UCH-L1 Inhibition Suppresses tau Aggresome Formation during Proteasomal Impairment

In conditions of proteasomal impairment, the damaged or misfolded proteins, collectively known as aggresome, can accumulate in the perinuclear space and be subsequently eliminated by autophagy. Abnormal aggregation of microtubule-associated protein tau in the cytoplasm is a common neuropathological feature of tauopathies. The deficiency in ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), a proteasomal deubiquitinating enzyme, is closely related to tau aggregation; however, the associated mechanisms remain unclear. Here, we showed that UCH-L1 inhibition interrupts proteasomal impairment-induced tau aggresome formation. By reducing the production of lysine (K63)-linked ubiquitin chains, UCH-L1 inhibition decreases HDAC6 deacetylase activity and attenuates the interaction of HDAC6 and tau protein, finally leading to tau aggresome formation impairment. All these results indicated that UCH-L1 plays a key role in the process of tau aggresome formation by regulating HDAC6 deacetylase activity and implied that UCH-L1 may act as a signaling molecule to coordinate the effects of the ubiquitin-proteasome system and the autophagy-lysosome pathway, which mediate protein aggregates degradation in the cytoplasm.     

Protein aggregates are recruited to aggresome by histone deacetylase 6 via unanchored ubiquitin C termini

The aggresome pathway is activated when proteasomal clearance of misfolded proteins is hindered. Misfolded polyubiquitinated protein aggregates are recruited and transported to the aggresome via the microtubule network by a protein complex consisting of histone deacetylase 6 (HDAC6) and the dynein motor complex. The current model suggests that HDAC6 recognizes protein aggregates by binding directly to polyubiquitinated proteins. Here, we show that there are substantial amounts of unanchored ubiquitin in protein aggregates with solvent-accessible C termini. The ubiquitin-binding domain (ZnF-UBP) of HDAC6 binds exclusively to the unanchored C-terminal diglycine motif of ubiquitin instead of conjugated polyubiquitin. The unanchored ubiquitin C termini in the aggregates are generated in situ by aggregate-associated deubiquitinase ataxin-3. These results provide structural and mechanistic bases for the role of HDAC6 in aggresome formation and further suggest a novel ubiquitin-mediated signaling pathway, where the exposure of ubiquitin C termini within protein aggregates enables HDAC6 recognition and transport to the aggresome.     

HDAC6 Regulates Mutant SOD1 Aggregation through Two SMIR Motifs and Tubulin Acetylation

Histone deacetylase 6 (HDAC6) is a tubulin deacetylase that regulates protein aggregation and turnover. Mutations in Cu/Zn superoxide dismutase (SOD1) linked to familial amyotrophic lateral sclerosis (ALS) make the mutant protein prone to aggregation. However, the role of HDAC6 in mutant SOD1 aggregation and the ALS etiology is unclear. Here we report that HDAC6 knockdown increased mutant SOD1 aggregation in cultured cells. Different from its known role in mediating the degradation of poly-ubiquitinated proteins, HDAC6 selectively interacted with mutant SOD1 via two motifs similar to the SOD1 mutant interaction region (SMIR) that we identified previously in p62/sequestosome 1. Expression of the aggregation-prone mutant SOD1 increased α-tubulin acetylation, and the acetylation-mimicking K40Q α-tubulin mutant promoted mutant SOD1 aggregation. Our results suggest that ALS-linked mutant SOD1 can modulate HDAC6 activity and increase tubulin acetylation, which, in turn, facilitates the microtubule- and retrograde transport-dependent mutant SOD1 aggregation. HDAC6 impairment might be a common feature in various subtypes of ALS.     

Inter-relationship of Histone Deacetylase-6 with Tau-cytoskeletal organization and remodeling

Cytoskeletal elements are the key players in cellular integrity, structure, signalling and migration. Each cytoskeletal element comprises of properties with respect to its structure and stability, which serve a specific array of functions. These structures are highly dynamic and regulated by modulation via direct interaction or post-translational modifications. HDAC6 is a cytoplasmic deacetylase known to regulate a wide range of cellular functions either through its deacetylase activity or direct interaction via its C-terminal ZnF UBP domain. HDAC6 has been widely studied for its role in aggresome formation, which acts as a protective mechanism upon protein aggregation. HDAC6 is known to play a critical role in the regulation of cytoskeletal elements-microtubules and actin filaments. This review summarizes the regulatory role of HDAC6 in cytoskeletal remodeling and dynamics of neuronal cells and its significance in neurodegenerative diseases.     

