Our overall objective is to understand how methamphetamine (METH) kills neurons, and whether this can be ameliorated or eliminated with pharmacologic intervention. We have preliminary data that METH-induced subclinical repetitive electrographic seizure discharges (RESDs) produce acidophilic neurons in the same brain regions as generalized seizures. Since 57% of mice with RESDs have no behavioral evidence of seizures, EEG recording is mandatory.
Our Specific Aims i n this project are (1) to show that METH-induced RESDs are associated with necrotic, not apoptotic neurons, as is currently believed;(2) to determine if METH-induced RESDs are associated with calpain I activation and nuclear translocation of mitochondrial cytochrome c (cyt c) and lysosomal cathepsins B and D (cath-B and cath-D) as well as RESD-induced neuronal necrosis;(3) to show that stopping electrographic seizures with antiepileptic drugs 5 min after the onset of METH-induced RESDs inhibits calpain I activation, is neuroprotective and prevents or reduces nuclear translocation of cyt c, cath-B and cath-D;and (4) to show that specific inhibition of calpain I is neuroprotective and prevents or reduces nuclear translocation of cyt c, cath-B and cath-D. The methods that we will use include (1) continuous EEG recording to identify mice with RESDs and rectal temperature monitoring to keep mice given METH at 40.0 ? 1.0?C and control mice given normal saline at 37.0 ? 1.0?C;(2) use of the hematoxylin and eosin (H &E) stain at the light-microscopic level to identify dead (acidophilic) neurons with confirmation of morphological necrosis with transmission electron microscopy;(3) semi-quantitative scoring of 7 brain regions for acidophilic neurons with the H &E stain and nuclear translocation of cyt c, cath-B and cath-D with immunofluorescence microscopy;(4) stereological estimate of the numbers of acidophilic and normal neurons in the most damaged brain region (probably hippocampus) and the numbers of normal neurons and those showing nuclear cyt c, cath-B and cath-D;(4) fluorometric calpain activity assays and western blots of ?II-spectrin, a calpain substrate, to document calpain I activation;(5) parentera administration of diazepam and phenobarbital after METH administration to stop electrographic seizures, which we predict will also provide neuroprotection and inhibit calpain I activation and nuclear translocation of cyt c, cath-B and cath-D;(6) injection of the calpain I inhibitor MDL 28170, which we predict will be neuroprotective and will prevent or reduce nuclear translocation of cyt c, cath-B and cath-D;and (6) statistical analysis, using multifactorial analysis of devianc and multi- factorial ANOVA, with post-hoc t-tests with pooled standard deviations, and an ? of 0.05. With respect to relevance, METH abuse is a major health problem in the United States (Cho &Melega 2002, Rawson et al. 2002). Our goal is to reduce morbidity from brain damage caused by acute METH toxicity in humans. Our finding that METH-induced subclinical RESDs in mice are associated with widespread neuronal damage could have important translational applicability.

Public Health Relevance

Methamphetamine (METH) abuse is a major health problem in the United States. Acute, high-dose ingestion can kill nerve cells in the brain and result in chronic cognitive problems. We have found that METH-induced nerve cell death is associated with subclinical seizure activity. Our goal in this project is to determine if treatment with antiepileptic drugs prevents METH-induced nerve cell death and if the death-promoting enzyme calpain I is activated by subclinical seizures, is inhibited by treatment with antiepileptic drugs or by a specific calpain I inhibitor, nd if the latter also prevents METH-induced nerve cell death.

Agency
National Institute of Health (NIH)
Institute
Veterans Affairs (VA)
Type
Non-HHS Research Projects (I01)
Project #
1I01BX001858-01A1
Application #
8540280
Study Section
Neurobiology A (NURA)
Project Start
2013-07-01
Project End
2017-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
1
Fiscal Year
2013
Total Cost
Indirect Cost
Name
VA Greater Los Angels Healthcare System
Department
Type
DUNS #
066689118
City
Los Angeles
State
CA
Country
United States
Zip Code
90073