C41 FUNCTIONS OF THE PROTEASOME IN CELL REGULATION AND NEUROMUSCULAR DISEASE
Goldberg AL
Department of Cell Biology, Harvard Medical School, Boston, USA
Most proteins in mammalian cells are degraded by the ubiquitin‐proteasome pathway, where protein substrates are linked to ubiquitin molecules by one of the cell's many ubiquitin ligases (E3s). This modification marks the protein for rapid degradation by the 26S proteasome. Much has been learned about this large complex, which uses ATP to unfold proteins and to inject them into its 20S core proteasome where they are digested to small peptides. An important function of this system is to selectively destroy abnormally folded proteins as they unfortunately accumulate in various neurodegenerative diseases such as ALS. Protein inclusions in the affected neurons contain ubiquitin and proteasome. This observation and a number of studies suggest a failure of the degradation process (e.g. especially in HD and PD).
With denervation or disuse, in fasting, and in many systemic diseases (e.g. cancer, sepsis, diabetes, excessive glucocorticoids), muscles atrophy due to a general activation of the ubiquitin‐proteasome pathway in muscles. The atrophying muscles show a common pattern of changes in expression of specific genes (which we term ‘atrogenes’). The two proteins induced most dramatically are muscle‐specific ubiquitin ligases, atrogin‐1 and MuRF‐1, which trigger the atrophy process and the accelerated proteolysis. Their expression is normally inhibited by IGF‐1 and insulin, but rises in atrophying muscles due to activation of the transcription factor, Foxo3, which leads to excessive proteolysis and.
Much has been learned about the functions of this system by the use of inhibitors of the proteasome that enter cells and inhibit intracellular proteolysis. Blocking proteasome function eventually induces apoptosis, especially in cancer cells. One such inhibitor (Velcade PS341) has been approved by the FDA for treatment of multiple myeloma, but it is now in trials against diverse cancers.
Although most peptides released by proteasomes are rapidly digested to amino acids, some are transported through the ER to the cell surface, where they are presented to the immune system on MHC Class I molecules. This process enables circulating cytotoxic T‐cells to screen for and eliminate virally infected cells and cancers.
C42 ROLE OF DYSFUNCTION OF THE UBIQUITIN PROTEASOME PATHWAY IN THE MECHANISMS OF MOTOR NEURON DEGENERATION IN A MOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS
Cheroni C1, Peviani M1, Pozzi S1, Cascio P2, De Biasi S3, Monti C3, Dantuma N4 & Bendotti C1
1Department of Neuroscience, Institute for Pharmacological Research Mario Negri, Milan, 2Department of Morphophysiology, School of Veterinary Medicine, University of Turin, Turin, 3Department of Biomolecular Sciences and Biotechnologies, University of Milan, Milan, Italy, and 4Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden
E‐mail address for correspondence: [email protected]
Background: Accumulation of misfolded and ubiquitinated proteins in degenerating cells is a hallmark of both ALS patients and a murine model of the disease (SOD1G93A mice). The ubiquitin‐proteasome pathway (UPP) is the main proteolytic system in eukaryotic cells. Increasing evidence suggests a link between UPP impairment and neurodegenerative diseases.
Objective: The aim of the study was to investigate the possible role of UPP in the pathogenetic mechanisms of ALS in SOD1G93A mice.
Methods: Experiments were conducted on SOD1G93A transgenic mice at various stages of disease progression; non‐transgenic littermates were used as control. Biochemical quantification of proteasome activity was conducted by measuring the fluorescence of 7‐amido‐4‐methylcoumarin (AMC) generated from cleavage of specific peptide‐AMC linked substrates in tissue homogenates. The levels of inducible and constitutive proteasome subunits were analysed using Western blots. Immunohistochemical evaluation of 20S proteasome and ubiquitin levels and distribution was carried out on paraformaldehyde‐fixed spinal cord cryosections using diaminobenzidine or immunofluorescent staining.
To evaluate the UPP activity in vivo and at the cellular level, SOD1G93A mice were cross‐bred with a transgenic mouse model (GFP reporter mice) with constitutive and ubiquitous expression of UbG76V‐GFP reporter for proteasomal degradation; the functional status of the UPP was analysed in double transgenic mice (GFP/SOD1G93A) by the levels of the reporter substrate.
