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Rationale: The ubiquitin-proteasome system (UPS) and the autophagic-lysosomal pathway are pivotal to proteostasis. Targeting these pathways is emerging as an attractive strategy for treating cancer. However, a significant proportion of patients who receive a proteasome inhibitor-containing regime show cardiotoxicity. Moreover, UPS and autophagic-lysosomal pathway defects are implicated in cardiac pathogenesis. Hence, a better understanding of the cross-talk between the 2 catabolic pathways will help advance cardiac pathophysiology and medicine. Objective: Systemic proteasome inhibition (PSMI) was shown to increase p62/SQSTM1 expression and induce myocardial macroautophagy. Here we investigate how proteasome malfunction activates cardiac autophagic-lysosomal pathway. Methods and Results: Myocardial macroautophagy, TFEB (transcription factor EB) expression and activity, and p62 expression were markedly increased in mice with either cardiomyocyte-restricted ablation of Psmc1 (an essential proteasome subunit gene) or pharmacological PSMI. In cultured cardiomyocytes, PSMI-induced increases in TFEB activation and p62 expression were blunted by pharmacological and genetic calcineurin inhibition and by siRNA-mediated Molcn1 silencing. PSMI induced remarkable increases in myocardial autophagic flux in wild type mice but not p62 null (p62-KO) mice. Bortezomib-induced left ventricular wall thickening and diastolic malfunction was exacerbated by p62 deficiency. In cultured cardiomyocytes from wild type mice but not p62-KO mice, PSMI induced increases in LC3-II flux and the lysosomal removal of ubiquitinated proteins. Myocardial TFEB activation by PSMI as reflected by TFEB nuclear localization and target gene expression was strikingly less in p62-KO mice compared with wild type mice. Conclusions: (1) The activation of cardiac macroautophagy by proteasomal malfunction is mediated by the Mocln1-calcineurin-TFEB-p62 pathway; (2) p62 unexpectedly exerts a feed-forward effect on TFEB activation by proteasome malfunction; and (3) targeting the Mcoln1 (mucolipin1)-calcineurin-TFEB-p62 pathway may provide new means to intervene cardiac autophagic-lysosomal pathway activation during proteasome malfunction.

The ubiquitin–proteasome system (UPS) and macroautophagy (autophagy) are central to normal proteostasis and interdependent in that autophagy is known to compensate for the UPS to alleviate ensuing proteotoxic stress that impairs cell function. UPS and autophagy dysfunctions are believed to have a major role in the pathomechanisms of neurodegenerative disease. Here we show that continued 26S proteasome dysfunction in mouse brain cortical neurons causes paranuclear accumulation of fragmented dysfunctional mitochondria, associated with earlier recruitment of Parkin and lysine 48-linked ubiquitination of mitochondrial outer membrane (MOM) proteins, including Mitofusin-2. Early events also include phosphorylation of p62/SQSTM1 (p62) and increased optineurin, as well as autophagosomal LC3B and removal of some mitochondria, supporting the induction of selective autophagy. Inhibition of the degradation of ubiquitinated MOM proteins with continued 26S proteasome dysfunction at later stages may impede efficient mitophagy. However, continued 26S proteasome dysfunction also decreases the levels of essential autophagy proteins ATG9 and LC3B, which is characterised by decreases in their gene expression, ultimately leading to impaired autophagy. Intriguingly, serine 351 phosphorylation of p62 did not enhance its binding to Keap1 or stabilise the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor in this neuronal context. Nrf2 protein levels were markedly decreased despite transcriptional activation of the Nrf2 gene. Our study reveals novel insights into the interplay between the UPS and autophagy in neurons and is imperative to understanding neurodegenerative disease where long-term proteasome inhibition has been implicated.

