A role for an Hsp70 nucleotide exchange factor in the regulation of synaptic vesicle endocytosis.

Neurotransmission requires a continuously available pool of synaptic vesicles (SVs) that can fuse with the plasma membrane and release their neurotransmitter contents upon stimulation. After fusion, SV membranes and membrane proteins are retrieved from the presynaptic plasma membrane by clathrin-mediated endocytosis. After the internalization of a clathrin-coated vesicle, the vesicle must uncoat to replenish the pool of SVs. Clathrin-coated vesicle uncoating requires ATP and is mediated by the ubiquitous molecular chaperone Hsc70. In vitro, depolymerized clathrin forms a stable complex with Hsc70*ADP. This complex can be dissociated by nucleotide exchange factors (NEFs) that release ADP from Hsc70, allowing ATP to bind and induce disruption of the clathrin:Hsc70 association. Whether NEFs generally play similar roles in vesicle trafficking in vivo and whether they play such roles in SV endocytosis in particular is unknown. To address this question, we used information from recent structural and mechanistic studies of Hsp70:NEF and Hsp70:co-chaperone interactions to design a NEF inhibitor. Using acute perturbations at giant reticulospinal synapses of the sea lamprey (Petromyzon marinus), we found that this NEF inhibitor inhibited SV endocytosis. When this inhibitor was mutated so that it could no longer bind and inhibit Hsp110 (a NEF that we find to be highly abundant in brain cytosol), its ability to inhibit SV endocytosis was eliminated. These observations indicate that the action of a NEF, most likely Hsp110, is normally required during SV trafficking to release clathrin from Hsc70 and make it available for additional rounds of endocytosis.

Acute increase of α-synuclein inhibits synaptic vesicle recycling evoked during intense stimulation.

Parkinson's disease is associated with multiplication of the α-synuclein gene and abnormal accumulation of the protein. In animal models, α-synuclein overexpression broadly impairs synaptic vesicle trafficking. However, the exact steps of the vesicle trafficking pathway affected by excess α-synuclein and the underlying molecular mechanisms remain unknown. Therefore we acutely increased synuclein levels at a vertebrate synapse and performed a detailed ultrastructural analysis of the effects on presynaptic membranes. At stimulated synapses (20 Hz), excess synuclein caused a loss of synaptic vesicles and an expansion of the plasma membrane, indicating an impairment of vesicle recycling. The N-terminal domain (NTD) of synuclein, which folds into an α-helix, was sufficient to reproduce these effects. In contrast, α-synuclein mutants with a disrupted N-terminal α-helix (T6K and A30P) had little effect under identical conditions. Further supporting this model, another α-synuclein mutant (A53T) with a properly folded NTD phenocopied the synaptic vesicle recycling defects observed with wild type. Interestingly, the vesicle recycling defects were not observed when the stimulation frequency was reduced (5 Hz). Thus excess α-synuclein impairs synaptic vesicle recycling evoked during intense stimulation via a mechanism that requires a properly folded N-terminal α-helix.

Increased synapsin expression and neurite sprouting in lamprey brain after spinal cord injury.

Spinal cord injury induces structural plasticity throughout the mammalian nervous system, including distant locations in the brain. Several types of injury-induced plasticity have been identified, such as neurite sprouting, axon regeneration, and synaptic remodeling. However, the molecular mechanisms involved in injury-induced plasticity are unclear as is the extent to which injury-induced plasticity in brain is conserved across vertebrate lineages. Due to its robust roles in neurite outgrowth and synapse formation during developmental processes, we examined synapsin for its potential involvement in injury-induced plasticity. We used lamprey, a vertebrate that undergoes robust anatomical plasticity and functional recovery after spinal cord injury. At 3 and 11 weeks after spinal cord transection, synapsin I mRNA was upregulated >2-fold in lamprey brain, as assayed by semi-quantitative RT-PCR. Other synaptic vesicle-associated genes remained unchanged. In situ hybridization revealed that synapsin I mRNA was increased globally throughout the lamprey brain. Immunolabeling for synapsin I protein revealed a significant increase in both the intensity and density of synapsin I-positive structures in lamprey hindbrain at 11 weeks post-transection, relative to controls. Moreover, the number of structures immunolabeled for phospho-synapsin (serine 9) increased after injury, suggestive of neurite sprouting. Indeed, at the ultrastructural level, there was an increase in neurite density at 11 weeks post-transection. Taken together, these data show that neurite sprouting in the brain is an evolutionarily conserved response to a distant spinal cord injury and suggest that synapsin and its phosphorylation at serine 9 play key roles in the sprouting mechanism.

Regenerated synapses in lamprey spinal cord are sparse and small even after functional recovery from injury.

Despite the potential importance that synapse regeneration plays in restoring neuronal function after spinal cord injury (SCI), even the most basic questions about the morphology of regenerated synapses remain unanswered. Therefore, we set out to gain a better understanding of central synapse regeneration by examining the number, distribution, molecular composition, and ultrastructure of regenerated synapses under conditions in which behavioral recovery from SCI was robust. To do so, we used the giant reticulospinal (RS) neurons of lamprey spinal cord because they readily regenerate, are easily identifiable, and contain large synapses that serve as a classic model for vertebrate excitatory neurotransmission. Using a combination of light and electron microscopy, we found that regenerated giant RS synapses regained the basic structures and presynaptic organization observed at control giant RS synapses at a time when behavioral recovery was nearly complete. However, several obvious differences remained. Most strikingly, regenerated giant RS axons produced very few synapses. In addition, presynaptic sites within regenerated axons were less complex, had fewer vesicles, and had smaller active zones than normal. In contrast, the densities of presynapses and docked vesicles were nearly restored to control values. Thus, robust functional recovery from SCI can occur even when the structures of regenerated synapses are sparse and small, suggesting that functional recovery is due to a more complex set of compensatory changes throughout the spinal network.