The accumulation of aggregate-prone proteins in neurons is a hallmark of many neurodegenerative disorders, including the polyglutamine tract expansion diseases such as Huntington's disease and spinocerebellar ataxia type 3, familial forms of Parkinson's disease and amyotrophic lateral sclerosis [caused by point mutations in α-synuclein and superoxide dismutase 1 (SOD1), respectively]. These abnormal proteins are thought to cause disease via toxic gain-of-function mechanisms. Thus, one rational approach to combating their toxicity is to reduce the cellular content of the mutant protein by accelerating their degradation.
The two major routes for protein degradation within mammalian cells are macroautophagy and the ubiquitin–proteasome system. Degradation by the macroautophagy–lysosomal pathway begins with the formation of double-layered autophagosomes that enclose portions of cytoplasm. These vacuoles ultimately fuse with lysosomes, and the cytosolic components are degraded by its various lysosomal acid hydrolases. Macroautophagy (which we call here autophagy) is a key mechanism for the clearance of many aggregation-prone (or aggregated) proteins associated with neurodegenerative diseases, including mutant forms of huntingtin, SOD1 and α-synuclein (1
). Furthermore, activation of this autophagic process (e.g. by rapamycin) enhances the removal of the aggregate-prone proteins such as mutant huntingtin and attenuates its toxicity in cell and animal models (2
). The ubiquitin–proteasome pathway also plays a critical role in the selective degradation of misfolded, mutated or damaged proteins. Such proteins are targeted for rapid hydrolysis by a series of enzymes that covalently attach a chain of ubiquitin molecules onto lysine residues on the protein. This polyubiquitin chain serves as a recognition motif for binding of the protein to the 26S proteasome. The ubiquitinated proteins are digested to small peptides within the core 20S proteasome particle. This barrel-shaped particle contains three types of peptidase sites that can cleave nearly all peptide bonds in proteins. The short (2
) residue peptides typically released by the proteasome are then rapidly hydrolyzed to amino acids by cytosolic endo- and aminopeptidases.
The ubiquitin–proteasome pathway can efficiently digest soluble misfolded proteins, but once proteins such as huntingtin are aggregated, the autophagic/lysosomal process assumes primary importance in their clearance from the cytosol (3
). However, in the case of proteins containing polyglutamine tracts, eukaryotic proteasomes can cleave only very poorly (if at all) within polyglutamine sequences (6
). Consequently, in degrading huntingtin, the 26S proteasome appears to release polyglutamine-rich fragments for digestion by cytosolic peptidases (6
). Because they lack extensive flanking sequences, such peptides have a very strong tendency to aggregate (probably even stronger than that of the full-length protein). Therefore, the rapid hydrolysis of these polyglutamine-rich peptides seems likely to be important in preventing or retarding the progression of polyglutamine disorders. Most larger peptides released by proteasomes are initially digested by endopeptidases (8
), and the resulting shorter peptides are rapidly hydrolyzed to amino acids by various cytosolic aminopeptidases (11
Surprisingly, only one cytosolic peptidase, puromycin-sensitive aminopeptidase (PSA, also termed cytosol alanyl aminopeptidase, human gene symbol NPEPPS), was found to be able to digest short polyglutamine peptides (15
). PSA is a ubiquitous, 100 kDa, Zn2+
metallopeptidase present in high concentrations in the brain (especially in the striatum, the hippocampus and the cerebellum) (16
). Although PSA was initially described as an enkephalin-degrading enzyme (18
), its localization predominantly in the cytoplasm and its broad distribution in tissues argue against such a function. Instead, a role for PSA in digesting proteasome products to amino acids or antigenic peptides presented on MHC Class I molecules seems most likely based on its cytosolic location and its ability to digest diverse sequences (12
). In fact, we have found that PSA is the dominant intracellular peptidase in degrading a large variety of dipeptides (R.H. and A.L.G., unpublished data).
These observations suggest that a loss of PSA function could lead to a toxic accumulation of fragments of normal gene products and increase the susceptibility to polyglutamine diseases. In fact, PSA-deficient mice display behavioural and neurological abnormalities (17
) including movement disorders that perhaps are related to the failure to rapidly clear peptides released by the proteasomes which could have deleterious effects. Interestingly, the expression of PSA was found to be highly induced in PC12 cells upon expression of huntingtin exon 1 containing an expanded, but not a normal length, polyQ sequence (22
In addition, PSA is induced in the CNS in a mouse model of fronto-temporal dementia expressing a mutated tau (16
). Furthermore, overexpression of PSA had a remarkable ability to protect against neurodegeneration induced by misexpression of the longest isoform of human tau in Drosophila
The present studies were undertaken to test whether PSA may play an important role in the clearance of polyQ-rich fragments, polyQ-containing proteins or other mutant polypeptides associated with neurodegenerative diseases. We have tested whether altering the activity or content of PSA modifies aggregate accumulation and toxicity of different forms of mutant huntingtin exon 1 in cell culture and in vivo models of neurodegenerative diseases. Using a variety of approaches and experimental systems, we show that PSA is an important determinant of aggregate content and of huntingtin exon 1 toxicity. Surprisingly, this ability of PSA to reduce the content of aggregates formed by huntingtin exon 1, as well as another expanded polyQ protein, ataxin-3, correlated with an ability of PSA to promote autophagy. Indeed, PSA was found to enhance the clearance of a range of aggregation-prone proteins associated with neurodegenerative diseases, including some proteins lacking polyglutamine expansions, and to increase intracellular protein degradation by promoting autophagy. These studies have uncovered a surprising new function of this cytosolic peptidase in regulating autophagy and a remarkable ability of PSA activity to influence aggregate content and toxicity in a variety of neurodegenerative diseases.