Cyclin D1-Cdk4 regulates neuronal activity through phosphorylation of GABAA receptors.

Nuclear Cyclin D1 (Ccnd1) is a main regulator of cell cycle progression and cell proliferation. Interestingly, Ccnd1 moves to the cytoplasm at the onset of differentiation in neuronal precursors. However, cytoplasmic functions and targets of Ccnd1 in post-mitotic neurons are unknown. Here we identify the α4 subunit of gamma-aminobutyric acid (GABA) type A receptors (GABAARs) as an interactor and target of Ccnd1-Cdk4. Ccnd1 binds to an intracellular loop in α4 and, together with Cdk4, phosphorylates the α4 subunit at threonine 423 and serine 431. These modifications upregulate α4 surface levels, increasing the response of α4-containing GABAARs, measured in whole-cell patch-clamp recordings. In agreement with this role of Ccnd1-Cdk4 in neuronal signalling, inhibition of Cdk4 or expression of the non-phosphorylatable α4 decreases synaptic and extra-synaptic currents in the hippocampus of newborn rats. Moreover, according to α4 functions in synaptic pruning, CCND1 knockout mice display an altered pattern of dendritic spines that is rescued by the phosphomimetic α4. Overall, our findings molecularly link Ccnd1-Cdk4 to GABAARs activity in the central nervous system and highlight a novel role for this G1 cyclin in neuronal signalling.

Cognitive decline in diabetic mice predisposed to Alzheimer's disease is greater than in wild type.

In this work, we tested the hypothesis that the development of dementia in individuals with type 2 diabetes (T2DM) requires a genetic background of predisposition to neurodegenerative disease. As a proof of concept, we induced T2DM in middle-aged hAPP NL/F mice, a preclinical model of Alzheimer's disease. We show that T2DM produces more severe behavioral, electrophysiological, and structural alterations in these mice compared with wild-type mice. Mechanistically, the deficits are not paralleled by higher levels of toxic forms of Aß or by neuroinflammation but by a reduction in γ-secretase activity, lower levels of synaptic proteins, and by increased phosphorylation of tau. RNA-seq analysis of the cerebral cortex of hAPP NL/F and wild-type mice suggests that the former could be more susceptible to T2DM because of defects in trans-membrane transport. The results of this work, on the one hand, confirm the importance of the genetic background in the severity of the cognitive disorders in individuals with T2DM and, on the other hand, suggest, among the involved mechanisms, the inhibition of γ-secretase activity.

Perinatal exposure to pesticides alters synaptic plasticity signaling and induces behavioral deficits associated with neurodevelopmental disorders.

Increasing evidence from animal and epidemiological studies indicates that perinatal exposure to pesticides cause developmental neurotoxicity and may increase the risk for psychiatric disorders such as autism and intellectual disability. However, the underlying pathogenic mechanisms remain largely elusive. This work was aimed at testing the hypothesis that developmental exposure to different classes of pesticides hijacks intracellular neuronal signaling contributing to synaptic and behavioral alterations associated with neurodevelopmental disorders (NDD). Low concentrations of organochlorine (dieldrin, endosulfan, and chlordane) and organophosphate (chlorpyrifos and its oxon metabolite) pesticides were chronically dosed ex vivo (organotypic rat hippocampal slices) or in vivo (perinatal exposure in rats), and then biochemical, electrophysiological, behavioral, and proteomic studies were performed. All the pesticides tested caused prolonged activation of MAPK/ERK pathway in a concentration-dependent manner. Additionally, some of them impaired metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD). In the case of the pesticide chlordane, the effect was attributed to chronic modulation of MAPK/ERK signaling. These synaptic alterations were reproduced following developmental in vivo exposure to chlordane and chlorpyrifos-oxon, and were also associated with prototypical behavioral phenotypes of NDD, including impaired motor development, increased anxiety, and social and memory deficits. Lastly, proteomic analysis revealed that these pesticides differentially regulate the expression of proteins in the hippocampus with pivotal roles in brain development and synaptic signaling, some of which are associated with NDD. Based on these results, we propose a novel mechanism of synaptic dysfunction, involving chronic overactivation of MAPK and impaired mGluR-LTD, shared by different pesticides which may have important implications for NDD.

Functional specialization of different PI3K isoforms for the control of neuronal architecture, synaptic plasticity, and cognition.

