Enhanced hippocampal LTP but normal NMDA receptor and AMPA receptor function in a rat model of CDKL5 deficiency disorder.

BACKGROUND: Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) cause a severe neurological disorder characterised by early-onset epileptic seizures, autism and intellectual disability (ID). Impaired hippocampal function has been implicated in other models of monogenic forms of autism spectrum disorders and ID and is often linked to epilepsy and behavioural abnormalities. Many individuals with CDKL5 deficiency disorder (CDD) have null mutations and complete loss of CDKL5 protein, therefore in the current study we used a Cdkl5-/y rat model to elucidate the impact of CDKL5 loss on cellular excitability and synaptic function of CA1 pyramidal cells (PCs). We hypothesised abnormal pre and/or post synaptic function and plasticity would be observed in the hippocampus of Cdkl5-/y rats. METHODS: To allow cross-species comparisons of phenotypes associated with the loss of CDKL5, we generated a loss of function mutation in exon 8 of the rat Cdkl5 gene and assessed the impact of the loss of CDLK5 using a combination of extracellular and whole-cell electrophysiological recordings, biochemistry, and histology. RESULTS: Our results indicate that CA1 hippocampal long-term potentiation (LTP) is enhanced in slices prepared from juvenile, but not adult, Cdkl5-/y rats. Enhanced LTP does not result from changes in NMDA receptor function or subunit expression as these remain unaltered throughout development. Furthermore, Ca2+ permeable AMPA receptor mediated currents are unchanged in Cdkl5-/y rats. We observe reduced mEPSC frequency accompanied by increased spine density in basal dendrites of CA1 PCs, however we find no evidence supporting an increase in silent synapses when assessed using a minimal stimulation protocol in slices. Additionally, we found no change in paired-pulse ratio, consistent with normal release probability at Schaffer collateral to CA1 PC synapses. CONCLUSIONS: Our data indicate a role for CDKL5 in hippocampal synaptic function and raise the possibility that altered intracellular signalling rather than synaptic deficits contribute to the altered plasticity. LIMITATIONS: This study has focussed on the electrophysiological and anatomical properties of hippocampal CA1 PCs across early postnatal development. Studies involving other brain regions, older animals and behavioural phenotypes associated with the loss of CDKL5 are needed to understand the pathophysiology of CDD.

Reversal of cell, circuit and seizure phenotypes in a mouse model of DNM1 epileptic encephalopathy.

Dynamin-1 is a large GTPase with an obligatory role in synaptic vesicle endocytosis at mammalian nerve terminals. Heterozygous missense mutations in the dynamin-1 gene (DNM1) cause a novel form of epileptic encephalopathy, with pathogenic mutations clustering within regions required for its essential GTPase activity. We reveal the most prevalent pathogenic DNM1 mutation, R237W, disrupts dynamin-1 enzyme activity and endocytosis when overexpressed in central neurons. To determine how this mutation impacted cell, circuit and behavioural function, we generated a mouse carrying the R237W mutation. Neurons from heterozygous mice display dysfunctional endocytosis, in addition to altered excitatory neurotransmission and seizure-like phenotypes. Importantly, these phenotypes are corrected at the cell, circuit and in vivo level by the drug, BMS-204352, which accelerates endocytosis. Here, we demonstrate a credible link between dysfunctional endocytosis and epileptic encephalopathy, and importantly reveal that synaptic vesicle recycling may be a viable therapeutic target for monogenic intractable epilepsies.

Phosphatidylinositol 4-kinase IIα is a glycogen synthase kinase 3-regulated interaction hub for activity-dependent bulk endocytosis.

Phosphatidylinositol 4-kinase IIα (PI4KIIα) generates essential phospholipids and is a cargo for endosomal adaptor proteins. Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle endocytosis mode during high neuronal activity and is sustained by glycogen synthase kinase 3ß (GSK3ß) activity. We reveal the GSK3ß substrate PI4KIIα is essential for ADBE via its depletion in primary neuronal cultures. Kinase-dead PI4KIIα rescues ADBE in these neurons but not a phosphomimetic form mutated at the GSK3ß site, Ser-47. Ser-47 phosphomimetic peptides inhibit ADBE in a dominant-negative manner, confirming that Ser-47 phosphorylation is essential for ADBE. Phosphomimetic PI4KIIα interacts with a specific cohort of presynaptic molecules, two of which, AGAP2 and CAMKV, are also essential for ADBE when depleted in neurons. Thus, PI4KIIα is a GSK3ß-dependent interaction hub that silos essential ADBE molecules for liberation during neuronal activity.

