Proteomics of mouse cortex following conditional deletion of Psmc1 proteasomal subunit in neurones

Neurodegenerative diseases are characterized by progressive degeneration of selective neurones in the nervous system and the formation of protein inclusions in surviving neurones. The mechanisms underlying neurodegeneration and neuroprotection in the nervous system remain elusive. Ubiquitin is one of the hallmarks of neuropathological inclusions in the majority of neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. Therefore, dysfunction of the ubiquitin proteasome system has been implicated in disease cause and/or progression. This thesis investigates a unique conditional genetic mouse model of neurodegeneration caused by conditional genetic 26S proteasomal depletion in mouse forebrain neurones. We have identified potential proteins targeted for ubiquitination in brain using bio-affinity chromatography of zinc finger protein ZNF216 coupled with mass spectrometry. This lead to the identification of several potential ubiquitinated proteins involved in gene expression and regulation. We have also investigated the global brain proteome in response to 26S proteasomal depletion in neurones using two-dimensional fluorescence difference in-gel electrophoresis coupled to mass spectrometry for protein identification. Several differentially expressed proteins were identified in the 26S proteasome-depleted cortex. Astrocytic intermediate filament proteins glial acid fibrillary protein and vimentin, as well as the antioxidant peroxiredxoin-6, were upregulated. Mitochondrial fumarate hydratase and stathmin-1, involved in the tricarboxylic acid cycle and cytoskeletal microtubule dynamics respectively, were downregulated. These proteins have been validated by biochemical and immunohistochemical approaches. Further analysis of oxidative stress revealed increased lipid and protein oxidation that may be involved in the neurodegeneration associated with 26S proteasomal depletion. However, we also show increased phospholipase A2 activity associated with peroxiredoxin-6 expression that may have additional roles in neurodegenerative and/or neuroprotective functions. Interestingly, the levels of reactive oxygen species were inversely correlated with the upregulation of peroxiredoxin-6. We suggest that peroxiredoxin-6 may play an important role in the brain in the protection against oxidative stress and our studies may improve our physiological and pathological understanding of neurodegenerative disease

Proteomics of mouse cortex following conditional deletion of Psmc1 proteasomal subunit in neurones

Neurodegenerative diseases are characterized by progressive degeneration of selective neurones in the nervous system and the formation of protein inclusions in surviving neurones. The mechanisms underlying neurodegeneration and neuroprotection in the nervous system remain elusive. Ubiquitin is one of the hallmarks of neuropathological inclusions in the majority of neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. Therefore, dysfunction of the ubiquitin proteasome system has been implicated in disease cause and/or progression. This thesis investigates a unique conditional genetic mouse model of neurodegeneration caused by conditional genetic 26S proteasomal depletion in mouse forebrain neurones. We have identified potential proteins targeted for ubiquitination in brain using bio-affinity chromatography of zinc finger protein ZNF216 coupled with mass spectrometry. This lead to the identification of several potential ubiquitinated proteins involved in gene expression and regulation. We have also investigated the global brain proteome in response to 26S proteasomal depletion in neurones using two-dimensional fluorescence difference in-gel electrophoresis coupled to mass spectrometry for protein identification. Several differentially expressed proteins were identified in the 26S proteasome-depleted cortex. Astrocytic intermediate filament proteins glial acid fibrillary protein and vimentin, as well as the antioxidant peroxiredxoin-6, were upregulated. Mitochondrial fumarate hydratase and stathmin-1, involved in the tricarboxylic acid cycle and cytoskeletal microtubule dynamics respectively, were downregulated. These proteins have been validated by biochemical and immunohistochemical approaches. Further analysis of oxidative stress revealed increased lipid and protein oxidation that may be involved in the neurodegeneration associated with 26S proteasomal depletion. However, we also show increased phospholipase A2 activity associated with peroxiredoxin-6 expression that may have additional roles in neurodegenerative and/or neuroprotective functions. Interestingly, the levels of reactive oxygen species were inversely correlated with the upregulation of peroxiredoxin-6. We suggest that peroxiredoxin-6 may play an important role in the brain in the protection against oxidative stress and our studies may improve our physiological and pathological understanding of neurodegenerative disease

Implications for oxidative stress and astrocytes following 26S proteasomal depletion in mouse forebrain neurones

Neurodegenerative diseases are characterized by progressive degeneration of selective neurones in the nervous system, but the underlying mechanisms involved in neuroprotection and neurodegeneration remain unclear. Dysfunction of the ubiquitin proteasome system is one of the proposed hypotheses for the cause and progression of neuronal loss. We have performed quantitative two-dimensional fluorescence difference in-gel electrophoresis combined with peptide mass fingerprinting to reveal proteome changes associated with neurodegeneration following 26S proteasomal depletion in mouse forebrain neurones. Differentially expressed proteins were validated by Western blotting, biochemical assays and immunohistochemistry. Of significance was increased expression of the antioxidant enzyme peroxiredoxin 6 (PRDX6) in astrocytes, associated with oxidative stress. Interestingly, PRDX6 is a bifunctional enzyme with antioxidant peroxidase and phospholipase A2 (PLA2) activities. The PLA2 activity of PRDX6 was also increased following 26S proteasomal depletion and may be involved in neuroprotective or neurodegenerative mechanisms. This is the first in vivo report of oxidative stress caused directly by neuronal proteasome dysfunction in the mammalian brain. The results contribute to understanding neuronal–glial interactions in disease pathogenesis, provide an in vivo link between prominent disease hypotheses and importantly, are of relevance to a heterogeneous spectrum of neurodegenerative diseases