Proteotoxic stress increases nuclear localization of ataxin-3.

Spinocerebellar ataxia type 3 (SCA3)/Machado Joseph disease results from expansion of the polyglutamine domain in ataxin-3 (Atx3). Atx3 is a transcriptional co-repressor, as well as a deubiquitinating enzyme that appears to function in cellular pathways involved in protein homeostasis. In this study, we show that interactions of Atx3 with valosin-containing protein and hHR23B are dynamic and modulated by proteotoxic stresses. Heat shock, a general proteotoxic stress, also induced wild-type and pathogenic Atx3 to accumulate in the nucleus. Mapping studies showed that two regions of Atx3, the Josephin domain and the C-terminus, regulated heat shock-induced nuclear localization. Heat shock-induced nuclear localization of Atx3 was not affected by a casein kinase-2 inhibitor or by mutating a predicted nuclear localization signal. However, serine-111 of Atx3 was required for nuclear localization of the Josephin domain and regulated nuclear localization of full-length Atx3. Atx3 null cells were more sensitive to toxic effects of heat shock suggesting that Atx3 had a protective function in the cellular response to heat shock. Importantly, we found that oxidative stress also induced nuclear localization of Atx3; both wild-type and pathogenic Atx3 accumulated in the nucleus of SCA3 patient fibroblasts following oxidative stress. Heat shock and oxidative stress are the first processes identified that increase nuclear localization of Atx3. Observations in this study provide new and important insights for understanding SCA3 pathology as the nucleus is likely a key site for early pathogenesis.

Basal and stress-induced Hsp70 are modulated by ataxin-3.

Regulation of basal and induced levels of hsp70 is critical for cellular homeostasis. Ataxin-3 is a deubiquitinase with several cellular functions including transcriptional regulation and maintenance of protein homeostasis. While investigating potential roles of ataxin-3 in response to cellular stress, it appeared that ataxin-3 regulated hsp70. Basal levels of hsp70 were lower in ataxin-3 knockout (KO) mouse brain from 2 to 63 weeks of age and hsp70 was also lower in fibroblasts from ataxin-3 KO mice. Transfecting KO cells with ataxin-3 rescued basal levels of hsp70 protein. Western blots of representative chaperones including hsp110, hsp90, hsp70, hsc70, hsp60, hsp40/hdj2, and hsp25 indicated that only hsp70 was appreciably altered in KO fibroblasts and KO mouse brain. Turnover of hsp70 protein was similar in wild-type (WT) and KO cells; however, basal hsp70 promoter reporter activity was decreased in ataxin-3 KO cells. Transfecting ataxin-3 restored hsp70 basal promoter activity in KO fibroblasts to levels of promoter activity in WT cells; however, mutations that inactivated deubiquitinase activity or the ubiquitin interacting motifs did not restore full activity to hsp70 basal promoter activity. Hsp70 protein and promoter activity were higher in WT compared to KO cells exposed to heat shock and azetidine-2-carboxylic acid, but WT and KO cells had similar levels in response to cadmium. Heat shock factor-1 had decreased levels and increased turnover in ataxin-3 KO fibroblasts. Data in this study are consistent with ataxin-3 regulating basal level of hsp70 as well as modulating hsp70 in response to a subset of cellular stresses.

Ataxin-3 binds VCP/p97 and regulates retrotranslocation of ERAD substrates.

Expansion of a polyglutamine tract in ataxin-3 (AT3) results in spinocerebellar ataxia type 3/Machado-Joseph disease, one of the nine polyglutamine neurodegenerative diseases. Understanding the normal functions of AT3 as well as its function in the context of expansion of the polyglutamine tract is critical for understanding the disease process. AT3 is a deubiquitylating enzyme with limited information on its cellular functions. We find that transfecting cells with AT3 increases cellular levels of endoplasmic reticulum-associated degradation (ERAD) substrates, CD3delta and TCRalpha, but does not alter levels of several non-ERAD substrates. AT3 increases the level of CD3delta by decreasing its degradation; pathogenic AT3 decreases degradation to a greater extent than wild-type AT3. Knock-down of endogenous AT3 decreases levels of CD3delta, suggesting that a normal function of AT3 is to regulate levels of ERAD substrates. AT3 binds VCP/p97, a key protein responsible for extracting ERAD substrates from the ER; binding is modulated by the size of the polyglutamine tract, and mutating a sequence adjacent to the polyglutamine tract inhibits the AT3-VCP interaction and AT3-dependent accumulation of CD3delta. AT3 and Ufd1 bind VCP in a mutually exclusive manner; AT3 decreases the interaction of VCP with Ufd1 as well as with ubiquitylated proteins. Using a reconstituted system, AT3 inhibits retrotranslocation of an ERAD substrate from the ER. These data suggest that a normal function of AT3 is to regulate flow through the ERAD pathway by modulating VCP-dependent extraction of proteins from the ER.

