Amyotrophic Lateral Sclerosis (ALS) is a fatal disease characterized by progressive paralysis due to motor neuron degeneration. Many therapies have been tested but no cure or effective therapy is available. Mutations of superoxide dismutase-1 (SOD1) are a cause of familial ALS and it is widely held that oxidative injury is likely to be a major contributor to disease pathogenesis. Despite this, the cause of motor neuron death is unknown and the role of mutant SOD1 in the generation of oxidative stress in ALS has not been established. We recently reported that the amino acid L-arginine slows disease progression in mutant SOD1 (G93A) transgenic ALS mice. The basis for this neuroprotective effect is not known but one possible mechanism may involve the production of neuroprotective polyamines that are potential substrates for LSD1, a key enzyme regulating protein methylation that shares considerable homology with FAD-dependent polyamine oxidases. We found that LSD1 expression suppresses transcription in transiently transfected SH-SY5Y neuroblastoma cells suggesting that LSD1 expression can regulate transcriptional activity in neurons. We also found that LSD1 enzyme activity is increased the spinal cords of G93A mice and that levels of dimethylated histone H3 Lys4 (DMH3K4), a substrate of LSD1, were reduced. In contrast, levels of trimethylated histone H3 Lys4 (TMH3K4), which is not demethylated by LSD1, were unchanged. Because of this, we examined the relationship between the expression of LSD1 and the neuroprotective effects of the polyamine, spermidine. We found that spermidine treatment reduced neuronal loss and gliosis in G93A mice and restored LSD1 activity to normal levels. To directly test the role of LSD1 in the pathogenesis of oxidative injury in neurons, we treated NSC-34 motor neuronal cells transfected with LSD1 shRNA with hydrogen peroxide and found that reduced LSD1 expression was associated with resistance to oxidative injury further supporting a possible link between oxidative injury, LSD1 expression and neurodegeneration in G93A mice. Based on these observations we hypothesize that increased LSD1 expression may contribute to motor neuron degeneration in ALS by altering histone and/or non-histone protein methylation. Interventions that reduce LSD1 expression may therefore protect neurons from degeneration in G93A mice. In the current proposal we plan to study the relationship between LSD1 expression, protein methylation and neurodegeneration in the G93A mice. We will also test the effects of LSD1 inhibitors on the behavioral and neuropathological phenotype of G93A mice.
The specific aims of the study are: 1) To determine the relationship between LSD1 induction in motor neurons and cell death mediated by oxidative stress in vitro;2) To determine the role of LSD1 in the survival of motor neurons harboring the G93A SOD1 mutation;and 3) To investigate the therapeutic effect of LSD1 modulators on motor neuron degeneration, neuropathology, clinical progression, and the survival of G93A mice. A variety of complementary methods will be used to accomplish our aims. We will determine the importance of LSD1 in oxidative injury and G93A-mediated neurotoxicity by transiently knocking down LSD1 expression with shRNA in NSC-34 cells and in cultured primary motor neurons and in NSC-34 cells stably transfected with either Tet- inducible LSD1 or a shRNA expression system to suppress LSD1 translation. We will measure LSD1 gene expression and activity levels using Real-Time PCR, Western blotting, immunofluorescent staining and by assessing transcriptional activity using a luciferase assay and the methylation of histone H3K4 using specific antibodies. We will determine the effect of spermidine and tranylcypromine, an LSD1 inhibitor on LSD1 activity and DMH3K4 levels and anti-oxidant gene expression in G93A mice. In addition, we will investigate the effects of these drugs on neuropathology, behavior and survival of G93A mice. We hope that our studies will lay the foundation for the development of better treatments for this fatal condition that disproportionately affects veterans who have been deployed to the Persian Gulf region.

Public Health Relevance

Amyotrophic lateral sclerosis (ALS or Lou Gehrig disease) is an untreatable fatal progressive neurological disease characterized by degeneration of the motor system. Gulf War veterans may have an increased risk of ALS compared to veterans who did not serve in the Gulf. The reasons for this increased risk are not known but it is thought to be due to exposure to a neurotoxin in the environment. The studies we propose are designed to better understand the causes of motor neuron degeneration in a well-characterized mouse model of ALS. This transgenic mouse is produced by adding the abnormal human gene that causes ALS to the mouse embryo. These mice develop progressive weakness and die prematurely at about 4 months of age. We expect that our studies on how motor neurons degenerate will lay the foundation for the development of better treatments for this irreversible progressive and fatal condition. The urgent need for such studies is highlighted by the prospect that hundreds of thousands of future veterans may be at increased risk for this devastating disease.

Agency
National Institute of Health (NIH)
Institute
Veterans Affairs (VA)
Type
Non-HHS Research Projects (I01)
Project #
5I01BX000856-03
Application #
8397585
Study Section
Neurobiology E (NURE)
Project Start
2011-01-01
Project End
2014-12-31
Budget Start
2013-01-01
Budget End
2013-12-31
Support Year
3
Fiscal Year
2013
Total Cost
Indirect Cost
Name
VA Boston Health Care System
Department
Type
DUNS #
034432265
City
Boston
State
MA
Country
United States
Zip Code
02130