Characterization and dynamics of aggresome formation by a cytosolic GFP-chimera.

Formation of a novel structure, the aggresome, has been proposed to represent a general cellular response to the presence of misfolded proteins (Johnston, J.A., C.L. Ward, and R.R. Kopito. 1998. J. Cell Biol. 143:1883-1898; Wigley, W.C., R.P. Fabunmi, M.G. Lee, C.R. Marino, S. Muallem, G.N. DeMartino, and P.J. Thomas. 1999. J. Cell Biol. 145:481-490). To test the generality of this finding and characterize aspects of aggresome composition and its formation, we investigated the effects of overexpressing a cytosolic protein chimera (GFP-250) in cells. Overexpression of GFP-250 caused formation of aggresomes and was paralleled by the redistribution of the intermediate filament protein vimentin as well as by the recruitment of the proteasome, and the Hsp70 and the chaperonin systems of chaperones. Interestingly, GFP-250 within the aggresome appeared not to be ubiquitinated. In vivo time-lapse analysis of aggresome dynamics showed that small aggregates form within the periphery of the cell and travel on microtubules to the MTOC region where they remain as distinct but closely apposed particulate structures. Overexpression of p50/dynamitin, which causes the dissociation of the dynactin complex, significantly inhibited the formation of aggresomes, suggesting that the minus-end-directed motor activities of cytoplasmic dynein are required for aggresome formation. Perinuclear aggresomes interfered with correct Golgi localization and disrupted the normal astral distribution of microtubules. However, ER-to-Golgi protein transport occurred normally in aggresome containing cells. Our results suggest that aggresomes can be formed by soluble, nonubiquitinated proteins as well as by integral transmembrane ubiquitinated ones, supporting the hypothesis that aggresome formation might be a general cellular response to the presence of misfolded proteins.     

The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress

The efficient clearance of cytotoxic misfolded protein aggregates is critical for cell survival. Misfolded protein aggregates are transported and removed from the cytoplasm by dynein motors via the microtubule network to a novel organelle termed the aggresome where they are processed. However, the means by which dynein motors recognize misfolded protein cargo, and the cellular factors that regulate aggresome formation, remain unknown. We have discovered that HDAC6, a microtubule-associated deacetylase, is a component of the aggresome. We demonstrate that HDAC6 has the capacity to bind both polyubiquitinated misfolded proteins and dynein motors, thereby acting to recruit misfolded protein cargo to dynein motors for transport to aggresomes. Indeed, cells deficient in HDAC6 fail to clear misfolded protein aggregates from the cytoplasm, cannot form aggresomes properly, and are hypersensitive to the accumulation of misfolded proteins. These findings identify HDAC6 as a crucial player in the cellular management of misfolded protein-induced stress.     

Impaired protein aggregate handling and clearance underlie the pathogenesis of p97/VCP-associated disease

Mutations in p97/VCP cause the multisystem disease inclusion body myopathy, Paget disease of the bone and frontotemporal dementia (IBMPFD). p97/VCP is a member of the AAA+ (ATPase associated with a variety of activities) protein family and has been implicated in multiple cellular processes. One pathologic feature in IBMPFD is ubiquitinated inclusions, suggesting that mutations in p97/VCP may affect protein degradation. The present study shows that IBMPFD mutant expression increases ubiquitinated proteins and susceptibility to proteasome inhibition. Co-expression of an aggregate prone protein such as expanded polyglutamine in IBMPFD mutant cells results in an increase in aggregated protein that localizes to small inclusions instead of a single perinuclear aggresome. These small inclusions fail to co-localize with autophagic machinery. IBMPFD mutants avidly bind to these small inclusions and may not allow them to traffic to an aggresome. This is rescued by HDAC6, a p97/VCP-binding protein that facilitates the autophagic degradation of protein aggregates. Expression of HDAC6 improves aggresome formation and protects IBMPFD mutant cells from polyglutamine-induced cell death. Our study emphasizes the importance of protein aggregate trafficking to inclusion bodies in degenerative diseases and the therapeutic benefit of inclusion body formation.     

