Mutations in cytoplasmic dynein or its activator dynactin are causative for neuronal diseases including heritable forms of motor neuron degeneration and CharcotMarieTooth disease. More broadly, we know that defects in dyneindriven functions such as retrograde axonal transport are involved in the pathogenic mechanisms of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), Huntington?s, and Alzheimer?s. However, the specific mechanisms involved remain unclear. Dynein is a pleiotropic cellular motor with multiple distinct roles in the neuron. Here we will focus on the hypothesis that defects in the dyneindriven retrograde transport of degradative organelles including lysosomes and autophagosomes are major contributors to the axonal degeneration that characterize these diseases. The goal of this proposal is to understand the specific mechanisms linking defects in dynein function to neurodegeneration, focusing on the following three aims: (1) How is retrograde axonal transport altered during neurodegeneration? We hypothesize that pathological alterations in the JNK and Cdk5 pathways lead to the dysregulation of opposing microtubule motors during axonal transport. We will test this hypothesis using quantitative live cell imaging of vesicular transport in primary neurons from multiple models of ALS. Then, we will mechanistically dissect how kinase misregulation affects motor function using in vitro reconstitution approaches with single molecule resolution. These studies will test the model that a disruption in the coordination of oppositelyoriented motors is the primary defect leading to altered transport along the axon. (2) What are the pathways for autophagosome biogenesis and cargoloading in the neuron? We hypothesize that autophagy in the neuron follows a stereotypical and spatially regulated pathway that is required to maintain cellular homeostasis. We will examine autophagosome biogenesis and cargoloading in primary sensory and motor neurons using quantitative live cell imaging, focusing on the roles of dynein and optineurin. Then we will determine how this pathway responds to cellular stressors, to address the hypothesis that this pathway has a limited ability to upregulate in response to cellular stress. (3) How do defects in dyneindriven autophagy lead to degeneration of the axon? We hypothesize that the active, dyneindriven transport of autophagosomes is tightly linked to function, and that defects in transport will lead to defective degradation of aging organelles and aggregated proteins. We will use live imaging and biochemical and cellular assays to determine how defects in autophagosome transport along the axon contribute to neurodegeneration and how distinct dynein mutations differentially perturb cellular functions, leading to disparate clinical manifestations. Mutations in cytoplasmic dynein are sufficient to cause human neurodegenerative diseases including spinal muscular atrophy (SMALED) and CharcotMarieTooth disease (Type 2O), but the mechanisms involved remain to be determined. Progress on these aims should offer new opportunities for therapeutic approaches or clinical intervention.

Public Health Relevance

The active movement of proteins, vesicles, and organelles along the extended axons of neurons is called axonal transport. This transport is essential to maintain healthy motor and sensory neurons, which have axons that can extend for a meter. Defects in axonal transport cause neurodegeneration, and occur in diseases such as Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig?s Disease). Here, we propose to investigate the mechanisms by which defects in axonal transport lead to degenerative disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
4R37NS060698-10
Application #
9617503
Study Section
Special Emphasis Panel (NSS)
Program Officer
Gubitz, Amelie
Project Start
2018-04-01
Project End
2021-03-31
Budget Start
2018-04-01
Budget End
2019-03-31
Support Year
10
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Physiology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Stavoe, Andrea K H; Holzbaur, Erika L F (2018) Axonal autophagy: Mini-review for autophagy in the CNS. Neurosci Lett :
Gopal, Pallavi P; Nirschl, Jeffrey J; Klinman, Eva et al. (2017) Amyotrophic lateral sclerosis-linked mutations increase the viscosity of liquid-like TDP-43 RNP granules in neurons. Proc Natl Acad Sci U S A 114:E2466-E2475
Nirschl, Jeffrey J; Ghiretti, Amy E; Holzbaur, Erika L F (2017) The impact of cytoskeletal organization on the local regulation of neuronal transport. Nat Rev Neurosci 18:585-597
Guedes-Dias, Pedro; Holzbaur, Erika L F (2017) Huntingtin Fibrils Poke Membranes. Cell 171:32-33
Klinman, Eva; Tokito, Mariko; Holzbaur, Erika L F (2017) CDK5-dependent activation of dynein in the axon initial segment regulates polarized cargo transport in neurons. Traffic 18:808-824
Maday, Sandra; Holzbaur, Erika L F (2016) Compartment-Specific Regulation of Autophagy in Primary Neurons. J Neurosci 36:5933-45
Moore, Andrew S; Holzbaur, Erika L F (2016) Spatiotemporal dynamics of autophagy receptors in selective mitophagy. Autophagy 12:1956-1957
Muhia, Mary; Thies, Edda; Labonté, Dorthe et al. (2016) The Kinesin KIF21B Regulates Microtubule Dynamics and Is Essential for Neuronal Morphology, Synapse Function, and Learning and Memory. Cell Rep 15:968-977
Klinman, Eva; Holzbaur, Erika L F (2016) Comparative analysis of axonal transport markers in primary mammalian neurons. Methods Cell Biol 131:409-24
Moore, Andrew S; Holzbaur, Erika L F (2016) Dynamic recruitment and activation of ALS-associated TBK1 with its target optineurin are required for efficient mitophagy. Proc Natl Acad Sci U S A 113:E3349-58

Showing the most recent 10 out of 21 publications