Epilepsy-Related CDKL5 Deficiency Slows Synaptic Vesicle Endocytosis in Central Nerve Terminals.

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a severe early-onset epileptic encephalopathy resulting mainly from de novo mutations in the X-linked CDKL5 gene. To determine whether loss of presynaptic CDKL5 function contributes to CDD, we examined synaptic vesicle (SV) recycling in primary hippocampal neurons generated from Cdkl5 knockout rat males. Using a genetically encoded reporter, we revealed that CDKL5 is selectively required for efficient SV endocytosis. We showed that CDKL5 kinase activity is both necessary and sufficient for optimal SV endocytosis, since kinase-inactive mutations failed to correct endocytosis in Cdkl5 knockout neurons, whereas the isolated CDKL5 kinase domain fully restored SV endocytosis kinetics. Finally, we demonstrated that CDKL5-mediated phosphorylation of amphiphysin 1, a putative presynaptic target, is not required for CDKL5-dependent control of SV endocytosis. Overall, our findings reveal a key presynaptic role for CDKL5 kinase activity and enhance our insight into how its dysfunction may culminate in CDD.SIGNIFICANCE STATEMENT Loss of cyclin-dependent kinase like 5 (CDKL5) function is a leading cause of monogenic childhood epileptic encephalopathy. However, information regarding its biological role is scarce. In this study, we reveal a selective presynaptic role for CDKL5 in synaptic vesicle endocytosis and that its protein kinase activity is both necessary and sufficient for this role. The isolated protein kinase domain is sufficient to correct this loss of function, which may facilitate future gene therapy strategies if presynaptic dysfunction is proven to be central to the disorder. It also reveals that a CDKL5-specific substrate is located at the presynapse, the phosphorylation of which is required for optimal SV endocytosis.

Control of synaptic vesicle release probability via VAMP4 targeting to endolysosomes.

Synaptic vesicle (SV) release probability (Pr), determines the steady state and plastic control of neurotransmitter release. However, how diversity in SV composition arises and regulates the Pr of individual SVs is not understood. We found that modulation of the copy number of the noncanonical vesicular SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor), vesicle-associated membrane protein 4 (VAMP4), on SVs is key for regulating Pr. Mechanistically, this is underpinned by its reduced ability to form an efficient SNARE complex with canonical plasma membrane SNAREs. VAMP4 has unusually high synaptic turnover and is selectively sorted to endolysosomes during activity-dependent bulk endocytosis. Disruption of endolysosomal trafficking and function markedly increased the abundance of VAMP4 in the SV pool and inhibited SV fusion. Together, our results unravel a new mechanism for generating SV heterogeneity and control of Pr through coupling of SV recycling to a major clearing system that regulates protein homeostasis.

Presynaptic dysfunction in neurodevelopmental disorders: Insights from the synaptic vesicle life cycle.

The activity-dependent fusion, retrieval and recycling of synaptic vesicles is essential for the maintenance of neurotransmission. Until relatively recently it was believed that most mutations in genes that were essential for this process would be incompatible with life, because of this fundamental role. However, an ever-expanding number of mutations in this very cohort of genes are being identified in individuals with neurodevelopmental disorders, including autism, intellectual disability and epilepsy. This article will summarize the current state of knowledge linking mutations in presynaptic genes to neurodevelopmental disorders by sequentially covering the various stages of the synaptic vesicle life cycle. It will also discuss how perturbations of specific stages within this recycling process could translate into human disease. Finally, it will also provide perspectives on the potential for future therapy that are targeted to presynaptic function.

Loss of Phosphatidylinositol 3-Kinase Activity in Regulatory T Cells Leads to Neuronal Inflammation.