Autophagy inhibition enhances vorinostat‐induced apoptosis via ubiquitinated protein accumulation

Autophagy is an evolutionarily conserved cell survival pathway that enables cells to recoup ATP and other critical biosynthetic molecules during nutrient deprivation or exposure to hypoxia, which are hallmarks of the tumour microenvironment. Autophagy has been implicated as a potential mechanism of resistance to anticancer agents as it can promote cell survival in the face of stress induced by chemotherapeutic agents by breaking down cellular components to generate alternative sources of energy. Disruption of autophagy with chloroquine (CQ) induces the accumulation of ubiquitin‐conjugated proteins in a manner similar to the proteasome inhibitor bortezomib (BZ). However, CQ‐induced protein accumulation occurs at a slower rate and is localized to lysosomes in contrast to BZ, which stimulates rapid buildup of ubiquitinated proteins and aggresome formation in the cytosol. The histone deacetylase (HDAC) inhibitor vorinostat (VOR) blocked BZ‐induced aggresome formation, but promoted CQ‐mediated ubiquitinated protein accumulation. Disruption of autophagy with CQ strongly enhanced VOR‐mediated apoptosis in colon cancer cells. Accordingly, knockdown of the essential autophagy gene Atg7 also sensitized cells to VOR‐induced apoptosis. Knockdown of HDAC6 greatly enhanced BZ‐induced apoptosis, but only marginally sensitized cells to CQ. Subsequent studies determined that the CQ/VOR combination promoted a large increase in superoxide generation that was required for ubiquitinated protein accumulation and cell death. Finally, treatment with the CQ/VOR combination significantly reduced tumour burden and induced apoptosis in a colon cancer xenograft model. Collectively, our results establish that inhibition of autophagy with CQ induces ubiquitinated protein accumulation and VOR potentiates CQ‐mediated aggregate formation, superoxide generation and apoptosis.     

Aggresome-like structure induced by isothiocyanates is novel proteasome-dependent degradation machinery

Unwanted or misfolded proteins are either refolded by chaperones or degraded by the ubiquitin-proteasome system (UPS). When UPS is impaired, misfolded proteins form aggregates, which are transported along microtubules by motor protein dynein towards the juxta-nuclear microtubule-organizing center to form aggresome, a single cellular garbage disposal complex. Because aggresome formation results from proteasome failure, aggresome components are degraded through the autophagy/lysosome pathway. Here we report that small molecule isothiocyanates (ITCs) can induce formation of aggresome-like structure (ALS) through covalent modification of cytoplasmic α- and β-tubulin. The formation of ALS is related to neither proteasome inhibition nor oxidative stress. ITC-induced ALS is a proteasome-dependent assembly for emergent removal of misfolded proteins, suggesting that the cell may have a previously unknown strategy to cope with misfolded proteins.     

The Guanine Nucleotide Exchange Factor Kalirin-7 Is a Novel Synphilin-1 Interacting Protein and Modifies Synphilin-1 Aggregate Transport and Formation

Synphilin-1 has been identified as an interaction partner of α-synuclein, a key protein in the pathogenesis of Parkinson disease (PD). To further explore novel binding partners of synphilin-1, a yeast two hybrid screening was performed and kalirin-7 was identified as a novel interactor. We then investigated the effect of kalirin-7 on synphilin-1 aggregate formation. Coexpression of kalirin-7 and synphilin-1 caused a dramatic relocation of synphilin-1 cytoplasmic small inclusions to a single prominent, perinuclear inclusion. These perinuclear inclusions were characterized as being aggresomes according to their colocalization with microtubule organization center markers, and their formation was microtubule-dependent. Furthermore, kalirin-7 increased the susceptibility of synphilin-1 inclusions to be degraded as demonstrated by live cell imaging and quantification of aggregates. However, the kalirin-7-mediated synphilin-1 aggresome response was not dependent on the GEF activity of kalirin-7 since various dominant negative small GTPases could not inhibit the formation of aggresomes. Interestingly, the aggresome response was blocked by HDAC6 catalytic mutants and the HDAC inhibitor trichostatin A (TSA). Moreover, kalirin-7 decreased the level of acetylated α-tubulin in response to TSA, which suggests an effect of kalirin-7 on HDAC6-mediated protein transportation and aggresome formation. In summary, this is the first report demonstrating that kalirin-7 leads to the recruitment of synphilin-1 into aggresomes in a HDAC6-dependent manner and also links kalirin-7 to microtubule dynamics.     