The ubiquitin proteasome system (UPS) is a fundamental cellular pathway, degrading most unwanted intracellular soluble proteins. Dysfunction of the UPS has been associated with normal aging as well as various age-related pathological conditions, including chronic human neurodegenerative diseases such as Alzheimer's and Parkinson's diseases, leading to a significant interest in the involvement of this degradative system in neurones. We previously reported that the 26S proteasome was essential for neuronal homeostasis and survival in mouse brains following conditional genetic homozygous knockout of a key subunit of the multi-meric 26S proteasome (19S ATPase Psmc1). Here, we investigated the effects of Psmc1 heterozygosity in the mouse brain and primary mouse embryonic fibroblasts. Neuropathologically and biochemically, Psmc1 heterozygous (Psmc1(+/-)) knockout mice were indistinguishable from wild-type mice. However, we report a novel age-related accumulation of intraneuronal lysine 48-specific polyubiquitin-positive granular staining in both wild-type and heterozygous Psmc1 knockout mouse brain. In Psmc1(+/-) MEFs, we found a significant decrease in PSMC1 levels, altered 26S proteasome assembly and a notable G2/M cell cycle arrest that was not associated with an increase in the cell cycle regulatory protein p21. The disturbance in cell cycle progression may be responsible for the growth inhibitory effects in Psmc1(+/-) MEFs.

Collapsin response mediator protein 2 (CRMP2) is an abundant brain-enriched protein that regulates neurite outgrowth. It is phosphorylated by Cdk5 and GSK3, and these modifications are abnormally high in the brains of Alzheimer's disease (AD) patients. Increased phosphorylation of CRMP2 is also apparent in mouse models of AD that express mutated AβPP and PSEN1, but not AβPP or tau alone, where it is detectable before the appearance of amyloid plaques and neurofibrillary tangles, suggesting it is an early event in AD pathogenesis. Here, we have extended these observations by showing that CRMP2 is not hyperphosphorylated in mice overexpressing mutated PSEN1 alone, or in cultured neurons treated with soluble, oligomeric Aβ42 peptide. Similarly, CRMP2 phosphorylation was not increased in a mouse model of severe neurodegeneration (PMSC-1 knockout) or in cultured neurons subjected to neurotoxic concentrations of NMDA or staurosporine. Most interestingly, CRMP2 phosphorylation was not increased in frontal cortex from patients with frontotemporal lobar degeneration associated with mutations in MAPT or with Pick bodies. Together, these observations are consistent with the hypothesis that abnormal phosphorylation of CRMP2 is specific to AD and occurs downstream of excessive processing of AβPP, but that neither excessive Aβ42 peptide nor neurotoxicity alone are sufficient to promote hyperphosphorylation.

The major human neurodegenerative diseases are characterised by ubiquitin-positive intraneuronal inclusions, however the precise nature of the ubiquitin modifications in these structures is unclear. Using a monoclonal antibody specific for Lys63-linked polyubiquitin we have performed the first immunohistochemical analysis of linkage-specific ubiquitination in vivo associated with neurodegeneration. Immunoreactivity was detected within the pathological lesions of Alzheimer's, Huntington's and Parkinson's disease brains, although staining of Lewy bodies in the substantia nigra in Parkinson's disease was rare, indicating a selective involvement of Lys63-linked polyubiquitin in inclusion biogenesis in this disorder. Immunoreactivity was also a feature in neurons of proteasome-depleted mice, suggesting a proteasomal contribution to the degradation of Lys63-linked polyubiquitinated proteins in vivo.

In the past twenty years, evidence has accumulated to show that ubiquitinated proteins are a consistent feature of the intraneuronal protein aggregates (inclusions) that characterize chronic neurodegenerative disease. These findings may indicate that age-related dysfunction of the 26S proteasome may be central to disease pathogenesis. The aggregate-prone proteins can also be eliminated by autophagy. We have used the Cre-recombinase/loxP genetic approach to ablate the proteasomal Psmc1 ATPase gene and deplete 26S proteasomes in neurons in different regions of the brain to mimic neurodegeneration. Deletion of the gene in dopaminergic neurons in the substantia nigra generates a new model of Parkinson disease. Ablation of the gene in the forebrain creates the first model of dementia with Lewy bodies. In both neuroanatomical regions, gene ablation causes the formation of Lewy-like inclusions together with extensive neurodegeneration. There is some evidence for neuronal autophagy in areas adjacent to inclusions. The models indicate that neuronal loss in neurodegenerative diseases can be attributed to proteasomal malfunction accompanied by Lewy-like inclusions as seen in dementia with Lewy bodies and Parkinson disease.

