A Method to Inflict Closed Head Traumatic Brain Injury in <em>Drosophila</em>

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

University of Wisconsin-Madison

Synaptotagmin I is a synaptic vesicle protein postulated to mediate vesicle docking, vesicle recycling, and the Ca(2+) sensing required to trigger vesicle fusion. Analysis of synaptotagmin I knockouts (sytI(NULL) mutants) in both Drosophila and mice led to these hypotheses. Although much research on the mechanisms of synaptic transmission in Drosophila is performed at the third instar neuromuscular junction, the ultrastructure of this synapse has never been analyzed in sytI(NULL) mutants. Here we report severe synaptic vesicle depletion, an accumulation of large vesicles, and decreased vesicle docking at sytI(NULL) third instar neuromuscular junctions. Mutations in synaptotagmin I's C(2)B Ca(2+)-binding motif nearly abolish synaptic transmission and decrease the apparent Ca(2+) affinity of neurotransmitter release. Although this result is consistent with disruption of the Ca(2+) sensor, synaptic vesicle depletion and/or redistribution away from the site of Ca(2+) influx could produce a similar phenotype. To address this question, we examined vesicle distributions at neuromuscular junctions from third instar C(2)B Ca(2+)-binding motif mutants and transgenic wild-type controls. The number of docked vesicles and the overall number of synaptic vesicles in the vicinity of active zones was unchanged in the mutants. We conclude that the near elimination of synaptic transmission and the decrease in the Ca(2+) affinity of release observed in C(2)B Ca(2+)-binding motif mutants is not due to altered synaptic vesicle distribution but rather is a direct result of disrupting synaptotagmin I's ability to bind Ca(2+). Thus, Ca(2+) binding by the C(2)B domain mediates a post-docking step in fusion.

Drosophila synaptotagmin I null mutants show severe alterations in vesicle populations but calcium-binding motif mutants do not.

Synaptotagmin I, a synaptic vesicle protein required for efficient synaptic transmission, contains a highly conserved polylysine motif necessary for function. Using Drosophila, we examined in which step of the synaptic vesicle cycle this motif functions. Polylysine motif mutants exhibited an apparent decreased Ca2+ affinity of release, and, at low Ca2+, an increased failure rate, increased facilitation, and increased augmentation, indicative of a decreased release probability. Disruption of Ca2+ binding, however, cannot account for all of the deficits in the mutants; rather, the decreased release probability is probably due to a disruption in the coupling of synaptotagmin to the release machinery. Mutants exhibited a major slowing of recovery from synaptic depression, which suggests that membrane trafficking before fusion is disrupted. The disrupted process is not endocytosis because the rate of FM 1-43 uptake was unchanged in the mutants, and the polylysine motif mutant synaptotagmin was able to rescue the synaptic vesicle depletion normally found in syt(null) mutants. Thus, the polylysine motif functions after endocytosis and before fusion. Finally, mutation of the polylysine motif inhibits the Ca2+-independent ability of synaptotagmin to accelerate SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated fusion. Together, our results demonstrate that the polylysine motif is required for efficient Ca2+-independent docking and/or priming of synaptic vesicles in vivo.

C2B polylysine motif of synaptotagmin facilitates a Ca2+-independent stage of synaptic vesicle priming in vivo.

Expansion of the lysosomal system, including cathepsin D upregulation, is an early and prominent finding in Alzheimer's disease brain. Cell culture studies, however, have provided differing perspectives on the lysosomal connection to Alzheimer's disease, including both protective and detrimental influences. We sought to clarify and molecularly define the connection in vivo in a genetically tractable model organism. Cathepsin D is upregulated with age in a Drosophila model of Alzheimer's disease and related tauopathies. Genetic analysis reveals that cathepsin D plays a neuroprotective role because genetic ablation of cathepsin D markedly potentiates tau-induced neurotoxicity. Further, generation of a C-terminally truncated form of tau found in Alzheimer's disease patients is significantly increased in the absence of cathepsin D. We show that truncated tau has markedly increased neurotoxicity, while solubility of truncated tau is decreased. Importantly, the toxicity of truncated tau is not affected by removal of cathepsin D, providing genetic evidence that modulation of neurotoxicity by cathepsin D is mediated through C-terminal cleavage of tau. We demonstrate that removing cathepsin D in adult postmitotic neurons leads to aberrant lysosomal expansion and caspase activation in vivo, suggesting a mechanism for C-terminal truncation of tau. We also demonstrate that both cathepsin D knockout mice and cathepsin D-deficient sheep show abnormal C-terminal truncation of tau and accompanying caspase activation. Thus, caspase cleavage of tau may be a molecular mechanism through which lysosomal dysfunction and neurodegeneration are causally linked in Alzheimer's disease.

Lysosomal dysfunction promotes cleavage and neurotoxicity of tau in vivo.

The unfolded protein response is a critical system by which the cell handles excess misfolded protein in the secretory pathway. The role of the system in modulating the effects of aggregation prone cytosolic proteins has received less attention. We use genetic reporters to demonstrate activation of the unfolded protein response in a transgenic Drosophila model of Alzheimer's disease and related tauopathies. We then use loss of function genetic reagents to support a role for the unfolded protein response in protecting from tau neurotoxicity. Our findings suggest that the unfolded protein response can ameliorate the toxicity of tau in vivo.

The unfolded protein response protects from tau neurotoxicity in vivo.

Traumatic brain injury (TBI) is a substantial health issue worldwide, yet the mechanisms responsible for its complex spectrum of pathologies remains largely unknown. To investigate the mechanisms underlying TBI pathologies, we developed a model of TBI in Drosophila melanogaster. The model allows us to take advantage of the wealth of experimental tools available in flies. Closed head TBI was inflicted with a mechanical device that subjects flies to rapid acceleration and deceleration. Similar to humans with TBI, flies with TBI exhibited temporary incapacitation, ataxia, activation of the innate immune response, neurodegeneration, and death. Our data indicate that TBI results in death shortly after a primary injury only if the injury exceeds a certain threshold and that age and genetic background, but not sex, substantially affect this threshold. Furthermore, this threshold also appears to be dependent on the same cellular and molecular mechanisms that control normal longevity. This study demonstrates the potential of flies for providing key insights into human TBI that may ultimately provide unique opportunities for therapeutic intervention.

Carin A. Loewen

Department of Genetics

University of Wisconsin-Madison

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Carin A. Loewen

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Genetics

University of Wisconsin-Madison

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Department of Genetics