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Neuroinflammation is involved in the pathogenesis of several neurologic disorders, including epilepsy. Both changes in the input/output functions of synaptic circuits and cell Ca2+ dysregulation participate in neuroinflammation, but their impact on neuron function in epilepsy is still poorly understood. Lipopolysaccharide (LPS), a toxic byproduct of bacterial lysis, has been extensively used to stimulate inflammatory responses both in vivo and in vitro. LPS stimulates Toll-like receptor 4, an important mediator of the brain innate immune response that contributes to neuroinflammation processes. Although we report that Toll-like receptor 4 is expressed in both excitatory and inhibitory mouse hippocampal neurons (both sexes), its chronic stimulation by LPS induces a selective increase in the excitatory synaptic strength, characterized by enhanced synchronous and asynchronous glutamate release mechanisms. This effect is accompanied by a change in short-term plasticity with decreased facilitation, decreased post-tetanic potentiation, and increased depression. Quantal analysis demonstrated that the effects of LPS on excitatory transmission are attributable to an increase in the probability of release associated with an overall increased expression of L-type voltage-gated Ca2+ channels that, at presynaptic terminals, abnormally contributes to evoked glutamate release. Overall, these changes contribute to the excitatory/inhibitory imbalance that scales up neuronal network activity under inflammatory conditions. These results provide new molecular clues for treating hyperexcitability of hippocampal circuits associated with neuroinflammation in epilepsy and other neurologic disorders. [ABSTRACT FROM AUTHOR]

Synapsins (Syns) are synaptic vesicle (SV)-associated proteins involved in the regulation of synaptic transmission and plasticity, which display a highly conserved ATP binding site in the central C-domain, whose functional role is unknown. Using molecular dynamics simulations, we demonstrated that ATP binding to SynI is mediated by a conformational transition of a flexible loop that opens to make the binding site accessible; such transition, prevented in the K269Q mutant, is not significantly affected in the absence of Ca2+ or by the E373K mutation that abolishes Ca2+-binding. Indeed, the ATP binding to SynI also occurred under Ca2+-free conditions and increased its association with purified rat SVs regardless of the presence of Ca2+and promoted SynI oligomerization. However, although under Ca2+-free conditions, SynI dimerization andSVclustering were enhanced, Ca2+favored the formation of tetramers at the expense of dimers and did not affect SV clustering, indicating a role of Ca2+-dependent dimer/tetramer transitions in the regulation of ATP-dependent SV clustering. To elucidate the role of ATP/SynI binding in synaptic physiology, mouse SynI knock-out hippocampal neurons were transduced with either wild-type or K269Q mutant SynI and inhibitory transmission was studied by patch-clamp and electron microscopy. K269Q-SynI expressing inhibitory synapses showed increased synaptic strength due to an increase in the release probability, an increased vulnerability to synaptic depression and a dysregulation of SV trafficking, when compared with wild-type SynI-expressing terminals. The results suggest that the ATP-SynI binding plays predocking and postdocking roles in the modulation of SV clustering and plasticity of inhibitory synapses. [ABSTRACT FROM AUTHOR]

Cyclin-dependentkinase-5(Cdk5)wasreported todownscaleneurotransmissionbysequestering synaptic vesicles(SVs)in the release-reluctant resting pool, but the molecular targets mediating this activity remain unknown. Synapsin I (SynI), a major SV phosphoprotein involved in the regulation ofSVtrafficking and neurotransmitter release, is one of the presynaptic substrates of Cdk5, which phosphorylates it in its C-terminal region at Ser549 (site 6)andSer551 (site 7).Herewedemonstrate thatCdk5phosphorylation of SynI fine tunes the recruitment of SVsto the active recycling pool and contributes to the Cdk5-mediated homeostatic responses. Phosphorylation of SynI by Cdk5 is physiologically regulated and enhancesits binding to F-actin. The effects ofCdk5inhibitiononthe sizeanddepletion kinetics of the recycling pool, as well asonSVdistribution within the nerve terminal, are virtually abolished in mouse SynI knock-out (KO) neurons or in KO neurons expressing the dephosphomimetic SynI mutants at sites 6,7 or site 7 only. The observation that the single site-7 mutant phenocopies the effects of the deletion of SynI identifies this site as the central switch in mediating the synaptic effects ofCdk5anddemonstrates that SynI is necessaryandsufficient for achieving the effects of the kinase on SV trafficking. The phosphorylation state of SynI by Cdk5 at site 7 is regulated during chronic modification of neuronal activity and is an essential downstream effector for the Cdk5-mediated homeostatic scaling. [ABSTRACT FROM AUTHOR]

Sustained neurotransmitter release at synapses during high-frequency synaptic activity involves the mobilization of synaptic vesicles (SVs) from the tightly clustered reserve pool (RP). Synapsin I (Syn I), a brain-specific peripheral membrane protein that undergoes activity-dependent cycles of SV association and dissociation, is implicated in RP organization via its ability to cluster SVs. Although Syn I has affinity for phospholipids, the mechanism for the reversible association of synapsin with SV membranes remains enigmatic. Here, we show that rat Syn I is able to sense membrane curvature via an evolutionary conserved amphipathic lipid packing sensor motif (ALPS). Deletion or mutational inactivation of the ALPS impairs the ability of Syn I to associate with highly curved membranes and with SVs. Furthermore, a Syn I mutant lacking ALPS displays defects in its ability to undergo activity-induced cycles of dispersion and reclustering in neurons and fails to induce vesicle clustering in vitro. Our data suggest a crucial role for ALPS-mediated sensing of membrane curvature in regulating synapsin function. [ABSTRACT FROM AUTHOR]