Rho kinase regulates phagocytosis, surface expression of GlcNAc, and Golgi fragmentation of apoptotic PC12 cells.

Apoptotic cells undergo a number of changes to prepare for phagocytosis; most occur during the execution phase of apoptosis, when dying cells undergo shrinkage and/or fragmentation into apoptotic bodies and express phagocytic markers on their surface. Although events during the execution phase are important to prepare corpses for phagocytosis, the mechanisms that control most execution phase events are unknown. To understand regulation of execution events we focused on Rho kinase (ROCK), because one isoform of ROCK, ROCK-I, is constitutively activated by caspases during execution. Using apoptotic PC12 cells as a model, we find that inhibition of ROCK activity during apoptosis decreases surface expression of GlcNAc, a carbohydrate known to function as a phagocytic marker. In addition, inhibition of ROCK blocks Golgi fragmentation in apoptotic cells, and constitutively active ROCK induces Golgi fragmentation in the absence of apoptosis. Importantly, PC12 cells dying in the presence of a ROCK inhibitor are less efficiently phagocytized than those dying without the inhibitor. These data highlight the role of ROCK in multiple processes in the execution phase of apoptosis, and suggest that ROCK plays an important role in controlling the outcome of apoptosis, that is, preparation of corpses for phagocytosis.

Rho kinase regulates fragmentation and phagocytosis of apoptotic cells.

During the execution phase of apoptosis, a cell undergoes cytoplasmic and nuclear changes that prepare it for death and phagocytosis. The end-point of the execution phase is condensation into a single apoptotic body or fragmentation into multiple apoptotic bodies. Fragmentation is thought to facilitate phagocytosis; however, mechanisms regulating fragmentation are unknown. An isoform of Rho kinase, ROCK-I, drives membrane blebbing through its activation of actin-myosin contraction; this raises the possibility that ROCK-I may regulate other execution phase events, such as cellular fragmentation. Here, we show that COS-7 cells fragment into a number of small apoptotic bodies during apoptosis; treating with ROCK inhibitors (Y-27632 or H-1152) prevents fragmentation. Latrunculin B and blebbistatin, drugs that interfere with actin-myosin contraction, also inhibit fragmentation. During apoptosis, ROCK-I is cleaved and activated by caspases, while ROCK-II is not activated, but rather translocates to a cytoskeletal fraction. siRNA knock-down of ROCK-I but not ROCK-II inhibits fragmentation of dying cells, consistent with ROCK-I being required for apoptotic fragmentation. Finally, cells dying in the presence of the ROCK inhibitor Y-27632 are not efficiently phagocytized. These data show that ROCK plays an essential role in fragmentation and phagocytosis of apoptotic cells.

MicroRNA pathways modulate polyglutamine-induced neurodegeneration.

Nine human neurodegenerative diseases are due to expansion of a CAG repeat- encoding glutamine within the open reading frame of the respective genes. Polyglutamine (polyQ) expansion confers dominant toxicity, resulting in neuronal degeneration. MicroRNAs (miRNAs) have been shown to modulate programmed cell death during development. To address whether miRNA pathways play a role in neurodegeneration, we tested whether genes critical for miRNA processing modulated toxicity induced by the spinocerebellar ataxia type 3 (SCA3) protein. These studies revealed a striking enhancement of polyQ toxicity upon reduction of miRNA processing in Drosophila and human cells. In parallel genetic screens, we identified the miRNA bantam (ban) as a potent modulator of both polyQ and tau toxicity in flies. Our studies suggest that ban functions downstream of toxicity of the SCA3 protein, to prevent degeneration. These findings indicate that miRNA pathways dramatically modulate polyQ- and tau-induced neurodegeneration, providing the foundation for new insight into therapeutics.

The polyglutamine neurodegenerative protein ataxin 3 regulates aggresome formation.

The polyglutamine-containing neurodegenerative protein ataxin 3 (AT3) has deubiquitylating activity and binds ubiquitin chains with a preference for chains of four or more ubiquitins. Here we characterize the deubiquitylating activity of AT3 in vitro and show it trims/edits K48-linked ubiquitin chains. AT3 also edits polyubiquitylated (125)I-lysozyme and decreases its degradation by proteasomes. Cellular studies show that endogenous AT3 colocalizes with aggresomes and preaggresome particles of the misfolded cystic fibrosis transmembrane regulator (CFTR) mutant CFTRDeltaF508 and associates with histone deacetylase 6 and dynein, proteins required for aggresome formation and transport of misfolded protein. Small interfering RNA knockdown of AT3 greatly reduces aggresomes formed by CFTRDeltaF508, demonstrating a critical role of AT3 in this process. Wild-type AT3 restores aggresome formation; however, AT3 with mutations in the active site or ubiquitin interacting motifs cannot restore aggresome formation in AT3 knockdown cells. These same mutations decrease the association of AT3 and dynein. These data indicate that the deubiquitylating activity of AT3 and its ubiquitin interacting motifs as well play essential roles in CFTRDeltaF508 aggresome formation.

