BIN1 recovers tauopathy-induced long-term memory deficits in mice and interacts with Tau through Thr348 phosphorylation.

The bridging integrator 1 gene (BIN1) is a major genetic risk factor for Alzheimer's disease (AD). In this report, we investigated how BIN1-dependent pathophysiological processes might be associated with Tau. We first generated a cohort of control and transgenic mice either overexpressing human MAPT (TgMAPT) or both human MAPT and BIN1 (TgMAPT;TgBIN1), which we followed-up from 3 to 15 months. In TgMAPT;TgBIN1 mice short-term memory deficits appeared earlier than in TgMAPT mice; however-unlike TgMAPT mice-TgMAPT;TgBIN1 mice did not exhibit any long-term or spatial memory deficits for at least 15 months. After killing the cohort at 18 months, immunohistochemistry revealed that BIN1 overexpression prevents both Tau mislocalization and somatic inclusion in the hippocampus, where an increase in BIN1-Tau interaction was also observed. We then sought mechanisms controlling the BIN1-Tau interaction. We developed a high-content screening approach to characterize modulators of the BIN1-Tau interaction in an agnostic way (1,126 compounds targeting multiple pathways), and we identified-among others-an inhibitor of calcineurin, a Ser/Thr phosphatase. We determined that calcineurin dephosphorylates BIN1 on a cyclin-dependent kinase phosphorylation site at T348, promoting the open conformation of the neuronal BIN1 isoform. Phosphorylation of this site increases the availability of the BIN1 SH3 domain for Tau interaction, as demonstrated by nuclear magnetic resonance experiments and in primary neurons. Finally, we observed that although the levels of the neuronal BIN1 isoform were unchanged in AD brains, phospho-BIN1(T348):BIN1 ratio was increased, suggesting a compensatory mechanism. In conclusion, our data support the idea that BIN1 modulates the AD risk through an intricate regulation of its interaction with Tau. Alteration in BIN1 expression or activity may disrupt this regulatory balance with Tau and have direct effects on learning and memory.

APOE-Sensitive Cholinergic Sprouting Compensates for Hippocampal Dysfunctions Due to Reduced Entorhinal Input.

Brain mechanisms compensating for cerebral lesions may mitigate the progression of chronic neurodegenerative disorders such as Alzheimer's disease (AD). Mild cognitive impairment (MCI), which often precedes AD, is characterized by neuronal loss in the entorhinal cortex (EC). This loss leads to a hippocampal disconnection syndrome that drives clinical progression. The concomitant sprouting of cholinergic terminals in the hippocampus has been proposed to compensate for reduced EC glutamatergic input. However, in absence of direct experimental evidence, the compensatory nature of the cholinergic sprouting and its putative mechanisms remain elusive. Transgenic mice expressing the human APOE4 allele, the main genetic risk factor for sporadic MCI/AD, display impaired cholinergic sprouting after EC lesion. Using these mice as a tool to manipulate cholinergic sprouting in a disease-relevant way, we showed that this sprouting was necessary and sufficient for the acute compensation of EC lesion-induced spatial memory deficit before a slower glutamatergic reinnervation took place. We also found that partial EC lesion generates abnormal hyperactivity in EC/dentate networks. Dentate hyperactivity was abolished by optogenetic stimulation of cholinergic fibers. Therefore, control of dentate hyperactivity by cholinergic sprouting may be involved in functional compensation after entorhinal lesion. Our results also suggest that dentate hyperactivity in MCI patients may be directly related to EC neuronal loss. Impaired sprouting during the MCI stage may contribute to the faster cognitive decline reported in APOE4 carriers. Beyond the amyloid contribution, the potential role of both cholinergic sprouting and dentate hyperactivity in AD symptomatogenesis should be considered in designing new therapeutic approaches. SIGNIFICANCE STATEMENT: Currently, curative treatment trials for Alzheimer's disease (AD) have failed. The endogenous ability of the brain to cope with neuronal loss probably represents one of the most promising therapeutic targets, but the underlying mechanisms are still unclear. Here, we show that the mammalian brain is able to manage several deleterious consequences of the loss of entorhinal neurons on hippocampal activity and cognitive performance through a fast cholinergic sprouting followed by a slower glutamatergic reinnervation. The cholinergic sprouting is gender dependent and highly sensitive to the genetic risk factor APOE4 Our findings highlight the specific impact of early loss of entorhinal input on hippocampal hyperactivity and cognitive deficits characterizing early stages of AD, especially in APOE4 carriers.