Neuronal connectivity and activity-dependent synaptic plasticity are fundamental properties that support brain function and cognitive performance. Phosphatidylinositol 3-kinase (PI3K) intracellular signaling controls multiple mechanisms mediating neuronal growth, synaptic structure, and plasticity. However, it is still unclear how these pleiotropic functions are integrated at molecular and cellular levels. To address this issue, we used neuron-specific virally delivered Cre expression to delete either p110α or p110ß (the two major catalytic isoforms of type I PI3K) from the hippocampus of adult mice. We found that dendritic and postsynaptic structures are almost exclusively supported by p110α activity, whereas p110ß controls neurotransmitter release and metabotropic glutamate receptor-dependent long-term depression at the presynaptic terminal. In addition to these separate functions, p110α and p110ß jointly contribute to N-methyl-d-aspartate receptor-dependent postsynaptic long-term potentiation. This molecular and functional specialization is reflected in different proteomes controlled by each isoform and in distinct behavioral alterations for learning/memory and sociability in mice lacking p110α or p110ß.

Potentiation of amyloid beta phagocytosis and amelioration of synaptic dysfunction upon FAAH deletion in a mouse model of Alzheimer's disease.

BACKGROUND: The complex pathophysiology of Alzheimer's disease (AD) hampers the development of effective treatments. Attempts to prevent neurodegeneration in AD have failed so far, highlighting the need for further clarification of the underlying cellular and molecular mechanisms. Neuroinflammation seems to play a crucial role in disease progression, although its specific contribution to AD pathogenesis remains elusive. We have previously shown that the modulation of the endocannabinoid system (ECS) renders beneficial effects in a context of amyloidosis, which triggers neuroinflammation. In the 5xFAD model, the genetic inactivation of the enzyme that degrades anandamide (AEA), the fatty acid amide hydrolase (FAAH), was associated with a significant amelioration of the memory deficit. METHODS: In this work, we use electrophysiology, flow cytometry and molecular analysis to evaluate the cellular and molecular mechanisms underlying the improvement associated to the increased endocannabinoid tone in the 5xFAD mouse- model. RESULTS: We demonstrate that the chronic enhancement of the endocannabinoid tone rescues hippocampal synaptic plasticity in the 5xFAD mouse model. At the CA3-CA1 synapse, both basal synaptic transmission and long-term potentiation (LTP) of synaptic transmission are normalized upon FAAH genetic inactivation, in a CB1 receptor (CB1R)- and TRPV1 receptor-independent manner. Dendritic spine density in CA1 pyramidal neurons, which is notably decreased in 6-month-old 5xFAD animals, is also restored. Importantly, we reveal that the expression of microglial factors linked to phagocytic activity, such as TREM2 and CTSD, and other factors related to amyloid beta clearance and involved in neuron-glia crosstalk, such as complement component C3 and complement receptor C3AR, are specifically upregulated in 5xFAD/FAAH-/- animals. CONCLUSION: In summary, our findings support the therapeutic potential of modulating, rather than suppressing, neuroinflammation in Alzheimer's disease. In our model, the long-term enhancement of the endocannabinoid tone triggered augmented microglial activation and amyloid beta phagocytosis, and a consequent reversal in the neuronal phenotype associated to the disease.

A kinesin 1-protrudin complex mediates AMPA receptor synaptic removal during long-term depression.

The synaptic removal of AMPA-type glutamate receptors (AMPARs) is a core mechanism for hippocampal long-term depression (LTD). In this study, we address the role of microtubule-dependent transport of AMPARs as a driver for vesicular trafficking and sorting during LTD. Here, we show that the kinesin-1 motor KIF5A/C is strictly required for LTD expression in CA3-to-CA1 hippocampal synapses. Specifically, we find that KIF5 is required for an efficient internalization of AMPARs after NMDA receptor activation. We show that the KIF5/AMPAR complex is assembled in an activity-dependent manner and associates with microsomal membranes upon LTD induction. This interaction is facilitated by the vesicular adaptor protrudin, which is also required for LTD expression. We propose that protrudin links KIF5-dependent transport to endosomal sorting, preventing AMPAR recycling to synapses after LTD induction. Therefore, this work identifies an activity-dependent molecular motor and the vesicular adaptor protein that executes AMPAR synaptic removal during LTD.

KIF13A drives AMPA receptor synaptic delivery for long-term potentiation via endosomal remodeling.

