Intersectin associates with synapsin and regulates its nanoscale localization and function.

Neurotransmission is mediated by the exocytic release of neurotransmitters from readily releasable synaptic vesicles (SVs) at the active zone. To sustain neurotransmission during periods of elevated activity, release-ready vesicles need to be replenished from the reserve pool of SVs. The SV-associated synapsins are crucial for maintaining this reserve pool and regulate the mobilization of reserve pool SVs. How replenishment of release-ready SVs from the reserve pool is regulated and which other factors cooperate with synapsins in this process is unknown. Here we identify the endocytic multidomain scaffold protein intersectin as an important regulator of SV replenishment at hippocampal synapses. We found that intersectin directly associates with synapsin I through its Src-homology 3 A domain, and this association is regulated by an intramolecular switch within intersectin 1. Deletion of intersectin 1/2 in mice alters the presynaptic nanoscale distribution of synapsin I and causes defects in sustained neurotransmission due to defective SV replenishment. These phenotypes were rescued by wild-type intersectin 1 but not by a locked mutant of intersectin 1. Our data reveal intersectin as an autoinhibited scaffold that serves as a molecular linker between the synapsin-dependent reserve pool and the presynaptic endocytosis machinery

Neuronal autophagy regulates presynaptic neurotransmission by controlling the axonal endoplasmic reticulum

Neurons are known to rely on autophagy for removal of defective proteins or organelles to maintain synaptic neurotransmission and counteract neurodegeneration. In spite of its importance for neuronal health, the physiological substrates of neuronal autophagy in the absence of proteotoxic challenge have remained largely elusive. We use knockout mice conditionally lacking the essential autophagy protein ATG5 and quantitative proteomics to demonstrate that loss of neuronal autophagy causes selective accumulation of tubular endoplasmic reticulum (ER) in axons, resulting in increased excitatory neurotransmission and compromised postnatal viability in vivo. The gain in excitatory neurotransmission is shown to be a consequence of elevated calcium release from ER stores via ryanodine receptors accumulated in axons and at presynaptic sites. We propose a model where neuronal autophagy controls axonal ER calcium stores to regulate neurotransmission in healthy neurons and in the brain

Spermidine protects from age-related synaptic alterations at hippocampal mossy fiber-CA3 synapses

Aging is associated with functional alterations of synapses thought to contribute to age-dependent memory impairment (AMI). While therapeutic avenues to protect from AMI are largely elusive, supplementation of spermidine, a polyamine normally declining with age, has been shown to restore defective proteostasis and to protect from AMI in Drosophila. Here we demonstrate that dietary spermidine protects from age-related synaptic alterations at hippocampal mossy fiber (MF)-CA3 synapses and prevents the aging-induced loss of neuronal mitochondria. Dietary spermidine rescued age-dependent decreases in synaptic vesicle density and largely restored defective presynaptic MF-CA3 long-term potentiation (LTP) at MF-CA3 synapses (MF-CA3) in aged animals. In contrast, spermidine failed to protect CA3-CA1 hippocampal synapses characterized by postsynaptic LTP from age-related changes in function and morphology. Our data demonstrate that dietary spermidine attenuates age-associated deterioration of MF-CA3 synaptic transmission and plasticity. These findings provide a physiological and molecular basis for the future therapeutic usage of spermidine

Defective lipid signalling caused by mutations in PIK3C2B underlies focal epilepsy

Epilepsy is one of the most frequent neurological diseases, with focal epilepsy accounting for the largest number of cases. The genetic alterations involved in focal epilepsy are far from being fully elucidated. Here, we show that defective lipid signalling caused by heterozygous ultra-rare variants in PIK3C2B, encoding for the class II phosphatidylinositol 3-kinase PI3K-C2β, underlie focal epilepsy in humans. We demonstrate that patients' variants act as loss-of-function alleles, leading to impaired synthesis of the rare signalling lipid phosphatidylinositol 3,4-bisphosphate, resulting in mTORC1 hyperactivation. In vivo, mutant Pik3c2b alleles caused dose-dependent neuronal hyperexcitability and increased seizure susceptibility, indicating haploinsufficiency as a key driver of disease. Moreover, acute mTORC1 inhibition in mutant mice prevented experimentally induced seizures, providing a potential therapeutic option for a selective group of patients with focal epilepsy. Our findings reveal an unexpected role for class II PI3K-mediated lipid signalling in regulating mTORC1-dependent neuronal excitability in mice and humans

Selective endocytosis of Ca(2+)-permeable AMPARs by the Alzheimer's disease risk factor CALM bidirectionally controls synaptic plasticity