Class I PI3K enzymes are critical for the maintenance of effective immunity. In T cells, PI3Kα and PI3Kδ are activated by the TCR and costimulatory receptors, whereas PI3Kγ is activated by G protein-coupled chemokine receptors. PI3Kδ is a key regulator of regulatory T (Treg) cell function. PI3K isoform-selective inhibitors are in development for the treatment of diseases associated with immune dysregulation, including chronic inflammatory conditions, cancer, and autoimmune diseases. Idelalisib (PI3Kδ), alpelisib (PI3Kα), duvelisib (PI3Kδ/γ), and copanlisib (pan-PI3K) have recently been approved for use in cancer treatment. Although effective, these therapies often have severe side effects associated with immune dysregulation and, in particular, loss of Treg cells. Therefore, it is important to gain a better understanding of the relative contribution of different PI3K isoforms under homeostatic and inflammatory conditions. Experimental autoimmune encephalitis is a mouse model of T cell-driven CNS inflammation, in which Treg cells play a key protective role. In this study, we show that PI3Kδ is required to maintain normal Treg cell development and phenotype under homeostatic conditions but that loss of PI3Kδ alone in Treg cells does not lead to autoimmunity. However, combined loss of PI3Kα and PI3Kδ signaling resulted in increased experimental autoimmune encephalitis disease severity. Moreover, mice lacking PI3Kα and PI3Kδ in Treg cells developed spontaneous peripheral nerve inflammation. These results show a key role for PI3K signaling in Treg cell-mediated protection against CNS inflammation.

An Epilepsy-Associated SV2A Mutation Disrupts Synaptotagmin-1 Expression and Activity-Dependent Trafficking.

The epilepsy-linked gene SV2A, has a number of potential roles in the synaptic vesicle (SV) life cycle. However, how loss of SV2A function translates into presynaptic dysfunction and ultimately seizure activity is still undetermined. In this study, we examined whether the first SV2A mutation identified in human disease (R383Q) could provide information regarding which SV2A-dependent events are critical in the translation to epilepsy. We utilized a molecular replacement strategy in which exogenous SV2A was expressed in mouse neuronal cultures of either sex, which had been depleted of endogenous SV2A to mimic the homozygous human condition. We found that the R383Q mutation resulted in a mislocalization of SV2A from SVs to the plasma membrane, but had no effect on its activity-dependent trafficking. This SV2A mutant displayed reduced mobility when stranded on the plasma membrane and reduced binding to its interaction partner synaptotagmin-1 (Syt1). Furthermore, the R383Q mutant failed to rescue reduced expression and dysfunctional activity-dependent trafficking of Syt1 in the absence of endogenous SV2A. This suggests that the inability to control Syt1 expression and trafficking at the presynapse may be key in the transition from loss of SV2A function to seizure activity.SIGNIFICANCE STATEMENT SV2A is a synaptic vesicle (SV) protein, the absence or dysfunction of which is linked to epilepsy. However, the series of molecular events that result in this neurological disorder is still undetermined. We demonstrate here that the first human mutation in SV2A identified in an individual with epilepsy displays reduced binding to synaptotagmin-1 (Syt1), an SV protein essential for synchronous neurotransmitter release. Furthermore, this mutant cannot correct alterations in both Syt1 expression and trafficking when expressed in the absence of endogenous SV2A (to mimic the homozygous human condition). This suggests that the inability to control Syt1 expression and trafficking may be key in the transition from loss of SV2A function to seizure activity.

Autism and Schizophrenia-Associated CYFIP1 Regulates the Balance of Synaptic Excitation and Inhibition.

Altered excitatory/inhibitory (E/I) balance is implicated in neuropsychiatric and neurodevelopmental disorders, but the underlying genetic etiology remains poorly understood. Copy number variations in CYFIP1 are associated with autism, schizophrenia, and intellectual disability, but its role in regulating synaptic inhibition or E/I balance remains unclear. We show that CYFIP1, and the paralog CYFIP2, are enriched at inhibitory postsynaptic sites. While CYFIP1 or CYFIP2 upregulation increases excitatory synapse number and the frequency of miniature excitatory postsynaptic currents (mEPSCs), it has the opposite effect at inhibitory synapses, decreasing their size and the amplitude of miniature inhibitory postsynaptic currents (mIPSCs). Contrary to CYFIP1 upregulation, its loss in vivo, upon conditional knockout in neocortical principal cells, increases expression of postsynaptic GABAA receptor ß2/3-subunits and neuroligin 3, enhancing synaptic inhibition. Thus, CYFIP1 dosage can bi-directionally impact inhibitory synaptic structure and function, potentially leading to altered E/I balance and circuit dysfunction in CYFIP1-associated neurological disorders.

