Glaucoma is a chronic neurodegenerative disease that makes up one of the leading causes of blindness worldwide. Despite increasing interest, therapies that directly address the neural dysfunction and loss remain elusive. In other neurodegenerative disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease, early pathologies occur in first in the axon - and this has been recently implicated in glaucoma. This proposal intends to examine the structural and functional defects in retinal ganglion cell axons in early glaucoma with the intention of identifying new targets for intervention in this debilitating disease. Using a combination of immunoblotting, tract tracing, histology, and quantitative immunochemistry this research enterprise will seek to determine the nature and time course of changes in cytoskeletal proteins within the axon, the enzymes responsible for these changes, and will examine different aspects of axonal transport in two rodent models of glaucoma - the DBA/2J mouse and the microbead occlusion model in rats.
Specific aim 1 will examine changes in the cytoskeleton compared to two major risk factors in glaucoma: age (DBA/2J mice) and exposure to elevated intraocular pressure (Microbead Occlusion Model). Retinas, optic nerves, the superior colliculus (SC), and cerebellum (as a control structure) will be harvested and immunoblots will be performed to examine the quantity, distribution, breakdown, and phosphoisoform of four major cytoskeletal proteins and three of the most common enzymes implicated in cytoskeletal alteration in neurodegenerative disorders.
Specific aim 2 will use techniques to identify cytoskeletal abnormalities in relation to neurobiological outcome measures including: 1) loss of anterograde axonal transport using intravitreal injections of the sensitive neuronal tracer cholera toxin beta subunit (CTB), and 2) axonal degeneration - immunofluorescence in the SC and optic nerve of the retinal ganglion cell (RGC) projection marker estrogen-related receptor beta (ERR?) and the RGC terminal marker vesicular glutamate transporter 2 (VGluT2) along with axon counts in semithin sections of optic nerve.
Specific aim 3 will focus on specific deficits in active axonal transport. First, the underlying causes of retrograde tracing loss in these models will be determined, i.e., to what degree it is due to functional loss of uptake/transport, blockade at the optic nerve head, axon loss, or RGC loss.
This aim will use a newly-validated method of applying retrograde tracer (Fluorogold) to the SC that was developed by the principle investigator to compare degeneration measures in specific aim 1 (see above) with RGC soma in the retina. Second, we will use anterograde and retrograde tracing methods (intravitreal CTB and SC-applied fluorogold) in the same animals to determine temporal differences in defects in anterograde vs. retrograde transport that have been suggested by the glaucoma literature, yet never directly tested. This proposal is designed with the goal of identifying new therapeutic targets for this disease as well as identifying the potential role that function-restoring therapies could play in glaucoma and other neurodegenerative diseases.

Public Health Relevance

Glaucoma is a leading cause of irreversible blindness worldwide. Despite increasing interest, therapies targeting the neurodegeneration that produces blindness remain elusive, but research is starting to uncover specific neuronal mechanisms related to the progression of this disease. This research proposal is designed to investigate new mechanisms proposed in other neurodegenerative diseases for relevance in glaucoma with the goal of identifying potential new therapeutic targets that will facilitate the treatment of this debilitating disorder.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY022358-03
Application #
8658094
Study Section
Anterior Eye Disease Study Section (AED)
Program Officer
Chin, Hemin R
Project Start
2012-05-01
Project End
2017-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
3
Fiscal Year
2014
Total Cost
$377,300
Indirect Cost
$132,300
Name
Northeast Ohio Medical University
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
077779882
City
Rootstown
State
OH
Country
United States
Zip Code
44272
DeToma, Alaina S; Dengler-Crish, Christine M; Deb, Aniruddha et al. (2014) Abnormal metal levels in the primary visual pathway of the DBA/2J mouse model of glaucoma. Biometals 27:1291-301