The Purkinje cell degeneration (pcd) mouse is a recessive model of neurodegeneration, involving the retina and cerebellum, with a phenotype of ataxia and blindness. Purkinje cell death in pcd is dramatic, as >99% of post-developmental Purkinje neurons are lost in three weeks. Loss-of-function of Nna1 causes pcd, and Nna1 is a highly conserved protease (zinc carboxypeptidase). In this project, we pursued a broad range of experiments to determine why Nna1 loss-of-function results in retinal and cerebellar degeneration in pcd mice. Each approach has yielded significant insights into Nna1 function, the cell death process in Purkinje cell neurons, and the biology of retinal photoreceptors. We discovered the molecular basis of the pcd-5J mutation, and proved that the carboxypeptidase activity of Nna1 is required to prevent both retinal photoreceptor loss and Purkinje cell degeneration in pcd. We found that autophagy activation and enhanced mitophagy are involved in pcd Purkinje cell death. We obtained loss-of-function alleles of the Drosophila Nna1 orthologue (NnaD) and discovered that reduced NnaD function yields a semi-lethal phenotype, with survivors displaying phenotypes that mirror the pathology observed in pcd mice. Finally, we linked Nna1-NnaD proteins with mitochondrial metabolic functions by quantitative proteomics studies of pcd mice and ultrastructural analysis of NnaD loss-of-function flies and pcd mice. Our discovery of a role for Nna1 loss-of-function in mitochondrial dysfunction and our documentation of increased mitophagy in degenerating neurons provide us with a unique opportunity to delineate mechanistic relationships between altered mitochondrial function and mitophagy. Furthermore, our recent observation that diminished Drp1-dependent mitochondrial fission can rescue degenerative phenotypes in NnaD flies suggests a connection between mitochondrial metabolic function, mitochondrial turnover, mitochondrial dynamics and neuron cell death. In this renewal, we will advance our knowledge of Nna1 protein function, pcd neuron degeneration, and neuronal mitochondrial biology. First, we will determine if Nna1-NnaD localizes to the mitochondria. Second, we will continue our studies of the Nna1 interactome by confirming proteomically predicted protein interactions and characterizing confirmed interactions, assaying interacting proteins as substrates for Nna1 carboxypeptidase activity, and testing if interacting proteins are involved in NnaD - Nna1 loss-of-function pathways. Third, we will evaluate the role of altered autophagy in pcd neurodegeneration, and fourth, we will determine if Nna1 plays a role in mitochondrial dynamics and if altered mitochondrial dynamics is contributing to neuron cell death in pcd mice. Thus, we will establish the intersection between Nna loss-of-function and mitochondrial pathways, in the hope that this will allow us to delineate how mitochondrial dysfunction occurs in a range of neurological diseases.
In our first cycle of R01 funding, we made huge strides towards understanding the molecular basis of Nna1 loss-of-function, and in the process, we unexpectedly linked Nna1 with mitochondrial metabolic function, mitochondrial autophagy (mitophagy), and mitochondrial dynamics. In the renewal project, we expect to uncover novel mechanisms underlying these pathways, by combining functional genomics and proteomics approaches with both Drosophila and mouse model systems. As mitochondrial dysfunction is often a primary cause of retinal degeneration and cerebellar degeneration, our studies could reveal potential candidates for involvement in human retinal diseases and cerebellar ataxias, and could provide powerful insights into the regulation of mitochondrial processes that presently remain poorly understood.
|Guyenet, Stephan J; Mookerjee, Shona S; Lin, Amy et al. (2015) Proteolytic cleavage of ataxin-7 promotes SCA7 retinal degeneration and neurological dysfunction. Hum Mol Genet 24:3908-17|
|Dubinsky, Amy N; Dastidar, Somasish Ghosh; Hsu, Cynthia L et al. (2014) Let-7 coordinately suppresses components of the amino acid sensing pathway to repress mTORC1 and induce autophagy. Cell Metab 20:626-38|
|Batlevi, Yakup; La Spada, Albert R (2011) Mitochondrial autophagy in neural function, neurodegenerative disease, neuron cell death, and aging. Neurobiol Dis 43:46-51|
|Guyenet, Stephan J; Furrer, Stephanie A; Damian, Vincent M et al. (2010) A simple composite phenotype scoring system for evaluating mouse models of cerebellar ataxia. J Vis Exp :|
|Chakrabarti, Lisa; Zahra, Rabaab; Jackson, Stephen M et al. (2010) Mitochondrial dysfunction in NnaD mutant flies and Purkinje cell degeneration mice reveals a role for Nna proteins in neuronal bioenergetics. Neuron 66:835-47|
|Young, Jessica E; Martinez, Refugio A; La Spada, Albert R (2009) Nutrient deprivation induces neuronal autophagy and implicates reduced insulin signaling in neuroprotective autophagy activation. J Biol Chem 284:2363-73|
|Young, Jessica E; La Spada, Albert R (2009) Development of selective nutrient deprivation as a system to study autophagy induction and regulation in neurons. Autophagy 5:555-7|
|Chakrabarti, Lisa; Eng, Jeremiah; Martinez, Refugio A et al. (2008) The zinc-binding domain of Nna1 is required to prevent retinal photoreceptor loss and cerebellar ataxia in Purkinje cell degeneration (pcd) mice. Vision Res 48:1999-2005|
|Chakrabarti, Lisa; Neal, James T; Miles, Michael et al. (2006) The Purkinje cell degeneration 5J mutation is a single amino acid insertion that destabilizes Nna1 protein. Mamm Genome 17:103-10|