The histone deacetylase inhibitor Scriptaid improves in vitro developmental competence of ovine somatic cell nuclear transferred embryos

Although the success rate of sheep cloning remains extremely low, using a histone deacetylase (HDAC) inhibitor to increase histone acetylation in SCNT embryos has significantly enhanced developmental competence in several species. The objective was to determine whether HDAC inhibitors trichostatin A (TSA) and the novel inhibitor Scriptaid enhance cloning efficiency in sheep cumulus cell (passage 2) reconstructed embryos. In this study, 0.2 μmol/L Scriptaid yielded a high blastocyst development rate, almost twice that of the untreated group (25/103 [24.3%] vs. 12/101 [11.9%]; P < 0.05). Furthermore, 0.2 μmol/L Scriptaid was more effective than 0.05 μmol/L TSA in terms of the blastocyst percentage for cloned ovine embryos in vitro (17/66 [25.7%] vs. 11/65 [16.8%]; P < 0.05). Furthermore, treatment with Scriptaid increased acetylation (compared with the Control, P < 0.05) at lysine residue 12 of histone H4 (acH4K12) and lysine residue 9 of histone H3 (acH3K9) in one-, two-, four-, and eight-cell stages, as well as blastocyst stages, in cloned embryos. In conclusion, Scriptaid was more effective than TSA to enhance in vitro developmental competence in ovine SCNT embryos; furthermore, Scriptaid improved epigenetic status.     

Chaperone-mediated hierarchical control in targeting misfolded proteins to aggresomes

Protein misfolding is a common event in living cells. Molecular chaperones not only assist protein folding; they also facilitate the degradation of misfolded polypeptides. When the intracellular degradative capacity is exceeded, juxtanuclear aggresomes are formed to sequester misfolded proteins. Despite the well-established role of chaperones in both protein folding and degradation, how chaperones regulate the aggregation process remains controversial. Here we investigate the molecular mechanisms underlying aggresome formation in mammalian cells. Analysis of the chaperone requirements for the fate of misfolded proteins reveals an unexpected role of heat shock protein 70 (Hsp70) in promoting aggresome formation. This proaggregation function of Hsp70 relies on the interaction with the cochaperone ubiquitin ligase carboxyl terminal of Hsp70/Hsp90 interacting protein (CHIP). Disrupting Hsp70-CHIP interaction prevents the aggresome formation, whereas a dominant-negative CHIP mutant sensitizes the aggregation of misfolded protein. This accelerated aggresome formation also relies on the stress-induced cochaperone Bcl2-associated athanogene 3. Our results indicate that a hierarchy of cochaperone interaction controls different aspects of the intracellular protein triage decision, extending the function of Hsp70 from folding and degradation to aggregation.     

aPKC Phosphorylation of HDAC6 Results in Increased Deacetylation Activity

The Class II histone deacetylase, HDAC6, has been shown to be involved in cell motility, aggresome formation and mitochondria transport. HDAC6 deacetylase activity regulates α-tubulin acetylation levels and thus plays a critical role in these processes. In turn, HDAC6 activity can be regulated by interaction with various proteins including multiple kinases. Kinase mediated phosphorylation of HDAC6 can lead to either increased or reduced activity. Our previous research has shown that sequestosome1/p62 (SQSTM1/p62) interacts with HDAC6 and regulates its activity. As SQSTM1/p62 is a scaffolding protein known to interact directly with the zeta isoform of Protein Kinase C (PKCζ), we sought to examine if HDAC6 could be a substrate for PKCζ phosphorylation and if so, how its activity might be regulated. Our data demonstrate that HDAC6 is not only present in a protein complex with PKCζ but can also be phosphorylated by PKCζ. We also show that specific phosphorylation of HDAC6 by PKCζ increases HDAC6 deacetylase activity resulting in reduced acetylated tubulin levels. Our findings provide novel insight into the molecular mechanism by which HDAC6, PKCζ and SQSTM1/p62 function together in protein aggregate clearance. These results also highlight a new research direction which may prove fruitful for understanding the underlying cause of several neurodegenerative diseases.     