Neuropathological investigations have identified major hallmarks of chronic neurodegenerative disease. These include protein aggregates called Lewy bodies in dementia with Lewy bodies and Parkinson's disease. Mutations in the α-synuclein gene have been found in familial disease and this has led to intense focused research in vitro and in transgenic animals to mimic and understand Parkinson's disease. A decade of transgenesis has lead to overexpression of wild type and mutated α-synuclein, but without faithful reproduction of human neuropathology and movement disorder. In particular, widespread regional neuronal cell death in the substantia nigra associated with human disease has not been described. The intraneuronal protein aggregates (inclusions) in all of the human chronic neurodegenerative diseases contain ubiquitylated proteins. There could be several reasons for the accumulation of ubiquitylated proteins, including malfunction of the ubiquitin proteasome system (UPS). This hypothesis has been genetically tested in mice by conditional deletion of a proteasomal regulatory ATPase gene. The consequences of gene ablation in the forebrain include extensive neuronal death and the production of Lewy-like bodies containing ubiquitylated proteins as in dementia with Lewy bodies. Gene deletion in catecholaminergic neurons, including in the substantia nigra, recapitulates the neuropathology of Parkinson's disease.

Complex intrinsic and extrinsic mechanisms determine neural cell fate during development of the nervous system. Using Id4 deficient mice, we show that Id4 is required for normal development of the central nervous system (CNS), timing neural differentiation in the developing forebrain. In the absence of Id4, the ventricular zone of the neocortex, future hippocampus as well as lateral and medial ganglionic eminences exhibited a 20-30% reduction in mitotic neural precursor cells (NPCs). Although the number of apoptotic cells was significantly increased, the neocortex of Id4(-/-) embryos was consistently thicker due to premature neuronal differentiation, which resulted in an increase in early-born neurons in the adult Id4(-/-) cortex. Late-born cortical neurons and astrocytes in the cortex, septum, hippocampus and caudate putamen of Id4(-/-) adult brains were decreased, however, likely due to the depletion of the NPC pool. Consequently, adult Id4(-/-) brains were smaller and exhibited enlarged ventricles. In vitro analysis of neurosphere cultures revealed that proliferation of Id4-deficient NPCs was impaired and that BMP2-mediated astrocyte differentiation was accelerated in the absence of Id4. Together, these in vivo and in vitro data suggest a crucial role for Id4 in regulating NPC proliferation and differentiation.

The ubiquitin-proteasome system and autophagy are crucially important for proteostasis in cells. These pathways are interdependent, and dysfunction in either pathway causes accumulation of ubiquitin-positive aggregates, a hallmark of human pathological conditions. To elucidate in vivo compensatory action(s) against proteasomal dysfunction, we developed mice with reduced proteasome activity in their livers. The mutant mice exhibited severe liver damage, accompanied by formation of aggregates positive for ubiquitin and p62/Sqstm1, an adaptor protein for both selective autophagy and the anti-oxidative Keap1-Nrf2 pathway. These aggregates were selectively entrapped by autophagosomes, and pathological features of livers with impaired proteasome activity were exacerbated by simultaneous suppression of autophagy. In contrast, concomitant loss of p62/Sqstm1 had no apparent effect on the liver pathology though p62/Sqstm1 was indispensable for the aggregates formation. Furthermore, defective proteasome function led to transcriptional activation of the Nrf2, which served as a physiological adaptation. Our in vivo data suggest that cells contain networks of cellular defense mechanisms against defective proteostasis.

