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ABSTRACT: 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 previously identified 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.
Journal of Biological Chemistry 04/2013; · 4.77 Impact Factor
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ABSTRACT: BACKGOUND: Amyotrophic lateral sclerosis (ALS) is progressive neurodegenerative disease characterized by the loss of motor function. Several ALS genes have been identified as their mutations can lead to familial ALS, including the recently reported RNA-binding protein fused in sarcoma (Fus). However, it is not clear how mutations of Fus lead to motor neuron degeneration in ALS. In this study, we present a Drosophila model to examine the toxicity of Fus, its Drosophila orthologue Cabeza (Caz), and the ALS-related Fus mutants. RESULTS: Our results show that the expression of wild-type Fus/Caz or FusR521G induced progressive toxicity in multiple tissues of the transgenic flies in a dose- and age-dependent manner. The expression of Fus, Caz, or FusR521G in motor neurons significantly impaired the locomotive ability of fly larvae and adults. The presynaptic structures in neuromuscular junctions were disrupted and motor neurons in the ventral nerve cord (VNC) were disorganized and underwent apoptosis. Surprisingly, the interruption of Fus nuclear localization by either deleting its nuclear localization sequence (NLS) or adding a nuclear export signal (NES) blocked Fus toxicity. Moreover, we discovered that the loss of caz in Drosophila led to severe growth defects in the eyes and VNCs, caused locomotive disability and NMJ disruption, but did not induce apoptotic cell death. CONCLUSIONS: These data demonstrate that the overexpression of Fus/Caz causes in vivo toxicity by disrupting neuromuscular junctions (NMJs) and inducing apoptosis in motor neurons. In addition, the nuclear localization of Fus is essential for Fus to induce toxicity. Our findings also suggest that Fus overexpression and gene deletion can cause similar degenerative phenotypes but the underlying mechanisms are likely different.
Molecular Neurodegeneration 03/2012; 7:10. · 4.28 Impact Factor
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ABSTRACT: Mutations in fused in sarcoma (FUS) have been reported to cause a subset of familial amyotrophic lateral sclerosis (ALS) cases. Wild-type FUS is mostly localized in the nuclei of neurons, but the ALS mutants are partly mislocalized in the cytoplasm and can form inclusions. We demonstrate that the C-terminal 32 amino acid residues of FUS constitute an effective nuclear localization sequence (NLS) as it targeted beta-galactosidase (LacZ, 116 kDa) to the nucleus. Deletion of or the ALS mutations within the NLS caused cytoplasmic mislocalization of FUS. Moreover, we identified the poly-A binding protein (PABP1), a stress granule marker, as an interacting partner of FUS. Large PABP1-positive cytoplasmic foci (i.e. stress granules) colocalized with the mutant FUS inclusions but were absent in wild-type FUS-expressing cells. Processing bodies, which are functionally related to stress granules, were adjacent to but not colocalized with the mutant FUS inclusions. Our results suggest that the ALS mutations in FUS NLS can impair FUS nuclear localization, induce cytoplasmic inclusions and stress granules, and potentially perturb RNA metabolism.
Neurobiology of aging 12/2011; 32(12):2323.e27-40. · 5.94 Impact Factor
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Haining Zhu,
Jun Zhao,
Beibei Zhu,
Joanne Collazo, Jozsef Gal,
Ping Shi,
Li Liu,
Anna-Lena Ström,
Xiaoning Lu,
Richard O McCann,
Michal Toborek,
Natasha Kyprianou
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ABSTRACT: Proteins on cell surface play important roles during cancer progression and metastasis via their ability to mediate cell-to-cell interactions and navigate the communication between cells and the microenvironment.
In this study a targeted proteomic analysis was conducted to identify the differential expression of cell surface proteins in human benign (BPH-1) versus malignant (LNCaP and PC-3) prostate epithelial cells. We identified EMMPRIN (extracellular matrix metalloproteinase inducer) as a key candidate and shRNA functional approaches were subsequently applied to determine the role of EMMPRIN in prostate cancer cell adhesion, migration, invasion as well as cytoskeleton organization.