Aggresomes: A Cellular Response to Misfolded Proteins

Intracellular deposition of misfolded protein aggregates into ubiquitin-rich cytoplasmic inclusions is linked to the pathogenesis of many diseases. Why these aggregates form despite the existence of cellular machinery to recognize and degrade misfolded protein and how they are delivered to cytoplasmic inclusions are not known. We have investigated the intracellular fate of cystic fibrosis transmembrane conductance regulator (CFTR), an inefficiently folded integral membrane protein which is degraded by the cytoplasmic ubiquitin-proteasome pathway. Overexpression or inhibition of proteasome activity in transfected human embryonic kidney or Chinese hamster ovary cells led to the accumulation of stable, high molecular weight, detergent-insoluble, multiubiquitinated forms of CFTR. Using immunofluorescence and transmission electron microscopy with immunogold labeling, we demonstrate that undegraded CFTR molecules accumulate at a distinct pericentriolar structure which we have termed the aggresome. Aggresome formation is accompanied by redistribution of the intermediate filament protein vimentin to form a cage surrounding a pericentriolar core of aggregated, ubiquitinated protein. Disruption of microtubules blocks the formation of aggresomes. Similarly, inhibition of proteasome function also prevented the degradation of unassembled presenilin-1 molecules leading to their aggregation and deposition in aggresomes. These data lead us to propose that aggresome formation is a general response of cells which occurs when the capacity of the proteasome is exceeded by the production of aggregation-prone misfolded proteins.     

Inducible nitric oxide synthase aggresome formation is mediated by nitric oxide

Nitric oxide (NO) generated by inducible NO synthase (iNOS) contributes critically to inflammatory injury and host defense. While previously thought as a soluble protein, iNOS was recently reported to form aggresomes inside cells. But what causes iNOS aggresome formation is unknown. Here we provide evidence demonstrating that iNOS aggresome formation is mediated by its own product NO. Exposure to inflammatory stimuli (lipopolysaccharide and interferon-γ) induced robust iNOS expression in mouse macrophages. While initially existing as a soluble protein, iNOS progressively formed protein aggregates as a function of time. Aggregated iNOS was inactive. Treating the cells with the NOS inhibitor N-nitro-l-arginine methyl ester (L-NAME) blocked NO production from iNOS without affecting iNOS expression. However, iNOS aggregation in cells was prevented by L-NAME. The preventing effect of NO blockade on iNOS aggresome formation was directly observed in GFP-iNOS-transfected cells by fluorescence imaging. Moreover, iNOS aggresome formation could be recaptured by adding exogenous NO to L-NAME-treated cells. These studies demonstrate that iNOS aggresome formation is caused by NO. The finding that NO induces iNOS aggregation and inactivation suggests aggresome formation as a feedback inhibition mechanism in iNOS regulation.     

dnc-1/dynactin 1 Knockdown Disrupts Transport of Autophagosomes and Induces Motor Neuron Degeneration

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. We previously showed that the expression of dynactin 1, an axon motor protein regulating retrograde transport, is markedly reduced in spinal motor neurons of sporadic ALS patients, although the mechanisms by which decreased dynactin 1 levels cause neurodegeneration have yet to be elucidated. The accumulation of autophagosomes in degenerated motor neurons is another key pathological feature of sporadic ALS. Since autophagosomes are cargo of dynein/dynactin complexes and play a crucial role in the turnover of several organelles and proteins, we hypothesized that the quantitative loss of dynactin 1 disrupts the transport of autophagosomes and induces the degeneration of motor neuron. In the present study, we generated a Caenorhabditis elegans model in which the expression of DNC-1, the homolog of dynactin 1, is specifically knocked down in motor neurons. This model exhibited severe motor defects together with axonal and neuronal degeneration. We also observed impaired movement and increased number of autophagosomes in the degenerated neurons. Furthermore, the combination of rapamycin, an activator of autophagy, and trichostatin which facilitates axonal transport dramatically ameliorated the motor phenotype and axonal degeneration of this model. Thus, our results suggest that decreased expression of dynactin 1 induces motor neuron degeneration and that the transport of autophagosomes is a novel and substantial therapeutic target for motor neuron degeneration.     

The wobbler mouse

Various mutations in humans and animals lead to the selective and progressive degeneration of motoneurons, resulting in muscular weakness, subsequent paralysis, and death (1-3). Amyotrophic lateral sclerosis (ALS) is the most common adult human motoneuron disease, but the vast majority of sporadic and familial cases of ALS are still of unknown origin (4). Murine models of motoneuron diseases, derived from spontaneous mutations in the colonies, have been known for half a century. Prior to the first identifications of the mutated proteins in human ALS, they have largely been used to explore the disease etiology. The chromosomal localization of these mutations does not favor a genetic similarity between these murine models and the few human forms of the disease for which the mutation or the chromosomal localization is known. Yet the fact that most human ALS cases are of unknown etiology and the recent discovery of molecules with no known role in motoneuron survival (5-7), indicate that these murine mutants may still contribute to the understanding of motoneuronal degenerative processes. This can be exemplified by the work performed on the wobbler mouse, one of the oldest and most extensively studied models, which is reviewed here.