Chronic neurodegenerative disease is characterized by extensive regional loss of neurons in the brain and neuropathological hallmarks in surviving neurones. Genetic modelling by overexpression of hallmark proteins does not produce extensive neurodegeneration, whereas genetic deletion of neuronal 26S proteasomes does, as well as some hallmarks of human disease. Abbreviations: Aβ, amyloid β-peptide; AD, Alzheimer's disease; APP, amyloid precursor protein; TDP43, TAR DNA-binding protein 43

Key Points Ubiquitin is a highly conserved 76 amino-acid protein that covalently attaches to protein substrates targeted for degradation by the 26S proteasome. The coordinated effort of a series of enzymes, including an activating enzyme (E1), a conjugating enzyme (E2) and a ligase (E3), uses ATP to ultimately form an isopeptide bond between ubiquitin and a substrate.Another class of enzymes called deubiquitylating enzymes (DUBs) deconstruct these linkages and also have an essential role in ubiquitin function. In addition, ubiquitin-like proteins (UBLs), including NEDD8, SUMO and ISG15, share a characteristic three-dimensional fold with ubiquitin but have their own dedicated enzyme cascades and distinct (although sometimes overlapping) biological functions.The ubiquitin–proteasome system (UPS) and UBL conjugation pathways have important roles in various human diseases, including numerous types of cancer, cardiovascular disease, viral diseases and neurodegenerative disorders. The proteasome inhibitor bortezomib (Velcade; Millennium Pharmaceuticals) is the first clinically validated drug to target the UPS and is approved for the treatment of multiple myeloma. This suggests that other diseases may conceivably be targeted by modulating components of the UPS and UBL conjugation pathways using small-molecule inhibitors.A significant hurdle to identifying drug-like inhibitors of enzyme targets within the UPS and UBL conjugation pathways is the limited understanding of the molecular mechanisms and biological consequences of UBL conjugation.Here, we provide an overview of the enzyme classes in the UPS and UBL pathways that are potential therapeutic targets, and highlight considerations that are important for drug discovery. We also discuss the progress in the development of small-molecule inhibitors, and review developments in understanding of the role of the components of the UPS and the UBL pathways in disease and their potential for therapeutic intervention., The ubiquitin–proteasome system (UPS) and ubiquitin-like protein (UBL) conjugation pathways are integral to cellular protein homeostasis, and their functional importance in various diseases, including cancer, cardiovascular disease and neurodegenerative disorders, is now beginning to emerge. Brownell and colleagues review developments in understanding of the role of the components of the UPS and the UBL pathways in disease and their potential for therapeutic intervention., The ubiquitin–proteasome system (UPS) and ubiquitin-like protein (UBL) conjugation pathways are integral to cellular protein homeostasis. The growing recognition of the fundamental importance of these pathways to normal cell function and in disease has prompted an in-depth search for small-molecule inhibitors that selectively block the function of these pathways. However, our limited understanding of the molecular mechanisms and biological consequences of UBL conjugation is a significant hurdle to identifying drug-like inhibitors of enzyme targets within these pathways. Here, we highlight recent advances in understanding the role of some of these enzymes and how these new insights may be the key to developing novel therapeutics for diseases including immuno-inflammatory disorders, cancer, infectious diseases, cardiovascular disease and neurodegenerative disorders.

A generality has been that polyubiquitin chain linkage can differentially address proteins for various physiological processes. 26S proteasomal degradation is the most established function of ubiquitin signalling, classically linked to Lys48 polyubiquitin chains. The other well-characterised polyubiquitin linkage, via Lys63, mediates nonproteolytic functions. However, there are five other lysine residues and ubiquitin's amino terminus which can participate in polyubiquitination. Our 26S proteasome knockout mouse provides a unique opportunity to comprehensively investigate the ubiquitin signals in their physiological context in neurones following genetic inhibition of the proteasome, using quantitative mass spectrometry of ubiquitin linkage-specific signature peptides. We provide the first evidence for diverse polyubiquitin chains in mammalian neurones in vivo and show that polyubiquitin linked via Lys6, Lys11, Lys29 and Lys48, but not Lys63, accumulates upon 26S proteasome dysfunction. This adaptable nature of ubiquitin signals for proteasomal targeting could reflect the extensive cellular processes which are regulated by proteasome proteolysis and/or may involve specific ubiquitin linkage preferences for subsets of proteins in mammalian neurones. Our molecular pathological findings make a significant contribution to the understanding of ubiquitin signalling in ubiquitin-proteasome function.

