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Methamphetamine produces neuronal inclusions in the nigrostriatal system and in PC12 cells
Francesco Fornai1, Paola Lenzi, Marco Gesi
1Department of Human Morphology and Applied Biology, University of Pisa, Pisa, Italy. f.fornai@med.unipi.it
Abstract:
Mice treated with the psychostimulant methamphetamine (MA) showed the appearance of intracellular inclusions in the nucleus of medium sized striatal neurones and cytoplasm of neurones of the substantia nigra pars compacta but not in the frontal cortex. All inclusions contained ubiquitin, the ubiquitin activating enzyme (E1), the ubiquitin protein ligase (E3-like, parkin), low and high molecular weight heat shock proteins (HSP 40 and HSP 70). Inclusions found in nigral neurones stained for alpha-synuclein, a proteic hallmark of Lewy bodies that are frequently observed in Parkinson's disease and other degenerative disorders. However, differing from classic Lewy bodies, MA-induced neuronal inclusions appeared as multilamellar bodies resembling autophagic granules. Methamphetamine reproduced this effect in cultured PC12 cells, which offered the advantage of a simple cellular model for the study of the molecular determinants of neuronal inclusions. PC12 inclusions, similar to those observed in nigral neurones, were exclusively localized in the cytoplasm and stained for alpha-synuclein. Time-dependent experiments showed that inclusions underwent a progressive fusion of the external membranes and developed an electrodense core. Inhibition of dopamine synthesis by alpha-methyl-p-tyrosine (alphaMpT), or administering the antioxidant S-apomorphine largely attenuated the formation of inclusions in PC12 cells exposed to MA. Inclusions were again observed when alphaMpT-treated cells were loaded with l-DOPA, which restored intracellular dopamine levels.
Insights
Methamphetamine (MA) induces neuronal inclusions containing ubiquitin and alpha-synuclein in mice and PC12 cells. Dopamine synthesis inhibition and antioxidants reduce inclusion formation, suggesting a role for dopamine metabolism.
Area of Science:
- Neuroscience
- Cell Biology
- Toxicology
Background:
- Psychostimulant methamphetamine (MA) can induce cellular changes in neurons.
- Intracellular inclusions are implicated in neurodegenerative diseases like Parkinson's.
- Alpha-synuclein is a key protein marker in Lewy bodies.
Purpose of the Study:
- To investigate the formation and characteristics of methamphetamine-induced intracellular inclusions.
- To explore the molecular components and cellular localization of these inclusions.
- To identify cellular mechanisms and potential protective factors against MA-induced inclusions.
Main Methods:
- Administration of methamphetamine (MA) to mice and PC12 cell cultures.
- Immunohistochemical analysis for ubiquitin, parkin, HSPs, and alpha-synuclein.
- Electron microscopy to examine inclusion morphology.
- Pharmacological manipulation including dopamine synthesis inhibition (alpha-methyl-p-tyrosine) and antioxidant treatment (S-apomorphine).
Main Results:
- MA induced intracellular inclusions in mouse striatal and substantia nigra neurons, but not frontal cortex.
- Inclusions contained ubiquitin, E1, parkin, HSP 40, HSP 70, and alpha-synuclein.
- MA-induced inclusions in PC12 cells were cytoplasmic, alpha-synuclein-positive, and resembled autophagic granules.
- Inhibition of dopamine synthesis or antioxidant treatment attenuated inclusion formation.
- Restoring dopamine levels with l-DOPA in inhibited cells led to inclusion reappearance.
Conclusions:
- Methamphetamine induces unique neuronal inclusions characterized by ubiquitin and alpha-synuclein.
- These inclusions share some markers with Lewy bodies but have distinct morphology.
- Dopamine metabolism and oxidative stress appear to play critical roles in MA-induced inclusion formation.
- PC12 cells provide a valuable model for studying the molecular basis of these inclusions.