The regulated trafficking of AMPA-type glutamate receptors (AMPARs) from dendritic compartments to the synaptic membrane in response to neuronal activity is a core mechanism for long-term potentiation (LTP). However, the contribution of the microtubule cytoskeleton to this synaptic transport is still unknown. In this work, using electrophysiological, biochemical, and imaging techniques, we have found that one member of the kinesin-3 family of motor proteins, KIF13A, is specifically required for the delivery of AMPARs to the spine surface during LTP induction. Accordingly, KIF13A depletion from hippocampal slices abolishes LTP expression. We also identify the vesicular protein centaurin-α1 as part of a motor transport machinery that is engaged with KIF13A and AMPARs upon LTP induction. Finally, we determine that KIF13A is responsible for the remodeling of Rab11-FIP2 endosomal structures in the dendritic shaft during LTP. Overall, these results identify specific kinesin molecular motors and endosomal transport machinery that catalyzes the dendrite-to-synapse translocation of AMPA receptors during synaptic plasticity.

Generation of mitochondrial reactive oxygen species is controlled by ATPase inhibitory factor 1 and regulates cognition.

The mitochondrial ATP synthase emerges as key hub of cellular functions controlling the production of ATP, cellular signaling, and fate. It is regulated by the ATPase inhibitory factor 1 (IF1), which is highly abundant in neurons. Herein, we ablated or overexpressed IF1 in mouse neurons to show that IF1 dose defines the fraction of active/inactive enzyme in vivo, thereby controlling mitochondrial function and the production of mitochondrial reactive oxygen species (mtROS). Transcriptomic, proteomic, and metabolomic analyses indicate that IF1 dose regulates mitochondrial metabolism, synaptic function, and cognition. Ablation of IF1 impairs memory, whereas synaptic transmission and learning are enhanced by IF1 overexpression. Mechanistically, quenching the IF1-mediated increase in mtROS production in mice overexpressing IF1 reduces the increased synaptic transmission and obliterates the learning advantage afforded by the higher IF1 content. Overall, IF1 plays a key role in neuronal function by regulating the fraction of ATP synthase responsible for mitohormetic mtROS signaling.

Ligand-independent activity of the ghrelin receptor modulates AMPA receptor trafficking and supports memory formation.

The biological signals of hunger, satiety, and memory are interconnected. The role of the hormone ghrelin in regulating feeding and memory makes ghrelin receptors attractive targets for associated disorders. We investigated the effects of the high ligand-independent activity of the ghrelin receptor GHS-R1a on the physiology of excitatory synapses in the hippocampus. Blocking this activity produced a decrease in the synaptic content of AMPA receptors in hippocampal neurons and a reduction in GluA1 phosphorylation at Ser845 Reducing the ligand-independent activity of GHS-R1a increased the surface diffusion of AMPA receptors and impaired AMPA receptor-dependent synaptic delivery induced by chemical long-term potentiation. Accordingly, we found that blocking this GHS-R1a activity impaired spatial and recognition memory in mice. These observations support a role for the ligand-independent activity of GHS-R1a in regulating AMPA receptor trafficking under basal conditions and in the context of synaptic plasticity that underlies learning.

GSK3α, not GSK3ß, drives hippocampal NMDAR-dependent LTD via tau-mediated spine anchoring.

Glycogen synthase kinase-3 (GSK3) is an important signalling protein in the brain and modulates different forms of synaptic plasticity. Neuronal functions of GSK3 are typically attributed to one of its two isoforms, GSK3ß, simply because of its prevalent expression in the brain. Consequently, the importance of isoform-specific functions of GSK3 in synaptic plasticity has not been fully explored. We now directly address this question for NMDA receptor-dependent long-term depression (LTD) in the hippocampus. Here, we specifically target the GSK3 isoforms with shRNA knock-down in mouse hippocampus and with novel isoform-selective drugs to dissect their roles in LTD. Using electrophysiological and live imaging approaches, we find that GSK3α, but not GSK3ß, is required for LTD. The specific engagement of GSK3α occurs via its transient anchoring in dendritic spines during LTD induction. We find that the major GSK3 substrate, the microtubule-binding protein tau, is required for this spine anchoring of GSK3α and mediates GSK3α-induced LTD. These results link GSK3α and tau in a common mechanism for synaptic depression and rule out a major role for GSK3ß in this process.

PTEN Activity Defines an Axis for Plasticity at Cortico-Amygdala Synapses and Influences Social Behavior.