Results: Biochemical assays of spinal cord tissue did not reveal any change of proteasome activity in the spinal cord of SOD1G93A mice compared to non‐transgenic littermates. However, we observed a significant reduction in constitutive catalytic subunits of the proteasome and increase in their inducible counterparts at an advanced stage of the disease. We also detected a selective decrease of 20S proteasome levels and an increase of ubiquitin signal in the spinal motor neuron during disease progression. Immunohistochemical analysis GFP reporter/SOD1G93A mice revealed accumulation of the reporter for proteasome activity in some neurons of the lumbar spinal cord at the end stage of the disease.
Conclusions: Although no changes of proteasome activity were found in the spinal cord homogenates of SOD1G93A mice during disease progression, we revealed a reduction of 20S constitutive proteasome and the concomitant increase of immunoproteasome. In fact, analyses on GFP/SOD1G93A mice revealed decrease of UPP function in some neurons of lumbar spinal cord.
Acknowledgement
This work was supported by Telethon, Italian Ministry of Health, MIUR.
C43 MECHANISMS BY WHICH EXPRESSION OF MUTANT SOD1 IMPAIRS PROTEASOME FUNCTION IN VULNERABLE SPINAL CORD TISSUE
Kabashi E, Agar JN, Taylor DM, Minotti S & Durham HD
Montreal Neurological Institute, McGill University, Montreal, Canada
E‐mail address for correspondence: [email protected]
Background: In experimental models familial ALS caused by mutations in the Cu/Zn‐superoxide dismutase (SOD1) gene, death of motor neurons is preceded by aggregation of mutant SOD1 and formation of cytoplasmic inclusions. Proteinaceous inclusions are also a hallmark of sporadic ALS and other neurodegenerative disorders, and their occurrence suggests overload of proteolytic pathways. We previously reported impaired proteasomal function in the lumbar, but not thoracic, spinal cord of pre‐symptomatic G93A SOD1 transgenic mice relative to non‐transgenic littermates and wild‐type (WT) SOD1 transgenic mice (1). This compromise of proteasome function was compounded by reduction in the level of 20Sα subunits in lumbar spinal motor neurons despite maintenance of levels of 20Sα proteasomal subunits in the tissue as a whole. A subsequent study reported decrease in constitutive β5 and increase in β5i in symptomatic mice (2).
Objectives: To further investigate the mechanism of proteasome dysfunction by examining expression of other proteasomal subunits and proteasome assembly in tissues of G93A SOD1 transgenic mice compared to non‐transgenic littermates and WT SOD1 transgenic mice, and in tissues from ALS patients.
Methods: Expression of proteasomal subunits was examined by Western blotting of homogenized lumbar and thoracic spinal cord and expressed relative to actin. Composition and assembly of proteasomes is examined by 2‐D Blue Native/SDS‐PAGE.
Results: Levels of the constitutive β5 subunit, responsible for the chymotryptic‐like activity of the proteasome, and the structural β3 subunit of the 20S proteasome were significantly reduced specifically in the lumbar region of the spinal cord of G93ASOD1 transgenic mice by 45 days of age. The reduction was dramatic by 75 days of age. Inducible β5i immunoproteasome subunit and regulatory components (19S6b and 11Sα) of the 20S proteasome remained unchanged at these ages.
Conclusions: The results provide additional evidence that proteasome dysfunction contributes significantly to pathogenesis of motor neuron disease. The early presymptomatic reduction in constitutive β5 subunits in lumbar spinal cord is consistent with reduction in chymotrypsin‐like catalytic activity. The pronounced differences in expression of various proteasomal subunits in tissue vulnerable to the disease process indicates more complex changes than substitution of immunoproteasome subunits and suggests altered assembly of proteasomes and/or post‐translational modification of subunits. This is being resolved by 2‐D blue native/SDS‐PAGE.
Acknowledgement
This work was supported by CIHR‐ALS Society of Canada‐MDAC neuromuscular partnership and ALS Association USA.
References
- Kabashi E, Agar JN, Taylor DM, et al. Journal of Neurochemistry 2004; 89:1325–35.
- Cheroni C, Reviani P, Cascio S, et al. Neurobiology of Disease 2005;18;509–22.
