Elliot Imler Abstract
Elliot Imler
Ph.D. Candidate
Neuroscience GIDP
Cold Spring Harbor- Neurobiology of Drosophila
Cold Spring, NY
September 29- October 3, 2015
A Drosophila model of CLN4B, a neurodegenerative autosomal dominant adult-onset Neuronal Ceroid Lipofuscinosis, links CSPα’s chaperone activity to toxicity
Elliot Imler, Jin Pyon, Yongquan Zhang, Sreeganga Chandra, Konrad E Zinsmaier.
1) Dept. of Neuroscience University of Arizona, Tucson AZ; 2) Depts. of Molecular and Cellular Biology, Neurology, and Molecular Medicine and Developmental Biology Yale University, New Haven CT
Abstract
Synaptic vesicle (SV)-associated cysteine-string protein α (CSPα) maintains synaptic function and is neuroprotective. CSPα recruits Hsc70 and activates its ATPase activity ensuring proper activity of SNARE proteins, dynamin and a limited number of other synaptic client proteins. Deletion of CSP in flies and mice causes progressive synaptic failure, paralysis, neuronal loss and premature death. Mutations in human CSPα (hCSPα, L115R and L116Δ) cause a dominant adult-onset form of Neuronal Ceroid Lipofuscinoses (ANCL, CLN4B), a progressive neurodegenerative disease with lysosomal pathology causing seizures, dementia, and early death.
We have generated a Drosophila model of ANCL by transgenically expressing normal hCSPα and the CLN4B-causing mutations L115R and L116Δ in fly neurons. Normal hCSPα is functional in flies and can significantly restore defects of Drosophila CSP (dCSP) null mutants.
Neuronal expression of hCSPα in wild type flies has no significant detrimental effects while CLN4B-mutant hCSPα induces neurodegeneration and reduces lifespan is a dose-dependent manner. Expression of hCSP-L115R and L116Δ induces the formation of abnormal SDSresistant protein aggregates (>250 kD) containing both hCSPα and endogenous dCSP. CSP aggregates accumulate on late endosomes found at axon terminals, axons and neuronal somata in a dose-dependent manner and arrest the formation of functional lysosomes and/or autophagosomes. Electron microscopy images show numerous pathological structures including abnormally large autophagosome-like structures, multi-laminar membrane “whirls”, large osmophillic bodies and widespread degeneration.
To define mechanisms that contribute to CLN4B-induced neurodegeneration, we are conducting F1 dominant gain- and loss-of-function genetic modifier screens. So far, we have identified a number of candidate genes that enhance or suppress the phenotypic effects of hCSPL116Δ expression. Interestingly, some of these are associated with CSP’s synaptic function, indicating a tight link between ANCL/CLN4B disease pathology and CSP function.
Abstract (for Lay Audience)
Despite the many advances of modern medicine, neurodegenerative disorders still impose a huge burden on the lives of those affected and their families as well the enormous financial cost to society. As the population ages, the urgency to discover therapeutic strategies for these diseases increases. We believe the best strategy for combating these diseases is by understanding them through their basic genetic and molecular mechanisms.
The particular neurodegenerative disease we study is called autosomal dominant adult-onset Neuronal Ceroid Lipofuscinosis or CLN4B for short. Although this particular disease is rare, only afflicting about a dozen known patients in the world, the mechanisms behind this disease may give us insight into general mechanisms of neurodegeneration. In fact, the gene affected in CLN4B, CSPα, has been recently linked to mechanisms of Alzheimer’s, Parkinson’s, and Huntington’s disease.
To research this disease we have created an animal model using the fruit fly Drosophila melanogaster which contains the mutated form of the human protein which causes CLN4B. Although there are numerous caveats to fly models, they have proven incredibly useful in the past due to the ease of genetic manipulations. Our initial characterization of the fly model shows that is recapitulates many of the pathologies found in human patients and cell culture models, including protein aggregation and cellular death. We also have found novel effects of the mutant proteins that have never been shown before due to this being the first living animal model of the disease.
We have been able to take advantage of the short lifespan and genetic toolbox of the fruit fly to perform a genetic screen to look for potential enhancers and suppressors of the disease pathology. Although we are in the early stages of this screen we have already found numerous hits that imply the toxicity of the mutations is due to an interaction with the normal cellular function of CSPα, which is in contrast to previous studies. More than just finding genetic mechanisms of the disease many of the results from out screen could be investigated as future therapeutic for CLN4B and potentially other neurodegenerative diseases.