AMPA-type glutamate receptors (AMPARs) mediate fast excitatory neurotransmission, and the plastic modulation of their surface levels determines synaptic strength. AMPARs of different subunit compositions fulfill distinct roles in synaptic long-term potentiation (LTP) and depression (LTD) to enable learning. Largely unknown endocytic mechanisms mediate the subunit-selective regulation of the surface levels of GluA1-homomeric Ca(2+)-permeable (CP) versus heteromeric Ca(2+)-impermeable (CI) AMPARs. Here, we report that the Alzheimer's disease risk factor CALM controls the surface levels of CP-AMPARs and thereby reciprocally regulates LTP and LTD in vivo to modulate learning. We show that CALM selectively facilitates the endocytosis of ubiquitinated CP-AMPARs via a mechanism that depends on ubiquitin recognition by its ANTH domain but is independent of clathrin. Our data identify CALM and related ANTH domain-containing proteins as the core endocytic machinery that determines the surface levels of CP-AMPARs to bidirectionally control synaptic plasticity and modulate learning in the mammalian brain

Sensory Experience Differentially Modulates the mRNA Expression of the Polysialyltransferases ST8SiaII and ST8SiaIV in Postnatal Mouse Visual Cortex

Polysialic acid (PSA) is a unique carbohydrate composed of a linear homopolymer of α-2,8 linked sialic acid, and is mainly attached to the fifth immunoglobulin-like domain of the neural cell adhesion molecule (NCAM) in vertebrate neural system. In the brain, PSA is exclusively synthesized by the two polysialyltransferases ST8SiaII (also known as STX) and ST8SiaIV (also known as PST). By modulating adhesive property of NCAM, PSA plays a critical role in several neural development processes such as cell migration, neurite outgrowth, axon pathfinding, synaptogenesis and activity-dependent plasticity. The expression of PSA is temporally and spatially regulated during neural development and a tight regulation of PSA expression is essential to its biological function. In mouse visual cortex, PSA is downregulated following eye opening and its decrease allows the maturation of GABAergic synapses and the opening of the critical period for ocular dominance plasticity. Relatively little is known about how PSA levels are regulated by sensory experience and neuronal activity. Here, we demonstrate that while both ST8SiaII and ST8SiaIV mRNA levels decrease around the time of eye opening in mouse visual cortex, only ST8SiaII mRNA level reduction is regulated by sensory experience. Using an organotypic culture system from mouse visual cortex, we further show that ST8SiaII gene expression is regulated by spiking activity and NMDA-mediated excitation. Further, we show that both ST8SiaII and ST8SiaIV mRNA levels are positively regulated by PKC-mediated signaling. Therefore, sensory experience-dependent ST8SiaII gene expression regulates PSA levels in postnatal visual cortex, thus acting as molecular link between visual activity and PSA expression

Hyaluronan Export through Plasma Membranes Depends on Concurrent K+ Efflux by Kir Channels

Hyaluronan is synthesized within the cytoplasm and exported into the extracellular matrix through the cell membrane of fibroblasts by the MRP5 transporter. In order to meet the law of electroneutrality, a cation is required to neutralize the emerging negative hyaluronan charges. As we previously observed an inhibiting of hyaluronan export by inhibitors of K+ channels, hyaluronan export was now analysed by simultaneously measuring membrane potential in the presence of drugs. This was done by both hyaluronan import into inside-out vesicles and by inhibition with antisense siRNA. Hyaluronan export from fibroblast was particularly inhibited by glibenclamide, ropivacain and BaCl2 which all belong to ATP-sensitive inwardly-rectifying Kir channel inhibitors. Import of hyaluronan into vesicles was activated by 150 mM KCl and this activation was abolished by ATP. siRNA for the K+ channels Kir3.4 and Kir6.2 inhibited hyaluronan export. Collectively, these results indicated that hyaluronan export depends on concurrent K+ efflux

FGF receptor-mediated tyrosine phosphorylation of DHHC palmitoyltransferases: a mechanism to regulate the levels of NCAM palmitoylation?