The significance of the accumulation of ubiquitin-positive intraneuronal inclusions in the brains of those affected with different neurodegenerative diseases is currently unclear. While one interpretation is that the disease mechanism(s) involves dysfunction of an ubiquitin-mediated process, such as the ubiquitin-proteasome system, the inclusions are also found in surviving neurones, suggesting a possible neuroprotective role. Here we review recent evidence in support of these seemingly opposing notions gleaned from cell and animal models as well as investigations of patient samples, with particular emphasis on studies relevant to Parkinson's disease.

Ubiquitin-positive intraneuronal inclusions are a consistent feature of the major human neurodegenerative diseases, suggesting that dysfunction of the ubiquitin proteasome system is central to disease etiology. Research using inhibitors of the 20S proteasome to model Parkinson's disease is controversial. We report for the first time that specifically 26S proteasomal dysfunction is sufficient to trigger neurodegenerative disease. Here, we describe novel conditional genetic mouse models using the Cre/loxP system to spatially restrict inactivation ofPsmc1(Rpt2/S4) to neurons of either the substantia nigra or forebrain (e.g., cortex, hippocampus, and striatum). PSMC1 is an essential subunit of the 26S proteasome andPsmc1conditional knock-out mice display 26S proteasome depletion in targeted neurons, in which the 20S proteasome is not affected. Impairment of specifically ubiquitin-mediated protein degradation caused intraneuronal Lewy-like inclusions and extensive neurodegeneration in the nigrostriatal pathway and forebrain regions. Ubiquitin and α-synuclein neuropathology was evident, similar to human Lewy bodies, but interestingly, inclusion bodies contained mitochondria. We support this observation by demonstrating mitochondria in an early form of Lewy body (pale body) from Parkinson's disease patients. The results directly confirm that 26S dysfunction in neurons is involved in the pathology of neurodegenerative disease. The model demonstrates that 26S proteasomes are necessary for normal neuronal homeostasis and that 20S proteasome activity is insufficient for neuronal survival. Finally, we are providing the first reproducible genetic platform for identifying new therapeutic targets to slow or prevent neurodegeneration.

As in all other mammalian tissues, the UPS (ubiquitin–proteasome system) is fundamental to normal brain function. A consistent feature of the major human neurodegenerative disorders is the accumulation of disease-related proteins, in non-native conformations, as protein aggregates within neurons or glial cells. Often the proteins in these aggregates are post-translationally conjugated with ubiquitin, suggesting a possible link between pathological protein-aggregation events in the nervous system and dysfunction of the UPS. Genetic evidence clearly demonstrates that disruption of ubiquitin-mediated processes can lead to neurodegeneration; however, the relationship between the UPS and idiopathic neurodegenerative disorders is less clear. In the latter cases, although a number of different mechanisms could potentially contribute to dysfunction of the UPS and promote the neurodegenerative process, whether UPS dysfunction is causally related to disease pathogenesis, or alternatively arises as a result of the pathological state, and indeed whether ubiquitinated inclusions are harmful or beneficial to cells, remains to be clarified.

Parkinson's disease (PD) is characterized by the progressive degeneration of substantia nigra pars compacta (SNpc) dopaminergic neurones and the formation of Lewy bodies (LB) in a proportion of the remaining neurones. α-synuclein is the main component of LB, but the pathological mechanisms that lead to neurodegeneration associated with LB formation remain unclear. Three pivotal elements have emerged in the development of PD: α-synuclein, mitochondria and protein degradation systems. We previously reported a unique model, created by conditional genetic depletion of 26S proteasomes in the SNpc of mice, which mechanistically links these three elements with the neuropathology of PD: progressive neurodegeneration and intraneuronal inclusion formation. Using this model, we tested the hypothesis that α-synuclein was essential for the formation of inclusions and neurodegeneration caused by 26S proteasomal depletion. We found that both of these processes were independent of α-synuclein. This provides an important insight into the relationship between the proteasome, α-synuclein, inclusion formation and neurodegeneration. We also show that the autophagy-lysosomal pathway is not activated in 26S proteasome-depleted neurones. This leads us to suggest that the paranuclear accumulation of mitochondria in inclusions in our model may reflect a role for the ubiquitin proteasome system in mitochondrial homeostasis and that neurodegeneration may be mediated through mitochondrial factors linked to inclusion biogenesis.

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.