Phosphatase and tensin homolog on chromosome 10 (PTEN) is a tumor suppressor and autism-associated gene that exerts an important influence over neuronal structure and function during development. In addition, it participates in synaptic plasticity processes in adulthood. As an attempt to assess synaptic and developmental mechanisms by which PTEN can modulate cognitive function, we studied the consequences of 2 different genetic manipulations in mice: presence of additional genomic copies of the Pten gene (Ptentg) and knock-in of a truncated Pten gene lacking its PDZ motif (Pten-ΔPDZ), which is required for interaction with synaptic proteins. Ptentg mice exhibit substantial microcephaly, structural hypoconnectivity, enhanced synaptic depression at cortico-amygdala synapses, reduced anxiety, and intensified social interactions. In contrast, Pten-ΔPDZ mice have a much more restricted phenotype, with normal synaptic connectivity, but impaired synaptic depression at cortico-amygdala synapses and virtually abolished social interactions. These results suggest that synaptic actions of PTEN in the amygdala contribute to specific behavioral traits, such as sociability. Also, PTEN appears to function as a bidirectional rheostat in the amygdala: reduction in PTEN activity at synapses is associated with less sociability, whereas enhanced PTEN activity accompanies hypersocial behavior.

A retention-release mechanism based on RAB11FIP2 for AMPA receptor synaptic delivery during long-term potentiation.

It is well--established that Rab11-dependent recycling endosomes drive the activity-dependent delivery of AMPA receptors (AMPARs) into synapses during long-term potentiation (LTP). Nevertheless, the molecular basis for this specialized function of recycling endosomes is still unknown. Here, we have investigated RAB11FIP2 (FIP2 hereafter) as a potential effector of Rab11-dependent trafficking during LTP in rat hippocampal slices. Surprisingly, we found that FIP2 operates independently from Rab11 proteins, and acts as a negative regulator of AMPAR synaptic trafficking. Under basal conditions, FIP2 associates with AMPARs at immobile compartments, separately from recycling endosomes. Using shRNA-mediated knockdown, we found that FIP2 prevents GluA1 (encoded by the Gria1 gene) AMPARs from reaching the surface of dendritic spines in the absence of neuronal stimulation. Upon induction of LTP, FIP2 is rapidly mobilized, dissociates from AMPARs and undergoes dephosphorylation. Interestingly, this dissociation of the FIP2-AMPAR complex, together with FIP2 dephosphorylation, is required for LTP, but the interaction between FIP2 and Rab11 proteins is not. Based on these results, we propose a retention-release mechanism, where FIP2 acts as a gate that restricts the trafficking of AMPARs, until LTP induction triggers their release and allows synaptic delivery.

NPC1 enables cholesterol mobilization during long-term potentiation that can be restored in Niemann-Pick disease type C by CYP46A1 activation.

NPC is a neurodegenerative disorder characterized by cholesterol accumulation in endolysosomal compartments. It is caused by mutations in the gene encoding NPC1, an endolysosomal protein mediating intracellular cholesterol trafficking. Cognitive and psychiatric alterations are hallmarks in NPC patients pointing to synaptic defects. However, the role of NPC1 in synapses has not been explored. We show that NPC1 is present in the postsynaptic compartment and is locally translated during LTP. A mutation in a region of the NPC1 gene commonly altered in NPC patients reduces NPC1 levels at synapses due to enhanced NPC1 protein degradation. This leads to shorter postsynaptic densities, increased synaptic cholesterol and impaired LTP in NPC1nmf164 mice with cognitive deficits. NPC1 mediates cholesterol mobilization and enables surface delivery of CYP46A1 and GluA1 receptors necessary for LTP, which is defective in NPC1nmf164 mice. Pharmacological activation of CYP46A1 normalizes synaptic levels of cholesterol, LTP and cognitive abilities, and extends life span of NPC1nmf164 mice. Our results unveil NPC1 as a regulator of cholesterol dynamics in synapses contributing to synaptic plasticity, and provide a potential therapeutic strategy for NPC patients.

Inhibition of p38 MAPK in the brain through nasal administration of p38 inhibitor loaded in chitosan nanocapsules.

Aim: To determine whether a p38 MAPK inhibitor incorporated into nanoemulsion-based chitosan nanocapsules can reduce the activity of this kinase in the brain through their nasal administration in mice. Materials & methods: We selected the p38 MAPK inhibitor PH797804, an ATP-competitive inhibitor of p38α encapsulated in nanoemulsion-based chitosan nanocapsules. Biological effect was evaluated in microglial and neuronal cells in vitro and in ex vivo and in vivo systems, in a mouse model of Alzheimer's disease. Results: Encapsulated inhibitor retains enzymatic inhibitory activity and tissue penetration capacity in vitro, ex vivo and in vivo. Conclusion: Nasal administration of chitosan nanocapsules can be an effective approach for brain-restricted reduction of p38 MAPK activity, thus reducing the side effects of systemic administration.

