Autophagy is an essential lysosomal degradation pathway that removes damaged organelles and protein aggregates from the cell. Autophagy is essential in post-mitotic cells such as neurons since they are unable to dilute out proteotoxins by cell division. In fact, neuron-specific loss of autophagy is sufficient to cause neuron cell death. Further, multiple neurodegenerative diseases characterized by excessive protein aggregation exhibit pronounced defects in autophagy. Despite the clear implications of defective autophagy in disease, little is known about the basic mechanisms driving autophagy in neurons. Preliminary data indicate that autophagosomes are preferentially generated in the distal neurite. Initially, they exhibit bidirectional motility but then exit the distal region and undergo robust retrograde transport to the cell soma. This shift in motility is accompanied by fusion with late endosomes/lysosomes. As autophagosomes travel toward the cell soma, they continue to acidify and mature into autolysosomes that may more effectively degrade cargo. Potentially, delivery of autolysosomes to the cell soma ensures rapid and efficient recycling of degradation products to primary sites of protein synthesis. Based on the preliminary data, this proposal will test the hypothesis that autophagosome biogenesis and maturation are spatially and temporally regulated along the axon of primary neurons. Further, this study will also test the hypothesis that autophagosome function and transport are tightly linked. To examine these hypotheses, this proposal will (1) determine the mechanisms of autophagosome biogenesis in primary neurons (2) determine the relationship between autophagosome transport and maturation in primary neurons under basal versus stress conditions and (3) determine the mechanisms of cargo degradation by autophagy in primary neurons under basal versus stress conditions. Together, this proposal will determine the mechanisms of autophagosome biogenesis from birth to maturation into degradative and functional organelles and how this pathway becomes altered in response to cellular stressors such as mitochondrial damage and protein aggregation. These hypotheses will be tested using a multidisciplinary approach ranging from live-cell imaging to biophysical techniques in neurons isolated from wild type animals and models of neurodegenerative disease. During the K99 phase, new methodologies in neurobiology and biophysics will be established to examine the proposed hypotheses on a mechanistic level. These methodologies will then be utilized during the R00 phase to establish an innovative and independent research program. Results from this study will uncover novel information about the regulation of autophagy in primary neurons and have significant implications on understanding the progression of neurodegenerative disease.

Public Health Relevance

To maintain health, cells have an in-house cleaning staff to routinely take out their trash. In diseases such as familial ALS, Alzheimer's, Parkinson's and Huntington's Disease, however, the effectiveness of this cleaning crew becomes compromised and toxic trash accumulates inside the cell, causing the cell to malfunction and die. To develop more targeted therapies to treat neurodegenerative diseases, this proposal will determine how cells normally dispose of their trash and how this process becomes misregulated in disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Career Transition Award (K99)
Project #
5K99NS082619-02
Application #
8626456
Study Section
NST-2 Subcommittee (NST)
Program Officer
Gubitz, Amelie
Project Start
2013-03-01
Project End
2015-02-28
Budget Start
2014-03-01
Budget End
2015-02-28
Support Year
2
Fiscal Year
2014
Total Cost
$90,000
Indirect Cost
$6,667
Name
University of Pennsylvania
Department
Physiology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Maday, Sandra; Holzbaur, Erika L F (2014) Autophagosome biogenesis in primary neurons follows an ordered and spatially regulated pathway. Dev Cell 30:71-85
Maday, Sandra; Twelvetrees, Alison E; Moughamian, Armen J et al. (2014) Axonal transport: cargo-specific mechanisms of motility and regulation. Neuron 84:292-309
Moughamian, Armen J; Osborn, Gregory E; Lazarus, Jacob E et al. (2013) Ordered recruitment of dynactin to the microtubule plus-end is required for efficient initiation of retrograde axonal transport. J Neurosci 33:13190-203