Abstract:
In the hippocampus, dentate gyrus granule cells connect to CA3 pyramidal cells via their axons, the mossy fibers (Mf). The synaptic terminals of Mfs (Mf boutons, MfBs) form large and complex synapses with thorny excrescences (TE) on the proximal dendrites of CA3 pyramidal cells (PCs). MfB/TE synapses have distinctive 'detonator' properties due to low initial release probability and large presynaptic facilitation. The molecular mechanisms shaping the morpho-functional properties of MfB/TE synapses are still poorly understood, though alterations in their morphology are associated with Down syndrome, intellectual disabilities, and Alzheimer's disease. Here, we identify the core PCP gene Vangl2 as essential to the morphogenesis and function of MfB/TE synapses. Vangl2 colocalises with the presynaptic heparan sulfate proteoglycan glypican 4 (GPC4) to stabilise the postsynaptic orphan receptor GPR158. Embryonic loss of Vangl2 disrupts the morphology of MfBs and TEs, impairs ultrastructural and molecular organisation, resulting in defective synaptic transmission and plasticity. In adult, the early loss of Vangl2 results in a number of hippocampus-dependent memory deficits including characteristic flexibility of declarative memory, organisation and retention of working / everyday-like memory. These deficits also lead to abnormal generalisation of memories to salient cues and diminished ability to form detailed contextual memories. Together, these results establish Vangl2 as a key regulator of DG-CA3 connectivity and functions.

Abstract:
Fragile X syndrome (FXS) is an X-linked neurodevelopmental disorder characterized by several behavioral abnormalities, including hyperactivity, anxiety, sensory hyper-responsiveness, and autistic-like symptoms such as social deficits. Despite considerable efforts, effective pharmacological treatments are still lacking, prompting the need for exploring the therapeutic value of existing drugs beyond their original approved use. One such repurposed drug is chlorzoxazone which is classified as a large-conductance calcium-dependent potassium (BKCa) channel opener. Reduced BKCa channel functionality has been reported in FXS patients, suggesting that molecules activating these channels could serve as promising treatments for this syndrome. Here, we sought to characterize the therapeutic potential of chlorzoxazone using the Fmr1-KO mouse model of FXS which recapitulates the main phenotypes of FXS, including BKCa channel alterations. Chlorzoxazone, administered either acutely or chronically, rescued hyperactivity and acoustic hyper-responsiveness as well as impaired social interactions exhibited by Fmr1-KO mice. Chlorzoxazone was more efficacious in alleviating these phenotypes than gaboxadol and metformin, two repurposed treatments for FXS that do not target BKCa channels. Systemic administration of chlorzoxazone modulated the neuronal activity-dependent gene c-fos in selected brain areas of Fmr1-KO mice, corrected aberrant hippocampal dendritic spines, and was able to rescue impaired BKCa currents recorded from hippocampal and cortical neurons of these mutants. Collectively, these findings provide further preclinical support for BKCa channels as a valuable therapeutic target for treating FXS and encourage the repurposing of chlorzoxazone for clinical applications in FXS and other related neurodevelopmental diseases.

Abstract:
OBJECTIVE: Steroidogenic factor-1 (SF1) neurons of the ventromedial hypothalamus play key roles in the regulation of food intake, body weight and glucose metabolism. The bile acid receptor Takeda G protein-coupled receptor 5 (TGR5) is expressed in the hypothalamus, where it determines some of the actions of bile acids on food intake and body weight through still poorly defined neuronal mechanisms. Here, we examined the role of TGR5 in SF1 neurons in the regulation of energy balance and glucose metabolism. METHODS: We used a genetic approach combined with metabolic phenotyping and molecular analyses to establish the effect of TGR5 deletion in SF1 neurons on meal pattern, body weight, body composition, energy expenditure and use of energy substrates as well as on possible changes in glucose handling and insulin sensitivity. RESULTS: Our findings reveal that TGR5 in SF1 neurons does not play a major role in the regulation of food intake or body weight under standard chow, but it is involved in the adaptive feeding response to the acute exposure to cold or to a hypercaloric, high-fat diet, without changes in energy expenditure. Notably, TGR5 in SF1 neurons hinder glucose metabolism, since deletion of the receptor improves whole-body glucose uptake through heightened insulin signaling in the hypothalamus and in the brown adipose tissue. CONCLUSIONS: TGR5 in SF1 neurons favours satiety by differently modifying the meal pattern in response to specific metabolic cues. These studies also reveal a novel key function for TGR5 in SF1 neurons in the regulation of whole-body insulin sensitivity, providing new insight into the role played by neuronal TGR5 in the regulation of metabolism.