EMMPRIN was found to be highly expressed on the surface of prostate cancer cells compared to BPH-1 cells, consistent with a correlation between elevated EMMPRIN and metastasis found in other tumors. No significant changes in cell proliferation, cell cycle progression, or apoptosis were detected in EMMPRIN knockdown cells compared to the scramble controls. Furthermore, EMMPRIN silencing markedly decreased the ability of PC-3 cells to form filopodia, a critical feature of invasive behavior, while it increased expression of cell-cell adhesion and gap junction proteins.
Our results suggest that EMMPRIN regulates cell adhesion, invasion, and cytoskeleton reorganization in prostate cancer cells. This study identifies a new function for EMMPRIN as a contributor to prostate cancer cell-cell communication and cytoskeleton changes towards metastatic spread, and suggests its potential value as a marker of prostate cancer progression to metastasis.
The Prostate 05/2011; 72(1):72-81. · 3.48 Impact Factor
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ABSTRACT: MicroRNAs (miRNAs) are a novel class of small noncoding RNAs that regulate gene expression at the posttranscriptional level and play a critical role in many important biological processes and pathological development. In the past few years, miRNAs have been implicated to play an important role in cancer initiation and development. In this chapter, we describe a protocol for the analysis and characterization of miRNAs in prostate cancer cells using a simple but effective array platform. The array is composed of 553 nonredundant miRNAs encompassing the entire set of known miRNAs in humans and mice. As an example, profiling of miRNAs in four prostate cancer cell lines has revealed that a set of miRNAs were differentially expressed between androgen-dependent and androgen-independent metastatic prostate cancer cells. Among them, the differential expression of miR-205 and miR-200c were further validated by Northern blot analysis in these two types of prostate cancer cells. This comprehensive and easy-to-follow protocol will be useful for studying miRNAs in various cancers and can be readily adapted for miRNA analysis in a variety of human diseases.
Methods in molecular biology (Clifton, N.J.) 01/2011; 732:69-88.
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ABSTRACT: Transport of material and signals between extensive neuronal processes and the cell body is essential to neuronal physiology and survival. Slowing of axonal transport has been shown to occur before the onset of symptoms in amyotrophic lateral sclerosis (ALS). We have previously shown that several familial ALS-linked copper-zinc superoxide dismutase (SOD1) mutants (A4V, G85R, and G93A) interacted and colocalized with the retrograde dynein-dynactin motor complex in cultured cells and affected tissues of ALS mice. We also found that the interaction between mutant SOD1 and the dynein motor played a critical role in the formation of large inclusions containing mutant SOD1. In this study, we showed that, in contrast to the dynein situation, mutant SOD1 did not interact with anterograde transport motors of the kinesin-1 family (KIF5A, B and C). Using dynein and kinesin accumulation at the sciatic nerve ligation sites as a surrogate measurement of axonal transport, we also showed that dynein mediated retrograde transport was slower in G93A than in WT mice at an early presymptomatic stage. While no decrease in KIF5A-mediated anterograde transport was detected, the slowing of anterograde transport of dynein heavy chain as a cargo was observed in the presymptomatic G93A mice. The results from this study along with other recently published work support that mutant SOD1 might only interact with and interfere with some kinesin members, which, in turn, could result in the impairment of a selective subset of cargos. Although it remains to be further investigated how mutant SOD1 affects different axonal transport motor proteins and various cargos, it is evident that mutant SOD1 can induce defects in axonal transport, which, subsequently, contribute to the propagation of toxic effects and ultimately motor neuron death in ALS.