The polyglutamine neurodegenerative protein ataxin-3 binds polyubiquitylated proteins and has ubiquitin protease activity.

The ubiquitin-proteasome pathway is critically involved in the pathology of neurodegenerative diseases characterized by protein misfolding and aggregation. Data in the present study suggest that the polyglutamine neurodegenerative disease protein, ataxin-3 (AT3), functions in the ubiquitin-proteasome pathway. AT3 contains an ubiquitin interaction motif (UIM) domain that binds polyubiquitylated proteins with a strong preference for chains containing four or more ubiquitins. Mutating the conserved leucine in the first UIM (L229A) almost totally eliminates binding to polyubiquitin chains while a similar mutation in the second UIM (L249A) also inhibits binding to polyubiquitin chains but to a lesser extent. Both wild-type and pathological AT3 increase cellular levels of a short-lived GFP that is degraded by the ubiquitin-proteasome pathway. AT3 has several properties characteristic of ubiquitin proteases including decreasing polyubiquitylation of 125I-lysozyme by removing ubiquitin from polyubiquitin chains, cleaving a ubiquitin protease substrate, and binding the specific ubiquitin protease inhibitor, ubiquitin-aldehyde. Mutating the predicted catalytic cysteine in AT3 inhibits each of these ubiquitin protease activities. The ability to bind and cleave ubiquitylated proteins is consistent with AT3 playing a role in the ubiquitin-proteasome system. This raises the possibility that pathological AT3, which tends to misfold and aggregate, may be exposed to aggregate-prone misfolded/denatured proteins as part of its normal function.

Ataxin-3 is a histone-binding protein with two independent transcriptional corepressor activities.

The mechanisms of pathology for the family of polyglutamine disease proteins are unknown; however, recently it was shown that several of these proteins inhibit transcription suggesting that transcriptional repression may be a potential mechanism for pathology. In the present study we use cell transfections, in vitro binding, co-immunoprecipitations, and reporter assays to show that the polyglutamine disease protein, ataxin-3, interacts with the major histone acetyltransferases cAMP-response-element binding protein (CREB)-binding protein, p300, and p300/CREB-binding protein-associated factor and inhibits transcription by these coactivators. Importantly, endogenous ataxin-3 is co-immunoprecipitated with each of these coactivators in non-transfected cells. The C-terminal polyglutamine-containing domain of ataxin-3 inhibits coactivator-dependent transcription and is required for binding coactivators. The N-terminal domain of ataxin-3 inhibits histone acetylation by p300 in vitro and inhibits transcription in vivo. Histone binding and blocking access of coactivators to acetylation sites on histones appears to be the mechanism of inhibition. Together, our data provide a novel mechanism of transcriptional regulation by ataxin-3 that involves targeting histones, coactivators, and an independent mode of direct repression of transcription, and suggests that its physiological function and possibly pathological effects are linked to its interactions with these proteins.

Acute activation of mitogen-activated protein kinases following traumatic brain injury in the rat: implications for posttraumatic cell death.

The regional activation (via phosphorylation) of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways was examined using immunoblotting and immunohistochemistry following experimental brain injury. Anesthetized rats were subjected to lateral fluid-percussion brain injury of moderate severity (2.4-2.6 atm) and euthanized at 2, 6, 24, and 72 h after injury; sham-injured animals were surgically prepared but were not injured. Immunohistochemical evidence of activation of JNK and ERK1/2 pathways was observed predominantly in regions that exhibit neural cell apoptosis and axonal damage following brain trauma. Activation of the ERK1/2 pathway was observed as early as 2 h and up to 72 h postinjury in nonneuronal cells in all layers of the cortex at the site of maximal injury, in the white matter below the site of maximal cortical damage and in the thalamus. In contrast, activation of JNK signaling was observed only at 24 and 72 h postinjury in a few neurons at the core of the cortical injury site. However, robust JNK activation was observed between 2 and 72 h postinjury in both axons and nonneuronal cells in the white matter below the site of maximal cortical damage and in the thalamus. Activation of ERK1/2, but not JNK, was observed in cells in the dentate hilus in the hippocampus in both hemispheres between 2 and 24 h postinjury. Immunoblotting analyses of extracts from various brain regions did not reveal significant alterations in intensities of either total or phosphorylated proteins underscoring the focal nature of the immunohistochemical observations. However, these results suggest that activation of MAP kinase signaling pathways may be associated with posttraumatic cell damage and are indicative of the heterogeneous nature of the mechanisms underlying regional cell death following TBI.