechanism to maintaining the homeostasis of sumoylation at the mammalian synapse.

Fragile X syndrome (FXS), the most common form of inherited intellectual disability (ID) and a leading cause of autism, results from the loss of expression of the Fmr1 gene which encodes the RNA-binding protein Fragile X Mental Retardation Protein (FMRP). Among the thousands mRNA targets of FMRP, numerous encode regulators of ion homeostasis. It has also been described that FMRP directly interacts with Ca²⁺ channels modulating their activity. Collectively these findings suggest that FMRP plays critical roles in Ca²⁺ homeostasis during nervous system development. We carried out a functional analysis of Ca²⁺ regulation using a calcium imaging approach in Fmr1-KO cultured neurons and we show that these cells display impaired steady state Ca²⁺ concentration and an altered entry of Ca²⁺ after KCl-triggered depolarization. Consistent with these data, we show that the protein product of the Cacna1a gene, the pore-forming subunit of the Ca_v2.1 channel, is less expressed at the plasma membrane of Fmr1-KO neurons compared to wild-type (WT). Thus, our findings point out the critical role that Ca_v2.1 plays in the altered Ca²⁺ flux in Fmr1-KO neurons, impacting Ca²⁺ homeostasis of these cells. Remarkably, we highlight a new phenotype of cultured Fmr1-KO neurons that can be considered a novel cellular biomarker and is amenable to small molecule screening and identification of new drugs to treat FXS.

Fragile X syndrome (FXS) is the most frequent inherited cause of intellectual disability and the best-studied monogenic cause of autism. FXS results from the functional absence of the fragile X mental retardation protein (FMRP) leading to abnormal pruning and consequently to synaptic communication defects. Here we show that FMRP is a substrate of the small ubiquitin-like modifier (SUMO) pathway in the brain and identify its active SUMO sites. We unravel the functional consequences of FMRP sumoylation in neurons by combining molecular replacement strategy, biochemical reconstitution assays with advanced live-cell imaging. We first demonstrate that FMRP sumoylation is promoted by activation of metabotropic glutamate receptors. We then show that this increase in sumoylation controls the homomerization of FMRP within dendritic mRNA granules which, in turn, regulates spine elimination and maturation. Altogether, our findings reveal the sumoylation of FMRP as a critical activity-dependent regulatory mechanism of FMRP-mediated neuronal function.

The PCDH19 gene (Xp22.1) encodes the cell-adhesion protein protocadherin-19 (PCDH19) and is responsible for a neurodevelopmental pathology characterized by female-limited epilepsy, cognitive impairment and autistic features, the pathogenic mechanisms of which remain to be elucidated. Here, we identified a new interaction between PCDH19 and GABA_A receptor (GABA_AR) alpha subunits in the rat brain. PCDH19 shRNA-mediated downregulation reduces GABA_AR surface expression and affects the frequency and kinetics of miniature inhibitory postsynaptic currents (mIPSCs) in cultured hippocampal neurons. In vivo, PCDH19 downregulation impairs migration, orientation and dendritic arborization of CA1 hippocampal neurons and increases rat seizure susceptibility. In sum, these data indicate a role for PCDH19 in GABAergic transmission as well as migration and morphological maturation of neurons.

Intellectual disability affects 2–3% of the world's population and typically begins during childhood, causing impairments in social skills and cognitive abilities. Mutations in the TM4SF2 gene, which encodes the TSPAN7 protein, cause a severe form of intellectual disability, and currently, no therapy is able to ameliorate this cognitive impairment. We previously reported that, in cultured neurons, shRNA-mediated down-regulation of TSPAN7 affects AMPAR trafficking by enhancing PICK1–GluA2 interaction, thereby increasing the intracellular retention of AMPAR. Here, we found that loss of TSPAN7 function in mice causes alterations in hippocampal excitatory synapse structure and functionality as well as cognitive impairment. These changes occurred along with alterations in AMPAR expression levels. We also found that interfering with PICK1–GluA2 binding restored synaptic function in Tm4sf2^−/y mice. Moreover, potentiation of AMPAR activity via the administration of the ampakine CX516 reverted the neurological phenotype observed in Tm4sf2^−/y mice, suggesting that pharmacological modulation of AMPAR may represent a new approach for treating patients affected by TM4SF2 mutations and intellectual disability.

Myosin IXa (Myo9a) is a motor protein that is highly expressed in the brain. However, the role of Myo9a in neurons remains unknown. Here, we investigated Myo9a function in hippocampal synapses. In rat hippocampal neurons, Myo9a localizes to the postsynaptic density (PSD) and binds the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) GluA2 subunit. Myo9a+/- mice displayed a thicker PSD and increased levels of PSD95 and surface AMPAR expression. Furthermore, synaptic transmission, long-term potentiation (LTP) and cognitive functions were impaired in Myo9a+/- mice. Together, these results support a key role for Myo9a in controlling the molecular structure and function of hippocampal synapses.