An Essential Role for the Tetraspanin LHFPL4 in the Cell-Type-Specific Targeting and Clustering of Synaptic GABAA Receptors.

Inhibitory synaptic transmission requires the targeting and stabilization of GABAA receptors (GABAARs) at synapses. The mechanisms responsible remain poorly understood, and roles for transmembrane accessory proteins have not been established. Using molecular, imaging, and electrophysiological approaches, we identify the tetraspanin LHFPL4 as a critical regulator of postsynaptic GABAAR clustering in hippocampal pyramidal neurons. LHFPL4 interacts tightly with GABAAR subunits and is selectively enriched at inhibitory synapses. In LHFPL4 knockout mice, there is a dramatic cell-type-specific reduction in GABAAR and gephyrin clusters and an accumulation of large intracellular gephyrin aggregates in vivo. While GABAARs are still trafficked to the neuronal surface in pyramidal neurons, they are no longer localized at synapses, resulting in a profound loss of fast inhibitory postsynaptic currents. Hippocampal interneuron currents remain unaffected. Our results establish LHFPL4 as a synapse-specific tetraspanin essential for inhibitory synapse function and provide fresh insights into the molecular make-up of inhibitory synapses.

Multisite tyrosine phosphorylation of the N-terminus of Mint1/X11α by Src kinase regulates the trafficking of amyloid precursor protein.

Mint/X11 is one of the four neuronal trafficking adaptors that interact with amyloid precursor protein (APP) and are linked with its cleavage to generate ß-amyloid peptide, a key player in the pathology of Alzheimer's disease. How APP switches between adaptors at different stages of the secretory pathway is poorly understood. Here, we show that tyrosine phosphorylation of Mint1 regulates the destination of APP. A canonical SH2-binding motif ((202) YEEI) was identified in the N-terminus of Mint1 that is phosphorylated on tyrosine by C-Src and recruits the active kinase for sequential phosphorylation of further tyrosines (Y191 and Y187). A single Y202F mutation in the Mint1 N-terminus inhibits C-Src binding and tyrosine phosphorylation. Previous studies observed that co-expression of wild-type Mint1 and APP causes accumulation of APP in the trans-Golgi. Unphosphorylatable Mint1 (Y202F) or pharmacological inhibition of Src reduced the accumulation of APP in the trans-Golgi of heterologous cells. A similar result was observed in cultured rat hippocampal neurons where Mint1(Y202F) permitted the trafficking of APP to more distal neurites than the wild-type protein. These data underline the importance of the tyrosine phosphorylation of Mint1 as a critical switch for determining the destination of APP. The regulation of amyloid precursor protein (APP) trafficking is poorly understood. We have discovered that the APP adapter, Mint1, is phosphorylated by C-Src kinase. Mint1 causes APP accumulation in the trans-Golgi network, whereas inhibition of Src or mutation of Mint1-Y202 permits APP recycling. The phosphorylation status of Mint1 could impact on the pathological trafficking of APP in Alzheimer's disease.

GIT1 and ßPIX are essential for GABA(A) receptor synaptic stability and inhibitory neurotransmission.

Effective inhibitory synaptic transmission requires efficient stabilization of GABA(A) receptors (GABA(A)Rs) at synapses, which is essential for maintaining the correct excitatory-inhibitory balance in the brain. However, the signaling mechanisms that locally regulate synaptic GABA(A)R membrane dynamics remain poorly understood. Using a combination of molecular, imaging, and electrophysiological approaches, we delineate a GIT1/ßPIX/Rac1/PAK signaling pathway that modulates F-actin and is important for maintaining surface GABA(A)R levels, inhibitory synapse integrity, and synapse strength. We show that GIT1 and ßPIX are required for synaptic GABA(A)R surface stability through the activity of the GTPase Rac1 and downstream effector PAK. Manipulating this pathway using RNAi, dominant-negative and pharmacological approaches leads to a disruption of GABA(A)R clustering and decrease in the strength of synaptic inhibition. Thus, the GIT1/ßPIX/Rac1/PAK pathway plays a crucial role in regulating GABA(A)R synaptic stability and hence inhibitory synaptic transmission with important implications for inhibitory plasticity and information processing in the brain.