Biochimica et Biophysica Acta 09/2010; 1802(9):707-16. · 4.66 Impact Factor
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ABSTRACT: A better understanding of the etiology of amyotrophic lateral sclerosis (ALS) is needed to develop effective therapies for the treatment of this fatal neurodegenerative disease. Extensive studies have produced a general agreement that ALS is likely to be a multifactorial and multisystem disease. Many mechanisms have been postulated to be involved in the pathology of ALS, such as oxidative stress, glutamate excitotoxicity, mitochondrial damage, defective axonal transport, glia cell pathology, and aberrant RNA metabolism. Mitochondria have shown to be an early target in ALS pathogenesis and contribute to the disease progression. Morphological and functional defects in mitochondria were found in both human patients and ALS mice overexpressing mutant SOD1. Mutant SOD1 was found to be preferentially associated with mitochondria and subsequently impair mitochondrial function. Recent studies suggest that axonal transport of mitochondria along microtubules is disrupted in ALS. Furthermore, new evidence suggests that mitochondrial fission and fusion as well as mitophagy clearance may also be affected by mutant SOD1. These results also illustrate the critical importance of maintaining proper mitochondrial function in axons and neuromuscular junctions, supporting the emerging "dying-back" axonopathy model of ALS. In this review, we will discuss findings supporting that mitochondrial dysfunction is likely to be a converging point of multiple pathways underlying the ALS pathogenesis and progression.
Journal of Alzheimer's disease: JAD 01/2010; 20 Suppl 2:S311-24. · 3.74 Impact Factor
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ABSTRACT: The p62/sequestosome 1 protein has been identified as a component of pathological protein inclusions in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). P62 has also been implicated in autophagy, a process of mass degradation of intracellular proteins and organelles. Autophagy is a critical pathway for degrading misfolded and/or damaged proteins, including the copper-zinc superoxide dismutase (SOD1) mutants linked to familial ALS. We previously reported that p62 interacted with ALS mutants of SOD1 and that the ubiquitin-association domain of p62 was dispensable for the interaction. In this study, we identified two distinct regions of p62 that were essential to its binding to mutant SOD1: the N-terminal Phox and Bem1 (PB1) domain (residues 1-104) and a separate internal region (residues 178-224) termed here as SOD1 mutant interaction region (SMIR). The PB1 domain is required for appropriate oligomeric status of p62 and the SMIR is the actual region interacting with mutant SOD1. Within the SMIR, the conserved W184, H190 and positively charged R183, R186, K187, and K189 residues are critical to the p62-mutant SOD1 interaction as substitution of these residues with alanine resulted in significantly abolished binding. In addition, SMIR and the p62 sequence responsible for the interaction with LC3, a protein essential for autophagy activation, are independent of each other. In cells lacking p62, the existence of mutant SOD1 in acidic autolysosomes decreased, suggesting that p62 can function as an adaptor between mutant SOD1 and the autophagy machinery. This study provides a novel molecular mechanism by which mutant SOD1 can be recognized by p62 in an ubiquitin-independent fashion and targeted for the autophagy-lysosome degradation pathway.
Journal of Neurochemistry 09/2009; 111(4):1062-73. · 4.06 Impact Factor
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ABSTRACT: The etiology of motor neuron degeneration in amyotrophic lateral sclerosis (ALS) remains to be better understood. Based on the studies from ALS patients and transgenic animal models, it is believed that ALS is likely to be a multifactorial and multisystem disease. Many mechanisms have been postulated to be involved in the pathology of ALS, such as oxidative stress, glutamate excitotoxicity, mitochondrial damage, defective axonal transport, glia cell pathology and aberrant RNA metabolism. Mitochondria, which play crucial roles in excitotoxicity, apoptosis and cell survival, have shown to be an early target in ALS pathogenesis and contribute to the disease progression. Morphological and functional defects in mitochondria were found in both human patients and ALS mice overexpressing mutant SOD1. Mutant SOD1 was found to be preferentially associated with mitochondria and subsequently impair mitochondrial function. Recent studies suggest that axonal transport of mitochondria along microtubules and mitochondrial dynamics may also be disrupted in ALS. These results also illustrate the critical importance of maintaining proper mitochondrial function in axons and neuromuscular junctions, supporting the emerging "dying-back" axonopathy model of ALS. In this review, we will discuss how mitochondrial dysfunction has been linked to the ALS variants of SOD1 and the mechanisms by which mitochondrial damage contributes to the disease etiology.