Elevated levels of Secreted-Frizzled-Related-Protein 1 contribute to Alzheimer's disease pathogenesis.

The deposition of aggregated amyloid-ß peptides derived from the pro-amyloidogenic processing of the amyloid precurson protein (APP) into characteristic amyloid plaques (APs) is distinctive to Alzheimer's disease (AD). Alternative APP processing via the metalloprotease ADAM10 prevents amyloid-ß formation. We tested whether downregulation of ADAM10 activity by its secreted endogenous inhibitor secreted-frizzled-related protein 1 (SFRP1) is a common trait of sporadic AD. We demonstrate that SFRP1 is significantly increased in the brain and cerebrospinal fluid of patients with AD, accumulates in APs and binds to amyloid-ß, hindering amyloid-ß protofibril formation. Sfrp1 overexpression in an AD-like mouse model anticipates the appearance of APs and dystrophic neurites, whereas its genetic inactivation or the infusion of α-SFRP1-neutralizing antibodies favors non-amyloidogenic APP processing. Decreased Sfrp1 function lowers AP accumulation, improves AD-related histopathological traits and prevents long-term potentiation loss and cognitive deficits. Our study unveils SFRP1 as a crucial player in AD pathogenesis and a promising AD therapeutic target.

Astrocytic p38α MAPK drives NMDA receptor-dependent long-term depression and modulates long-term memory.

NMDA receptor-dependent long-term depression (LTD) in the hippocampus is a well-known form of synaptic plasticity that has been linked to different cognitive functions. The core mechanism for this form of plasticity is thought to be entirely neuronal. However, we now demonstrate that astrocytic activity drives LTD at CA3-CA1 synapses. We have found that LTD induction enhances astrocyte-to-neuron communication mediated by glutamate, and that Ca2+ signaling and SNARE-dependent vesicular release from the astrocyte are required for LTD expression. In addition, using optogenetic techniques, we show that low-frequency astrocytic activation, in the absence of presynaptic activity, is sufficient to induce postsynaptic AMPA receptor removal and LTD expression. Using cell-type-specific gene deletion, we show that astrocytic p38α MAPK is required for the increased astrocytic glutamate release and astrocyte-to-neuron communication during low-frequency stimulation. Accordingly, removal of astrocytic (but not neuronal) p38α abolishes LTD expression. Finally, this mechanism modulates long-term memory in vivo.

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Correction: Tetraspanin 6: A novel regulator of hippocampal synaptic transmission and long term plasticity.

[This corrects the article DOI: 10.1371/journal.pone.0171968.].

MAP1B Light Chain Modulates Synaptic Transmission via AMPA Receptor Intracellular Trapping.

The regulated transport of AMPA-type glutamate receptors (AMPARs) to the synaptic membrane is a key mechanism to determine the strength of excitatory synaptic transmission in the brain. In this work, we uncovered a new role for the microtubule-associated protein MAP1B in modulating access of AMPARs to the postsynaptic membrane. Using mice and rats of either sex, we show that MAP1B light chain (LC) accumulates in the somatodendritic compartment of hippocampal neurons, where it forms immobile complexes on microtubules that limit vesicular transport. These complexes restrict AMPAR dendritic mobility, leading to the intracellular trapping of receptors and impairing their access to the dendritic surface and spines. Accordingly, increasing MAP1B-LC expression depresses AMPAR-mediated synaptic transmission. This effect is specific for the GluA2 subunit of the AMPAR and requires glutamate receptor interacting protein 1 (GRIP1) interaction with MAP1B-LC. Therefore, MAP1B-LC represents an alternative link between GRIP1-AMPARs and microtubules that does not result in productive transport, but rather limits AMPAR availability for synaptic insertion, with a direct impact on synaptic transmission.SIGNIFICANCE STATEMENT The ability of neurons to modify their synaptic connections, known as synaptic plasticity, is accepted as the cellular basis for learning and memory. One mechanism for synaptic plasticity is the regulated addition and removal of AMPA-type glutamate receptors (AMPARs) at excitatory synapses. In this study, we found that a microtubule-associated protein, MAP1B light chain (MAP1B-LC), participates in this process. MAP1B-LC forms immobile complexes along dendrites. These complexes limit intracellular vesicular trafficking and trap AMPARs inside the dendritic shaft. In this manner, MAP1B restricts the access of AMPARs to dendritic spines and the postsynaptic membrane, contributing to downregulating synaptic transmission.