Protein palmitoylation, the common posttranslational modification with the lipid palmitate, plays a pivotal role in protein trafficking and function. Our previous studies revealed that activation of fibroblast growth factor (FGF) receptor(s) by FGF2 leads to palmitoylation of the neural cell adhesion molecule (NCAM), its translocation to GM1 ganglioside-enriched lipid rafts, and stimulation of neurite outgrowth of cultured hippocampal neurons (1). Now, we used the Acyl-Biotin-Exchange (ABE) method and show a 1.3-fold increase in NCAM palmitoylation in mice injected with FGF2 in vivo. Furthermore, we analyzed the mechanisms of this regulation. Two members of the DHHC family, DHHC3 and DHHC7, were identified to mediate palmitoylation of NCAM in heterologous N2a cells (1). Because FGF:FGFR interaction leads to the activation of the canonical Ras/MAPK pathway and to the recruitment and activation of PLC\u3b3 and Src family tyrosine kinases (2), we investigated the tyrosine phosphorylation of DHHCs. Here, we present data showing that DHHC3 is highly tyrosine phosphorylated by activated FGFR1, suggesting a potential role of DHHC3 phosphorylation for regulation of its palmitoylating activity. We also report that treatment with the Src inhibitor PP2 significantly reduces DHHC3 tyrosine phosphorylation. Based on this evidence, we performed co-immunoprecipitation assays and found that Src but not FGFR1 is pulled-down by DHHC3, suggesting a possible direct interaction between the two molecules. Furthermore, DHHC3 isolated from brain homogenates appeared tyrosine phosphorylated, thus confirming the data obtained in the cell lines. Ongoing mutagenesis experiments aim to identify DHHC3 regulatory tyrosines. The generated DHHC3 mutants are assayed with the non-radioactive Click-IT palmitoylation assay that we specifically developed for NCAM. In summary, we demonstrated the FGFR-dependent phosphorylation of DHHC proteins, which might regulate palmitoylation activities of these enzymes towards to their substrates, including NCAM

Role of DHHC3 tyrosine phosphorylation in the control of its expression and functional activity

S-palmitoylation is a postranslational addition of palmitate, a 16-carbon saturated fatty acid to cysteine residues through a labile thioester linkage, which is catalyzed by protein acyl transferases (PAT). Recently the zinc finger DHHC (Asp-His-His-Cys) type-containing protein family has emerged as a large family of palmitoyl acyltransferases with 23 members in the mouse and human genomes. Among them DHHC3 (also known as Golgi-specific DHHC zinc finger protein, GODZ) was shown as a relatively promiscuous palmitoyltransferase, which palmitoylate a broad range of substrates. These substrates includes signaling proteins (as several G protein \u3b1 subunits), synaptic vesicle proteins (as SNAP-25 and cysteine-signaling protein), scaffold proteins (PSD-95), ion channels (\u3b32 subunit of GABAA receptor; GluR1 and 2) and cell adhesion molecules (as \u3b16 and \u3b24 integrin subunits and NCAM). Palmitoylation of neurospecific substrates of DHHC3 was shown to be important for synaptic function, plasticity, neuronal migration and maturation of neurons. Considering this, lack or gain of DHHC3 function could be a key point in progression of neurodegenerative diseases. So the exploration of regulation of DHHC3 activity is of the particular interest. Tyrosine phosphorylation is one of the most common regulatory posttranslational modifications and turns many protein enzymes on and off, thereby altering their function and activity. DHHC3 has several tyrosines which are potential sites for src and FGFR mediated phosphorylation. First we have shown that WT DHHC3 cotransfected in N2a cells with src or FGFR1 became highly tyrosine phosphorylated. Treatment of the cells with PP2 \u2013 a selective src inhibitor - reduces the phosphorylation of DHHC3, which is even more decreased if PP2 is applied together with FGFR inhibitor PD 173074. The DHHC3 Y-F mutant in which all 5 tyrosines (Y) at the cytoplasmic domains were mutated to phenylalanines (F) shows tyrosine phosphorylation neither under basal conditions nor in response to src or FGFR1 overexpression. Generating single and triple Y-F mutants (two sites 295, 297 on the C-terminus of the protein were mutated together) we were able to dissect the contribution of each site to the basal level of tyrosine phosphorylation and also to src or FGFR1 mediated hyperphosphorylation. Two tyrosines 295, 297 at the C-teminus of DHHC3 are responsible for activation directly by src-mediated pathway, while tyrosine located at the N-terminus of the protein in the position 18 is important for phosphorylation in response to signaling downstream of FGFR1. Interestingly, tyrosines 295, 297 contribute probably also to stability of DHHC3, since mutants lacking this site are expressed at significantly lower level than WT. It appeared that DHHC3tyrosine phosphorylation interferes with its autopalmitoylation: the full DHHC3Y-F mutant is 2 times more palmitoylated than the WT. The autopalmitoylation gives insight into understanding the catalytically function of the enzyme, because in accordance with the two-step ping-pong theory autopalmitoylation is an intermediate step in transferring the palmitate to the protein substrate. Hence, our ongoing study aims to verify if a lack of tyrosine phosphorylation of DHHC3 would affect its activity towards its substrates and play a role in the neuronal development and plasticity