Presynaptic nerve terminals release neurotransmitters repeatedly, often at high frequency, and in relative isolation from neuronal cell bodies. Repeated release requires cycles of SNARE-complex assembly and disassembly, with continuous generation of reactive SNARE-protein intermediates. Although many forms of neurodegeneration initiate presynaptically, only few pathogenic mechanisms are known, and the functions of presynaptic proteins linked to neurodegeneration, such as α-synuclein, remain unclear. Here, we find that maintenance of continuous presynaptic SNARE-complex assembly required a non-classical chaperone activity mediated by synucleins. Specifically, α-synuclein directly bound to the SNARE-protein synaptobrevin-2/VAMP2, and promoted SNARE-complex assembly. Moreover, triple knockout mice lacking synucleins developed age-dependent neurological impairments, exhibited decreased SNARE-complex assembly, and perished prematurely. Thus, synucleins may function to sustain normal SNAREcomplex assembly in a presynaptic terminal during aging.In presynaptic terminals, neurotransmitter release requires a tightly coordinated membrane fusion machinery whose central components are soluble NSF attachment protein receptor (SNARE) and Sec1/Munc18-like proteins (1-3). Terminals release neurotransmitters thousands of times per minute; during each release reaction, SNARE-complex assembly and disassembly generates highly reactive unfolded SNARE protein intermediates, rendering the terminals potentially vulnerable to activity-dependent degeneration. Indeed, much evidence points to presynaptic terminals as an initiation site for neurodegeneration (4-6), and knockout (KO) of at least one presynaptic chaperone protein, cysteine string protein-α (CSPα), causes fulminant neurodegeneration in mice (7). Synucleins are abundant presynaptic proteins that are expressed from three genes (α-, β-and γ-synuclein; 8). α-Synuclein is involved in neurodegeneration (9-11), and γ-synuclein may contribute to progression of many types of cancer (12). Synucleins may modify neurotransmitter release (13,14), but their physiological functions remain unknown. Strikingly, transgenic expression of α-synuclein abolishes the lethal neurodegeneration induced by KO of CSPα, whereas deletion of endogenous synucleins accelerates this neurodegeneration (15). CSPα KO mice exhibit decreased levels of the SNARE protein SNAP-25 and impaired SNARE-complex assembly, suggesting a link between SNARE-complex assembly and neurodegeneration. α-* To whom correspondence should be addressed: tcs1@stanford.edu. HHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptSynuclein rescues SNARE-complex assembly but not SNAP-25 levels (15). This result indicates that α-synuclein may enhance SNARE-protein function, and thereby compensate for the CSPα deletion. To address this hypothesis, we here examine the role of α-synuclein in SNARE-complex assembly and in the maintenance of continuous SNARE-cycling in presynaptic terminals over the lifetime of an animal.We imm...

Hippocampal CA1 region neurons specifically target latrophilin-2 (Lphn2), an adhesion-type GPCR, to dendritic spines in the stratum lacunosum-moleculare. In this study, Lphn2 controls assembly of excitatory synapses formed by presynaptic entorhinal cortex afferents but not by Schaffer-collateral afferents, suggesting a synaptic recognition function.

Selective serotonin reuptake inhibitors (SSRIs) are widely used antidepressants, but the mechanisms by which they influence behavior are only partially resolved. Adult hippocampal neurogenesis is necessary for some of the responses to SSRIs, but it is unknown whether the mature dentate gyrus granule cells (mature DG GCs) also contribute. We deleted Serotonin 1A receptor (5HT1AR; a receptor required for the SSRI response) specifically from DG GCs and found that the effects of the SSRI fluoxetine on behavior and the Hypothalamic-Pituitary-Adrenal (HPA) axis were abolished. By contrast, mice lacking 5HT1ARs only in young adult born granule cells (abGCs) showed normal fluoxetine responses. Importantly, 5HT1AR deficient mice engineered to express functional 5HT1ARs only in DG GCs responded to fluoxetine, indicating that 5HT1ARs in DG GCs are sufficient to mediate an antidepressant response. Taken together, these data indicate that both mature DG GCs and young abGCs must be engaged for an antidepressant response.