C44 THE ROLE OF ENDOPLASMIC RETICULUM (ER) STRESS AND THE UNFOLDED PROTEIN RESPONSE (UPR) IN AMYOTROPHIC LATERAL SCLEROSIS (ALS)
Atkin JD, Farg M, Turner BJ, Patch J, Cheema SS & Horne MK
Howard Florey Institute, University of Melbourne, Melbourne, Australia
E‐mail address for correspondence: [email protected]
Background: Mutant SOD1 misfolded aggregates are a common feature of spinal cords from both human ALS patients and transgenic rodent models. Increasing evidence suggests that disturbances in the neuronal ER occur as an early event in the pathway to clinical ALS. It is known that in the presence of misfolded proteins in the lumen, the capacity of the ER to maintain protein folding can quickly become overwhelmed and the UPR may be initiated. This involves the up‐regulation of a number of key chaperone and other proteins and the inhibition of further protein synthesis. If not sufficiently inhibited, the ER stress may result in cell death by apoptosis regulated by caspase‐12.
Objectives: The purpose of this study was to examine spinal cord tissue in the G93A mouse and rat models of ALS, and in NSC34 cells transfected with WT and mutant SOD1 constructs, for the presence of UPR/ER stress proteins.
Methods: Proteomic analysis was carried out to look for differentially expressed proteins using spinal cord tissue from two male transgenic SOD1 rats and one male normal control at p60. 2‐DE gel electrophoresis was performed and differentially expressed spots were excised from the gel, digested with Trypsin for 16 h and subjected to MALDI‐TOF mass spectrometry. Western blotting was performed to verify these findings and to look for the differential regulation of other proteins.
Results: Proteomic analysis has revealed that Hsp60, protein disulphide isomerase and Grp58 were significantly up‐regulated in SOD1 rats and mice. Western blotting has also revealed up‐regulation of BiP, ATF6α, CHOP, and caspase‐12, the latter two proteins being thought to initiate entry into the apoptotic pathway. This is currently being investigated further.
Conclusion: These findings suggest that the UPR is initiated in ALS which may contribute to the initiation of the apoptotic pathway.
C45 MONOMERIC/MISFOLDED SOD1 DETECTED IN MOUSE MODELS OF ALS USING A DESIGNED ANTIBODY
Chakrabartty A, Rakhit R, Cashman NR, Robertson J, Griffin J & Horne P
University of Toronto, Toronto, Canada
E‐mail address for correspondence: [email protected]
Background: Like other neurodegenerative diseases, protein aggregation is an important feature of the disease pathology. Inclusion bodies that are immunoreactive for SOD1 are present in both human cases of SOD1‐ALS and in animal ALS models that express human mutant SOD1 on a normal mouse genetic background. Toxicity of these cytoplasmic aggregates is thought to arise from aberrant interactions with the protein‐folding chaperone system or from inhibition of proteasomes. Toxicity has also been proposed to result from aberrant interactions with mitochondrial proteins such as Tom20 or Bcl‐2 because SOD1 has been detected in mitochondria from the spinal cord and brain, and mitochondrial vacuolization is an early event in ALS models. These interactions constitute a gain‐of‐function since interactions with these proteins have been unreported under normal conditions. SOD1 must, by inference, expose novel sites of interaction, but this has not been demonstrated in vivo.
Objectives: To create an antibody that selectively recognizes misfolded SOD1, and to examine whether misfolded SOD1 exists in vivo.
Methods: We have previously shown that mutant SOD1 is more aggregation prone than wild‐type SOD1 in vitro and both go through a monomeric intermediate prior to aggregation (1). We identified an epitope that is exposed only in monomeric SOD1 and not in native dimeric SOD1. We then generated an antibody directed against this epitope and verified that it reacts only with monomeric/misfolded SOD1. Misfolding of SOD1 in the G93A and G37R SOD1 mutant mice was then probed using this antibody.
Results and discussion: Monomeric/misfolded SOD1 was found in both G93A and G37R SOD1 mutant expressing mice, but not in non‐transgenic littermates. This shows directly that SOD1‐ALS is a misfolding disease. Misfolded SOD1 localizes primarily to motor neurons. Misfolded SOD1 accumulates with age, appearing before onset of rear‐leg weakness. Mitochondrial localization of the majority of monomeric/misfolded SOD1 was confirmed by double staining with a Tom20 antibody. Extracellular misfolded SOD1 was also observed by exclusion of double staining with markers of CNS cell types (anti‐GFAP, anti‐Mac2).
References
- Rakhit R, Crow JP, Lepock JR, Kondejewski LH, Cashman NR, Chakrabartty A. J Biol Chem 2004;279:15499–504.