Disruption of axonal transport is thought to occur early in the course of many neurological diseases including peripheral neuropathies and neurodegenerative diseases. Proposed mechanisms whereby transport defects lead to disease include impairment of organelle delivery to distal axons, defective retrograde neurotrophic signaling, and disruption of the synaptic vesicle cycle within the synaptic terminal. However, the way alterations in axonal transport cause disease is unclear. Simple model organisms such as the fruitfly, Drosophila melanogaster, allow genetic manipulations to be combined with analysis of axonal transport and synaptic physiology, and they are providing insights into the pathophysiology of peripheral nerve and neurodegenerative diseases. The long-term goal of this proposal is to understand how intracellular trafficking events are altered in neurological diseases in order to identify novel targets for therapeutic development. Retrograde axonal transport is mediated by the dynein/dynactin protein complex. The p150Glued dynactin subunit is mutated in two distinct, non-overlapping autosomal dominant neurodegenerative diseases: one that causes a motor neuron disease called Hereditary Motor Neuropathy type 7B (HMN7B), and the other that is called Perry Syndrome, characterized by parkinsonism, depression, hypoventilation, and weight loss. The HMN7B and Perry mutations are as close as 12 residues apart within the p150Glued CAP-Gly microtubule (MT)-binding domain. Our Preliminary Results suggest that these mutations differentially affect p150 interactions with MTs and p150 function at synapses. Furthermore, we find that HMN7B mutations disrupt the initiation of retrograde MT-mediated transport at the distal-most end of synapses (called terminal boutons) and block neurotransmitter release at the neuromuscular junction (NMJ).
In Aim 1, we will determine if HMN7B and Perry Syndrome mutations disrupt distinct protein interactions with p150 binding partners and also whether they cause defects in axonal transport or retrograde signaling.
In Aim 2, we will study the initiation of retrograde transport at NMJ TBs to determine if initiation occur when dynamic MT plus-ends capture vesicles through interactions between p150 and end binding protein-1 (EB1), a "master regulator of MT plus-ends". We will also test the hypothesis that HMN7B but not Perry mutations in p150 disrupt retrograde initiation at TBs, and determine if this defect is due to an alteration in microtubule dynamics at TBs.
In Aim3, we will investigate how mutations in p150 alter synaptic transmission by combining calcium and FM1-43 imaging, ultrastructural analysis and electrophysiology. Together these studies will help elucidate the function of the p150 CAP-Gly domain in neurons, and hold promise for shedding light on the mechanisms of cell-type specificity of neurodegenerative disease.

Public Health Relevance

Blockage of transport along neuronal processes and at synapses occurs in neurodegenerative diseases and peripheral neuropathies, but the way that altered trafficking causes disease is unclear. This study will investigate a key regulator of neuronal intracellular transport that is mutated in two different neurodegenerative diseases to find out how altered interactions with other proteins and the cytoskeleton disrupt neuronal function.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
1R01NS082563-01
Application #
8482528
Study Section
Synapses, Cytoskeleton and Trafficking Study Section (SYN)
Program Officer
Gubitz, Amelie
Project Start
2013-04-15
Project End
2018-03-31
Budget Start
2013-04-15
Budget End
2014-03-31
Support Year
1
Fiscal Year
2013
Total Cost
$354,375
Indirect Cost
$135,625
Name
Johns Hopkins University
Department
Neurology
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Chung, Tae; Prasad, Kalpana; Lloyd, Thomas E (2014) Peripheral neuropathy: clinical and electrophysiological considerations. Neuroimaging Clin N Am 24:49-65
Machamer, James B; Collins, Sarah E; Lloyd, Thomas E (2014) The ALS gene FUS regulates synaptic transmission at the Drosophila neuromuscular junction. Hum Mol Genet 23:3810-22