Serotonin receptor 1A knockout (Htr1a(KO)) mice show increased anxiety-related behavior in tests measuring innate avoidance. Here we demonstrate that Htr1a(KO) mice show enhanced fear conditioning to ambiguous conditioned stimuli, a hallmark of human anxiety. To examine the involvement of specific forebrain circuits in this phenotype, we developed a pharmacogenetic technique for the rapid tissue- and cell type-specific silencing of neural activity in vivo. Inhibition of neurons in the central nucleus of the amygdala suppressed conditioned responses to both ambiguous and nonambiguous cues. In contrast, inhibition of hippocampal dentate gyrus granule cells selectively suppressed conditioned responses to ambiguous cues and reversed the knockout phenotype. These data demonstrate that Htr1a(KO) mice have a bias in the processing of threatening cues that is moderated by hippocampal mossy-fiber circuits, and suggest that the hippocampus is important in the response to ambiguous aversive stimuli.

Neurexins are well-characterized presynaptic cell adhesion molecules that engage multifarious postsynaptic ligands and organize diverse synapse properties. However, the precise synaptic localization of neurexins remains enigmatic. Using super-resolution microscopy, we demonstrate that neurexin-1 forms discrete nanoclusters at excitatory synapses, revealing a novel organizational feature of synaptic architecture. Synapses generally contain a single nanocluster that comprises more than four neurexin-1 molecules and that also includes neurexin-2 and/or neurexin-3 isoforms. Moreover, we find that neurexin-1 is physiologically cleaved by ADAM10 similar to its ligand neuroligin-1, with ∼4–6% of neurexin-1 and ∼2–3% of neuroligin-1 present in the adult brain as soluble ectodomain proteins. Blocking ADAM10-mediated neurexin-1 cleavage dramatically increased the synaptic neurexin-1 content, thereby elevating the percentage of Homer1(+) excitatory synapses containing neurexin-1 nanoclusters from 40–50% to ∼80%, and doubling the number of neurexin-1 molecules per nanocluster. Taken together, our results reveal an unexpected nanodomain organization of synapses in which neurexin-1 is assembled into discrete presynaptic nanoclusters that are dynamically regulated via ectodomain cleavage.

Dopamine release during reward-driven behaviors influences synaptic plasticity. However, dopamine innervation and release in the hippocampus and its role during aversive behaviors are controversial. Here we show that in vivo hippocampal synaptic plasticity in the CA3-CA1 circuit underlies contextual learning during inhibitory avoidance (IA) training. Immunohistochemistry and molecular techniques verified sparse dopaminergic innervation of the hippocampus from the midbrain. The long-term synaptic potentiation (LTP) underlying the learning of IA was assessed with a D1-like dopamine receptor agonist or antagonist in ex vivo hippocampal slices and in vivo in freely-moving mice. Inhibition of D1-like dopamine receptors impaired memory of the IA task and prevented the training-induced enhancement of both ex vivo and in vivo LTP induction. The results indicate that dopamine-receptor signaling during an aversive contextual task regulates aversive memory retention and regulates associated synaptic mechanisms in the hippocampus that likely underlie learning.

The central nucleus of the amygdala (CeA) serves as a major output of this structure and plays a critical role in the expression of conditioned fear. By combining cell- and tissue-specific pharmacogenetic inhibition with functional magnetic resonance imaging (fMRI), we identified circuits downstream of CeA that control fear expression in mice. Selective inhibition of a subset of neurons in CeA led to decreased conditioned freezing behavior and increased cortical arousal as visualized by fMRI. Correlation analysis of fMRI signals identified functional connectivity between CeA, cholinergic forebrain nuclei, and activated cortical structures, and cortical arousal was blocked by cholinergic antagonists. Importantly, inhibition of these neurons switched behavioral responses to the fear stimulus from passive to active responses. Our findings identify a neural circuit in CeA that biases fear responses toward either passive or active coping strategies.

Human kallikrein 6 (protease M/zyme/neurosin) is a serine protease that has been suggested to be a serum biomarker for ovarian cancer and may also be involved in pathologies of the CNS. The precursor form of human kallikrein 6 (pro-hK6) was overexpressed in Pichia pastoris and found to be autoprocessed to an active but unstable mature enzyme that subsequently yielded the inactive, self-cleavage product, hK6 (D81-K244). Site-directed mutagenesis was used to investigate the basis for the intrinsic catalytic activity and the activation mechanism of pro-hK6. A single substitution R80 --> Q stabilized the activity of the mature enzyme, while substitution of the active site serine (S197 --> A) resulted in complete loss of hK6 proteolytic activity and facilitated protein production. Our data suggest that the enzymatic activity of hK6 is regulated by an autoactivation/autoinactivation mechanism. Mature hK6 displayed a trypsin-like activity against synthetic substrates and human plasminogen was identified as a putative physiological substrate for hK6, as specific cleavage at the plasminogen internal bond S460-V461 resulted in the generation of angiostatin, an endogenous inhibitor of angiogenesis and metastatic growth.