Abstract:
Core planar cell polarity (PCP) genes, which are involved in various neurodevelopmental disorders such as neural tube closure, epilepsy, and autism spectrum disorder, have poorly defined molecular signatures in neurons, mostly synapse-centric. Here, we show that the core PCP protein Prickle-like protein 2 (Prickle2) controls neuronal polarity and is a previously unidentified member of the axonal initial segment (AIS) proteome. We found that Prickle2 is present and colocalizes with AnkG480, the AIS master organizer, in the earliest stages of axonal specification and AIS formation. Furthermore, by binding to and regulating AnkG480, Prickle2 modulates its ability to bundle microtubules, a crucial mechanism for establishing neuronal polarity and AIS formation. Prickle2 depletion alters cytoskeleton organization, and Prickle2 levels determine both axon number and AIS maturation. Last, early Prickle2 depletion produces impaired action potential firing.

Abstract:
Social behavior is a basic domain affected by several neurodevelopmental disorders, including ASD and a heterogeneous set of neuropsychiatric disorders. The SCRIB gene that codes for the polarity protein SCRIBBLE has been identified as a risk gene for spina bifida, the most common type of neural tube defect, found at high frequencies in autistic patients, as well as other congenital anomalies. The deletions and mutations of the 8q24.3 region encompassing SCRIB are also associated with multisyndromic and rare disorders. Nonetheless, the potential link between SCRIB and relevant social phenotypes has not been fully investigated. Hence, we show that Scrib(crc/+) mice, carrying a mutated version of Scrib, displayed reduced social motivation behavior and social habituation, while other behavioral domains were unaltered. Social deficits were associated with the upregulation of ERK phosphorylation, together with increased c-Fos activity. Importantly, the social alterations were rescued by both direct and indirect pERK inhibition. These results support a link between polarity genes, social behaviors and hippocampal functionality and suggest a role for SCRIB in the etiopathology of neurodevelopmental disorders. Furthermore, our data demonstrate the crucial role of the MAPK/ERK signaling pathway in underlying social motivation behavior, thus supporting its relevance as a therapeutic target.

Abstract:
Scribble (Scrib) is a conserved polarity protein acting as a scaffold involved in multiple cellular and developmental processes. Recent evidence from our group indicates that Scrib is also essential for brain development as early global deletion of Scrib in the dorsal telencephalon induced cortical thickness reduction and alteration of interhemispheric connectivity. In addition, Scrib conditional knockout (cKO) mice have behavioral deficits such as locomotor activity impairment and memory alterations. Given Scrib broad expression in multiple cell types in the brain, we decided to determine the neuronal contribution of Scrib for these phenotypes. In the present study, we further investigate the function of Scrib specifically in excitatory neurons on the forebrain formation and the control of locomotor behavior. To do so, we generated a novel neuronal glutamatergic specific Scrib cKO mouse line called Nex-Scrib (-/-) cKO. Remarkably, cortical layering and commissures were impaired in these mice and reproduced to some extent the previously described phenotype in global Scrib cKO. In addition and in contrast to our previous results using Emx1-Scrib (-/-) cKO, the Nex-Scrib (-/-) cKO mutant mice exhibited significantly reduced locomotion. Altogether, the novel cKO model described in this study further highlights an essential role for Scrib in forebrain development and locomotor behavior.

