Conference Agenda

The loss of UBE3A causes the Angelman Syndrome (AS), a severe neurodevelopmental disorder characterized by intellectual disability, motor delay and seizures. Although considerable efforts have been put to dissect the molecular underpinnings of UBE3A function in neurons, the pathogenic mechanisms of these neurodevelopmental disorders are still poorly understood. To this aim, we study the effects of UBE3A loss on the regulation of synaptic development at single-cell level in vivo. We combine cortex-directed in utero electroporation to inactivate UBE3A in sparse pyramidal neurons with advanced microscopy and biochemistry to investigate the role of UBE3A on the formation, maturation and functional organization of excitatory and inhibitory synapses. Our results indicate that UBE3A critically regulates the formation of excitatory synapses, and controls the assembly and the maturation of specific subtypes of inhibitory synapses, namely those located in the perisomatic region and in the axon initial segment. The in utero replacement of endogenous Ube3a with individual human UBE3A isoforms, which display different subcellular distributions, indicates that the development of specific subtypes of synapses is selectively controlled by distinct isoforms and highlight the importance of UBE3A nuclear function in the regulation of synapses. These results suggest that synaptic dysfunction in AS cannot be ascribed to just a single mechanism, but instead to a more global regulatory role of UBE3A. Under this perspective, we hypothesize that the loss of UBE3A might interfere with GABA signaling during development, which possesses a crucial trophic function driving fundamental developmental processes of the brain, including synaptogenesis. In line with this, we present here preliminary data suggesting that the switch of GABA from depolarizing to hyperpolarizing is indeed delayed upon UBE3A deletion.

Oxytocin (OT), a master regulator of social behavior, has been proposed to ameliorate social deficits in different neuropsychiatric conditions. OT also regulates key neurodevelopmental events, suggesting that OT can modify the onset and progression of these conditions if administered postnatally and/or in childhood. To identify the molecular targets and time window of OT action, we are working on different models of neurodevelopmental disorders (22q11DS, Magel2, Dysbindin, Oprm1, MeCP2 and Oxtr KO mice).

Here we report the action of OT in MeCP2 KO male mice which recapitulate many symptoms of Rett Syndrome, including early dysregulation in excitatory-inhibitory (E/I) balance and down-regulation of KCC2, which could be ameliorated by recombinant human IGF-1 (rhIGF-1) (Castro, 2016) and KCC2 enhancers (Tang 2019). Because we have previously shown that KCC2 can also be modulated by OT through its specific receptor OXTR (Leonzino, 2016), we hypothesized that OT could also modulate KCC2 in MeCP2 KO. We thus quantified KCC2, OXTR and IGF-1R levels in brain sections of MeCP2 KO male mice treated with OT, rhIGF-1 or vehicle and compared their levels to WT mice. Our data demonstrate that 1) in MeCP2 KO mice, alterations in KCC2 expression are region-specific: significantly reduced KCC2 expression levels were found in the prefrontal cortex (PFC), anterior olfactory nucleus (AON) and pyriform cortex (Pyr) while other regions did not show any alteration. 2) Treatment with OT and rhIGF-1 rescued KCC2 expression in a region-specific, complementary manner: rhIGF-1 rescued KCC2 in the Pyr, whereas OT normalizes KCC2 in the PFC and AON, suggesting that a combined treatment of rhIGF-1 and OT could lead to a more effective KCC2 normalization in the brain of MeCP2 KO mice. Our results also indicate that OT could be particularly powerful in turning GABA on when E/I unbalance and KCC2 dysregulation in the PFC are subtending a neurodevelopmental pathological condition.

The most common adverse effects of sleep deprivation (SD) include deficits in information processing and related executive functions. However, the molecular mechanisms underlying these alterations remain elusive. Previously, we found that these neurobehavioral complications are associated with increased levels of the neurosteroid allopregnanolone (AP) and Brain-Derived Neurotrophic Factor (BDNF) in the medial prefrontal cortex (mPFC).

Here, we employed single-unit extracellular recordings, immunohistochemical analysis, and behavioral assessments to investigate how imbalances in BDNF and AP levels in the mPFC may mediate the information-processing and executive deficits caused by SD. Male rats were subjected to the platform method, and at the end of the SD period, they were tested using well-established paradigms for studying information processing and executive functions in both humans and rodents: the prepulse inhibition and the 5-choice continuous performance test.

Our findings revealed that the increase in BDNF levels in the PFC due to SD was accompanied by a downregulation of KCC2, a transporter responsible for expelling chloride ions from the cells. Consequently, this downregulation led to the accumulation of chloride within the PFC pyramidal cells, resulting in a switch polarity of GABA-A receptors from inhibitory to excitatory. Notably, no differences were observed in KCC2 phosphorylation or NKCC1 expression in the PFC of sleep-deprived rats. Importantly, treatment with the NKCC1 blocker bumetanide normalized the reversal of chloride currents in pyramidal cells and rescued the neurobehavioral deficits observed in SD rats. These effects were similar to those achieved with the 5-alpha reductase inhibitor finasteride, suggesting that AP may contribute to the cognitive consequences of SD by acting on depolarizing GABA-A receptors in PFC pyramidal neurons.

To our knowledge, this is the first study to demonstrate that the synergy between chloride current reversal and imbalances in neurosteroid synthesis in the PFC is necessary to impair information processing and executive functions following SD.

The neuronal GABAergic switch represents a critical event occurring early in life before birth characterized by the excitatory-to-inhibitory transition of the GABAergic signaling. The process relies on chloride homeostasis and is mainly determined by developmentally-regulated changes in intracellular chloride concentration, via two chloride co-transporters, NKCC1 and KCC2. Impairments in the accomplishment of this event have been associated to a remarkable excitation/inhibition network imbalance, usually linked to cognitive disabilities and behavioral deficits, typical hallmarks of neurodevelopmental disorders, which are frequently related to inflammatory states. Here we investigated the impact of prenatal IL-6 elevation, a dominant proinflammatory cytokine during inflammatory states, on the forthcoming development of GABA developmental switch in the hippocampus. Using a combination of calcium and chloride imaging, molecular techniques, together with electrophysiological and multi-electrode array recordings, we found that IL-6 is able to accelerate the GABA switch in juvenile offspring born from IL-6-injected dams, associated to a reduction of the whole-neuronal network hippocampal activity. Our in vitro investigations revealed the engagement of Stat3, a transcription factor activated by IL-6 signaling, as the STAT3 inhibitor Stattic prevented IL-6–mediated GABA switch acceleration. qRT-PCR analysis revealed that Kcc2, but not Nkcc1, gene was upregulated upon IL-6 priming thus suggesting a prominent role of IL-6 pathway on targeting Kcc2 expression, and also KCC2 protein trafficking and surface expression. Taken together, our in vitro and in vivo evidences show that IL6 may regulate the GABAergic switch in early neuronal development by guiding transcriptional and cytoplasmic effects. Results from these studies will allow us to clarify the role of IL-6 in this fundamental process often related to the occurrence of neurodevelopmental diseases.