Human Molecular Genetics Advance Access originally published online on September 29, 2008
Human Molecular Genetics 2009 18(1):82-96; doi:10.1093/hmg/ddn319
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Functional alterations of the ubiquitin-proteasome system in motor neurons of a mouse model of familial amyotrophic lateral sclerosis
1 Laboratory of Molecular Neurobiology, Department of Neuroscience, Mario Negri Institute for Pharmacological Research, Via La Masa, 19, 20156 Milan, Italy 2 Department of Biomolecular Sciences and Biotechnologies, University of Milan, Milan, Italy 3 Department of Cell and Molecular Biology (CMB), Karolinska Institutet, Stockholm, Sweden
* To whom correspondence should be addressed. Tel: +39 0239014488; Fax: +39 023546277; Email: bendotti{at}marionegri.it
Received July 21, 2008; Accepted September 26, 2008
In familial and sporadic amyotrophic lateral sclerosis (ALS) and in rodent models of the disease, alterations in the ubiquitin-proteasome system (UPS) may be responsible for the accumulation of potentially harmful ubiquitinated proteins, leading to motor neuron death. In the spinal cord of transgenic mice expressing the familial ALS superoxide dismutase 1 (SOD1) gene mutation G93A (SOD1G93A), we found a decrease in constitutive proteasome subunits during disease progression, as assessed by real-time PCR and immunohistochemistry. In parallel, an increased immunoproteasome expression was observed, which correlated with a local inflammatory response due to glial activation. These findings support the existence of proteasome modifications in ALS vulnerable tissues. To functionally investigate the UPS in ALS motor neurons in vivo, we crossed SOD1G93A mice with transgenic mice that express a fluorescently tagged reporter substrate of the UPS. In double-transgenic UbG76V-GFP /SOD1G93A mice an increase in UbG76V-GFP reporter, indicative of UPS impairment, was detectable in a few spinal motor neurons and not in reactive astrocytes or microglia, at symptomatic stage but not before symptoms onset. The levels of reporter transcript were unaltered, suggesting that the accumulation of UbG76V-GFP was due to deficient reporter degradation. In some motor neurons the increase of UbG76V-GFP was accompanied by the accumulation of ubiquitin and phosphorylated neurofilaments, both markers of ALS pathology. These data suggest that UPS impairment occurs in motor neurons of mutant SOD1-linked ALS mice and may play a role in the disease progression.
Part of confocal microscopy experiments were carried out at the Centro Interdipartimentale di Microscopia Avanzata (CIMA) of the University of Milan.