Peter Bronk

Drosophila Hsc70-4 Is Critical for Neurotransmitter Exocytosis In Vivo

Previous in vitro studies of cysteine-string protein (CSP) imply a potential role for the clathrin-uncoating ATPase Hsc70 in exocytosis. We show that hypomorphic mutations in Drosophila Hsc70-4 (Hsc4) impair nerve-evoked neurotransmitter release, but not synaptic vesicle recycling in vivo. The loss of release can be restored by increasing external or internal Ca(2+) and is caused by a reduced Ca(2+) sensitivity of exocytosis downstream of Ca(2+) entry. Hsc4 and CSP are likely to act in common pathways, as indicated by their in vitro protein interaction, the similar loss of evoked release in individual and double mutants, and genetic interactions causing a loss of release in trans-heterozygous hsc4-csp double mutants. We suggest that Hsc4 and CSP cooperatively augment the probability of release by increasing the Ca(2+) sensitivity of vesicle fusion.

Molecular chaperones and the regulation of neurotransmitter exocytosis.

Regulated neurotransmitter release depends on a precise sequence of events that lead to repeated cycles of exocytosis and endocytosis. These events are mediated by a series of molecular interactions among vesicular, plasma membrane, and cytosolic proteins. An emerging theme has been that molecular chaperones may guide the sequential restructuring of stable or transient protein complexes to promote a temporal and spatial regulation of the endo- and exocytotic machinery and to ensure a vectorial passage through the vesicle cycle. Chaperones, specialized for a few substrates, are ideally suited to participate in regulatory processes that require some molecular dexterity to rearrange conformational or oligomeric protein structures. This article emphasizes the significance of three molecular chaperone systems in regulated neurotransmitter release: the regulation of soluble NSF attachment protein receptor (SNARE) complexes by N-ethylmaleimide-sensitive factor (NSF) and the soluble NSF attachment protein (SNAP), the uncoating of clathrin-coated vesicles by the 70 kDa heat-shock cognate protein (Hsc70), and the regulation of SNARE complex-associated protein interactions by cysteine-string protein and Hsc70.

CSPα knockout causes neurodegeneration by impairing SNAP-25 function.

At a synapse, the synaptic vesicle protein cysteine‐string protein‐α (CSPα) functions as a co‐chaperone for the SNARE protein SNAP‐25. Knockout (KO) of CSPα causes fulminant neurodegeneration that is rescued by α‐synuclein overexpression. The CSPα KO decreases SNAP‐25 levels and impairs SNARE‐complex assembly; only the latter but not the former is reversed by α‐synuclein. Thus, the question arises whether the CSPα KO phenotype is due to decreased SNAP‐25 function that then causes neurodegeneration, or due to the dysfunction of multiple as‐yet uncharacterized CSPα targets. Here, we demonstrate that decreasing SNAP‐25 levels in CSPα KO mice by either KO or knockdown of SNAP‐25 aggravated their phenotype. Conversely, increasing SNAP‐25 levels by overexpression rescued their phenotype. Inactive SNAP‐25 mutants were unable to rescue, showing that the rescue was specific. Under all conditions, the neurodegenerative phenotype precisely correlated with SNARE‐complex assembly, indicating that impaired SNARE‐complex assembly due to decreased SNAP‐25 levels is the ultimate correlate of neurodegeneration. Our findings suggest that the neurodegeneration in CSPα KO mice is primarily produced by defective SNAP‐25 function, which causes neurodegeneration by impairing SNARE‐complex assembly.

Presynaptic Regulation of Neurotransmission in Drosophila by the G Protein-Coupled Receptor Methuselah

Regulation of synaptic strength is essential for neuronal information processing, but the molecular mechanisms that control changes in neuroexocytosis are only partially known. Here we show that the putative G protein-coupled receptor Methuselah (Mth) is required in the presynaptic motor neuron to acutely upregulate neurotransmitter exocytosis at larval Drosophila NMJs. Mutations in the mth gene reduce evoked neurotransmitter release by approximately 50%, and decrease synaptic area and the density of docked and clustered vesicles. Pre- but not postsynaptic expression of normal Mth restored normal release in mth mutants. Conditional expression of Mth restored normal release and normal vesicle docking and clustering but not the reduced size of synaptic sites, suggesting that Mth acutely adjusts vesicle trafficking to synaptic sites.

The Multiple Functions of Cysteine-String Protein Analyzed at Drosophila Nerve Terminals

The synaptic vesicle-associated cysteine-string protein (CSP) is important for synaptic transmission. Previous studies revealed multiple defects at neuromuscular junctions (NMJs) of csp null-mutant Drosophila, but whether these defects are independent of each other or mechanistically linked through J domain mediated-interactions with heat-shock cognate protein 70 (Hsc70) has not been established. To resolve this issue, we genetically dissected the individual functions of CSP by an in vivo structure/function analysis. Expression of mutant CSP lacking the J domain at csp null-mutant NMJs fully restored normal thermo-tolerance of evoked transmitter release but did not completely restore evoked release at room temperature and failed to reverse the abnormal intraterminal Ca2+ levels. This suggests that J domain-mediated functions are essential for the regulation of intraterminal Ca2+ levels but only partially required for regulating evoked release and not required for protecting evoked release against thermal stress. Hence, CSP can also act as an Hsc70-independent chaperone protecting evoked release from thermal stress. Expression of mutant CSP lacking the L domain restored neurotransmission and partially reversed the abnormal intraterminal Ca2+ levels, suggesting that the L domain is important, although not essential, for the role of CSP in regulating intraterminal Ca2+ levels. We detected no effects of csp mutations on individual presynaptic Ca2+ signals triggered by action potentials, suggesting that presynaptic Ca2+ entry is not primarily impaired. Both the J and L domains were also required for the role of CSP in synaptic growth. Together, these results suggest that CSP has several independent synaptic functions, affecting synaptic growth, evoked release, thermal protection of evoked release, and intraterminal Ca2+ levels at rest and during stimulation.