Differential regulation of MeCP2 and PP1 in passive or voluntary administration of cocaine or food.

Cocaine exposure induces changes in the expression of numerous genes, in part through epigenetic modifications. We have initially shown that cocaine increases the expression of the chromatin remodeling protein methyl-CpG binding protein 2 (MeCP2) and characterized the protein phosphatase-1Cß (PP1Cß) gene, as repressed by passive i.p. cocaine injections through a Mecp2-mediated mechanism involving de novo DNA methylation. Both proteins being involved in learning and memory processes, we investigated whether voluntary cocaine administration would similarly affect their expression using an operant self-administration paradigm. Passive and voluntary i.v. cocaine intake was found to induce Mecp2 and to repress PP1Cß in the prefrontal cortex and the caudate putamen. This observation is consistent with the role of Mecp2 acting as a transcriptional repressor of PP1Cß and shows that passive intake was sufficient to alter their expression. Surprisingly, striking differences were observed under the same conditions in food-restricted rats tested for food pellet delivery. In the prefrontal cortex and throughout the striatum, both proteins were induced by food operant conditioning, but remained unaffected by passive food delivery. Although cocaine and food activate a common reward circuit, changes observed in the expression of other genes such as reelin and GAD67 provide new insights into molecular mechanisms differentiating neuroadaptations triggered by each reinforcer. The identification of hitherto unknown genes differentially regulated by drugs of abuse and a natural reinforcer should improve our understanding of how two rewarding stimuli differ in their ability to drive behavior.

Regulation of Brain DNA Methylation Factors and of the Orexinergic System by Cocaine and Food Self-Administration.

Inhibitors of DNA methylation and orexin type-1 receptor antagonists modulate the neurobiological effects driving drugs of abuse and natural reinforcers by activating common brain structures of the mesolimbic reward system. In this study, we applied a self-administration paradigm to assess the involvement of factors regulating DNA methylation processes and satiety or appetite signals. These factors include Dnmts and Tets, miR-212/132, orexins, and orx-R1 genes. The study focused on dopamine projection areas such as the prefrontal cortex (PFCx) and caudate putamen (CPu) and in the hypothalamus (HP) that is interconnected with the reward system. Striking changes were observed in response to both reinforcers, but differed depending on contingent and non-contingent delivery. Expression also differed in the PFCx and the CPu. Cocaine and food induced opposite effects on Dnmt3a expression in both brain structures, whereas they repressed both miRs to a different extent, without affecting their primary transcript in the CPu. Unexpectedly, orexin mRNAs were found in the CPu, suggesting a transport from their transcription site in the HP. The orexin receptor1 gene was found to be induced by cocaine in the PFCx, consistent with a regulation by DNA methylation. Global levels of 5-methylcytosines in the PFCx were not significantly altered by cocaine, suggesting that it is rather their distribution that contributes to long-lasting behaviors. Together, our data demonstrate that DNA methylation regulating factors are differentially altered by cocaine and food. At the molecular level, they support the idea that neural circuits activated by both reinforcers do not completely overlap.

Amphiphysin 2 modulation rescues myotubular myopathy and prevents focal adhesion defects in mice.

Centronuclear myopathies (CNMs) are severe diseases characterized by muscle weakness and myofiber atrophy. Currently, there are no approved treatments for these disorders. Mutations in the phosphoinositide 3-phosphatase myotubularin (MTM1) are responsible for X-linked CNM (XLCNM), also called myotubular myopathy, whereas mutations in the membrane remodeling Bin/amphiphysin/Rvs protein amphiphysin 2 [bridging integrator 1 (BIN1)] are responsible for an autosomal form of the disease. Here, we investigated the functional relationship between MTM1 and BIN1 in healthy skeletal muscle and in the physiopathology of CNM. Genetic overexpression of human BIN1 efficiently rescued the muscle weakness and life span in a mouse model of XLCNM. Exogenous human BIN1 expression with adeno-associated virus after birth also prevented the progression of the disease, suggesting that human BIN1 overexpression can compensate for the lack of MTM1 expression in this mouse model. Our results showed that MTM1 controls cell adhesion and integrin localization in mammalian muscle. Alterations in this pathway in Mtm1 -/y mice were associated with defects in myofiber shape and size. BIN1 expression rescued integrin and laminin alterations and restored myofiber integrity, supporting the idea that MTM1 and BIN1 are functionally linked and necessary for focal adhesions in skeletal muscle. The results suggest that BIN1 modulation might be an effective strategy for treating XLCNM.