Biochimica et Biophysica Acta 09/2009; 1802(1):45-51. · 4.66 Impact Factor
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ABSTRACT: Adenylate kinase 4 (AK4) is a unique member with no enzymatic activity in vitro in the adenylate kinase (AK) family although it shares high sequence homology with other AKs. It remains unclear what physiological function AK4 might play or why it is enzymatically inactive. In this study, we showed increased AK4 protein levels in cultured cells exposed to hypoxia and in an animal model of the neurodegenerative disease amyotrophic lateral sclerosis. We also showed that short hairpin RNA (shRNA)-mediated knockdown of AK4 in HEK293 cells with high levels of endogenous AK4 resulted in reduced cell proliferation and increased cell death. Furthermore, we found that AK4 over-expression in the neuronal cell line SH-SY5Y with low endogenous levels of AK4 protected cells from H(2)O(2) induced cell death. Proteomic studies revealed that the mitochondrial ADP/ATP translocases (ANTs) interacted with AK4 and higher amount of ANT was co-precipitated with AK4 when cells were exposed to H(2)O(2) treatment. In addition, structural analysis revealed that, while AK4 retains the capability of binding nucleotides, AK4 has a glutamine residue instead of a key arginine residue in the active site well conserved in other AKs. Mutation of the glutamine residue to arginine (Q159R) restored the adenylate kinase activity with GTP as substrate. Collectively, these results indicate that the enzymatically inactive AK4 is a stress responsive protein critical to cell survival and proliferation. It is likely that the interaction with the mitochondrial inner membrane protein ANT is important for AK4 to exert the protective benefits to cells under stress.
The international journal of biochemistry & cell biology 01/2009; 41(6):1371-80. · 4.89 Impact Factor
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ABSTRACT: An important consequence of protein misfolding related to neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), is the formation of proteinaceous inclusions or aggregates within the central nervous system. We have previously shown that several familial ALS-linked copper-zinc superoxide dismutase (SOD1) mutants (A4V, G85R, and G93A) interact and co-localize with the dynein-dynactin complex in cultured cells and affected tissues of ALS mice. In this study, we report that the interaction between mutant SOD1 and the dynein motor plays a critical role in the formation of large inclusions containing mutant SOD1. Disruption of the motor by overexpression of the p50 subunit of dynactin in neuronal and non-neuronal cell cultures abolished the association between aggregation-prone SOD1 mutants and the dynein-dynactin complex. The p50 overexpression also prevented mutant SOD1 inclusion formation and improved the survival of cells expressing A4V SOD1. Furthermore, we observed that two ALS-linked SOD1 mutants, H46R and H48Q, which showed a lower propensity to interact with the dynein motor, also produced less aggregation and fewer large inclusions. Overall, these data suggest that formation of large inclusions depends upon association of the abnormal SOD1s with the dynein motor. Whether the misfolded SOD1s directly perturb axonal transport or impair other functional properties of the dynein motor, this interaction could propagate a toxic effect that ultimately causes motor neuron death in ALS.
Journal of Biological Chemistry 06/2008; 283(33):22795-805. · 4.77 Impact Factor
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ABSTRACT: Transport of material between extensive neuronal processes and the cell body is crucial for neuronal function and survival. Growing evidence shows that deficits in axonal transport contribute to the pathogenesis of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Here we review recent data indicating that defects in dynein-mediated retrograde axonal transport are involved in ALS etiology. We discuss how mutant copper-zinc superoxide dismutase (SOD1) and an aberrant interaction between mutant SOD1 and dynein could perturb retrograde transport of neurotrophic factors and mitochondria. A possible contribution of axonal transport to the aggregation and degradation processes of mutant SOD1 is also reviewed. We further consider how the interference with axonal transport and protein turnover by mutant SOD1 could influence the function and viability of motor neurons in ALS.