Abstract:
Neurodevelopmental disorders arise from combined defects in processes including cell proliferation, differentiation, migration and commissure formation. The evolutionarily conserved tumor-suppressor protein Scribble (Scrib) serves as a nexus to transduce signals for the establishment of apicobasal and planar cell polarity during these processes. Human SCRIB gene mutations are associated with neural tube defects and this gene is located in the minimal critical region deleted in the rare Verheij syndrome. In this study, we generated brain-specific conditional cKO mouse mutants and assessed the impact of the Scrib deletion on brain morphogenesis and behavior. We showed that embryonic deletion of Scrib in the telencephalon leads to cortical thickness reduction (microcephaly) and partial corpus callosum and hippocampal commissure agenesis. We correlated these phenotypes with a disruption in various developmental mechanisms of corticogenesis including neurogenesis, neuronal migration and axonal connectivity. Finally, we show that Scrib cKO mice have psychomotor deficits such as locomotor activity impairment and memory alterations. Altogether, our results show that Scrib is essential for early brain development due to its role in several developmental cellular mechanisms that could underlie some of the deficits observed in complex neurodevelopmental pathologies.

Abstract:
The presence of an inactivating HSP110 mutation in colorectal cancers has been correlated with an excellent prognosis and with the ability of HSP110 to favor the formation of tolerogenic (M2-like) macrophages. These clinical and experimental results suggest a potentially powerful new strategy against colorectal cancer: the inhibition of HSP110. In this work, as an alternative to neutralizing antibodies, Nanofitins (scaffold ~7 kDa-proteins) targeting HSP110 were isolated from the screening of a synthetic Nanofitin library, and their capacity to bind (immunoprecipitation, biolayer interferometry) and to inhibit HSP110 was analyzed in vitro and in vivo. Three Nanofitins were found to inhibit HSP110 chaperone activity. Interestingly, they share a high degree of homology in their variable domain and target the peptide-binding domain of HSP110. In vitro, they inhibited the ability of HSP110 to favor M2-like macrophages. The Nanofitin with the highest affinity, A-C2, was studied in the CT26 colorectal cancer mice model. Our PET/Scan experiments demonstrate that A-C2 may localized within the tumor area, in accord with the reported HSP110 abundance in the tumor microenvironment. A-C2 treatment reduced tumor growth, and was associated with an increase in immune cells infiltrating the tumor and particularly cytotoxic macrophages. These results were confirmed in a chicken chorioallantoic membrane tumor model. Finally, we showed the complementarity between A-C2 and an anti-PD-L1 strategy in the in vivo and in ovo tumor models. Altogether, Nanofitins appear to be promising new immunotherapeutic lead compounds.

Abstract:
The organization of spatial information, including pattern completion and pattern separation processes, relies on the hippocampal circuits, yet the molecular and cellular mechanisms underlying these two processes are elusive. Here, we find that loss of Vangl2, a core PCP gene, results in opposite effects on pattern completion and pattern separation processes. Mechanistically, we show that Vangl2 loss maintains young postmitotic granule cells in an immature state, providing increased cellular input for pattern separation. The genetic ablation of Vangl2 disrupts granule cell morpho-functional maturation and further prevents CaMKII and GluA1 phosphorylation, disrupting the stabilization of AMPA receptors. As a functional consequence, LTP at lateral perforant path-GC synapses is impaired, leading to defects in pattern completion behavior. In conclusion, we show that Vangl2 exerts a bimodal regulation on young and mature GCs, and its disruption leads to an imbalance in hippocampus-dependent pattern completion and separation processes.

Abstract:
Dynamic mechanical interactions between adhesion complexes and the cytoskeleton are essential for axon outgrowth and guidance. Whether planar cell polarity (PCP) proteins, which regulate cytoskeleton dynamics and appear necessary for some axon guidance, also mediate interactions with membrane adhesion is still unclear. Here we show that Vangl2 controls growth cone velocity by regulating the internal retrograde actin flow in an N-cadherin-dependent fashion. Single molecule tracking experiments show that the loss of Vangl2 decreased fast-diffusing N-cadherin membrane molecules and increased confined N-cadherin trajectories. Using optically manipulated N-cadherin-coated microspheres, we correlated this behavior to a stronger mechanical coupling of N-cadherin with the actin cytoskeleton. Lastly, we show that the spatial distribution of Vangl2 within the growth cone is selectively affected by an N-cadherin-coated substrate. Altogether, our data show that Vangl2 acts as a negative regulator of axonal outgrowth by regulating the strength of the molecular clutch between N-cadherin and the actin cytoskeleton.