Journal of Neurochemistry 05/2008; 106(2):495-505. · 4.06 Impact Factor
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ABSTRACT: MicroRNAs (miRNAs) are a novel class of small noncoding RNAs that regulate gene expression at the post-transcriptional level and play a critical role in many important biological processes. Most miRNAs are conserved between humans and mice, which makes it possible to analyze their expressions with a set of selected array probes. Here, we report a simple array platform that can detect 553 nonredundant miRNAs encompassing the entire set of miRNAs for humans and mice. The platform features carefully selected and designed probes with optimized hybridization parameters. Potential cross-reaction between mature miRNAs and their precursors was investigated. The array platform was used to analyze miRNAs in the mouse central nervous system (CNS, spinal cord and brain), and two other non-CNS organs (liver and heart). Two types of miRNAs, differentially expressed organ/tissue-associated miRNAs and ubiquitously expressed miRNAs, were detected in the array analysis. In addition to the previously reported neuron-related miR-124a, liver-related miR-122a, and muscle-related miR-133a, we also detected new tissue-associated miRNAs (e.g., liver-associated miR-194). Interestingly, while the majority of pre-miRNAs were undetectable, miR690, miR709, and miR720 were clearly detected at both mature and precursor levels by the array analysis, indicating a limited cross-reaction between pre-miRNAs and their mature miRNAs. The reliability of this array technology was validated by comparing the results with independent Northern blot analyses and published data. A new approach of data normalization based on Northern blot analysis of one ubiquitously expressed miRNA is introduced and compared with traditional approaches. We expect this miRNA array platform to be useful for a wide variety of biological studies.
RNA 11/2007; 13(10):1803-22. · 5.09 Impact Factor
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ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a progressive neurode-generative disease characterized by motor neuron death. A hallmark of the disease is the appearance of protein aggregates in the affected motor neurons. We have found that p62, a protein implicated in protein aggregate formation, accumulated progressively in the G93A mouse spinal cord. The accumulation of p62 was in parallel to the increase of polyubiquitinated proteins and mutant SOD1 aggregates. Immunostaining studies showed that p62, ubiquitin, and mutant SOD1 co-localized in the protein aggregates in affected cells in G93A mouse spinal cord. The p62 protein selectively interacted with familial ALS mutants, but not WT SOD1. When p62 was co-expressed with SOD1 in NSC34 cells, it greatly enhanced the formation of aggregates of the ALS-linked SOD1 mutants, but not wild-type SOD1. Cell viability was measured in the presence and absence of overexpressed p62, and the results suggest that the large aggregates facilitated by p62 were not directly toxic to cells under the conditions in this study. Deletion of the ubiquitin-association (UBA) domain of p62 significantly decreased the p62-facilitated aggregate formation, but did not completely inhibit it. Further protein interaction experiments also showed that the truncated p62 with the UBA domain deletion remained capable of interacting with mutant SOD1. The findings of this study show that p62 plays a critical role in forming protein aggregates in familial ALS, likely by linking misfolded mutant SOD1 molecules and other cellular proteins together.
Journal of Biological Chemistry 05/2007; 282(15):11068-77. · 4.77 Impact Factor
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ABSTRACT: To identify essential host genes affecting replication of Tomato bushy stunt virus (TBSV), a small model plant virus, we screened 800 yeast genes present in the yeast Tet promoters Hughes Collection. In total, we have identified 30 new host genes whose down-regulation either increased or decreased the accumulation of a TBSV replicon RNA. The identified essential yeast genes are involved in RNA transcription/metabolism, protein metabolism/transport, or other cellular processes. Detailed analysis of the effects of some of the identified yeast genes revealed that they might affect RNA replication by altering (i) the amounts/functions of p33 and p92(pol) viral replication proteins, (ii) the standard 10 to 20:1 ratio between p33 and p92(pol) in the viral replicase, (iii) the activity of the tombusvirus replicase, and (iv) the ratio of plus- versus minus-stranded RNA replication products. Altogether, this and previous genetic screening of yeast (Panavas et al., Proc. Natl. Acad. Sci. USA 102:7326-7331, 2005) led to the identification of 126 host genes (out of approximately 5,600 genes that represent approximately 95% of all the known and predicted yeast genes) that affected the accumulation of tombusvirus RNA.
Journal of Virology 09/2006; 80(15):7394-404. · 5.40 Impact Factor
