Lentivector-mediated rescue from cerebellar ataxia in a mouse model of spinocerebellar ataxia

Polyglutamine disorders are inherited neurodegenerative diseases caused by the accumulation of expanded polyglutamine protein (polyQ). Previously, we identified a new guanosine triphosphatase, CRAG, which facilitates the degradation of polyQ aggregates through the ubiquitin-proteasome pathway in cultured cells. Because expression of CRAG decreases in the adult brain, a reduced level of CRAG could underlie the onset of polyglutamine diseases. To examine the potential of CRAG expression for treating polyglutamine diseases, we generated model mice expressing polyQ predominantly in Purkinje cells. The model mice showed poor dendritic arborization of Purkinje cells, a markedly atrophied cerebellum and severe ataxia. Lentivector-mediated expression of CRAG in Purkinje cells of model mice extensively cleared polyQ aggregates and re-activated dendritic differentiation, resulting in a striking rescue from ataxia. Our in vivo data substantiate previous cell-culture-based results and extend further the usefulness of targeted delivery of CRAG as a gene therapy for polyglutamine diseases.     

PML clastosomes prevent nuclear accumulation of mutant ataxin-7 and other polyglutamine proteins

The pathogenesis of spinocerebellar ataxia type 7 and other neurodegenerative polyglutamine (polyQ) disorders correlates with the aberrant accumulation of toxic polyQ-expanded proteins in the nucleus. Promyelocytic leukemia protein (PML) nuclear bodies are often present in polyQ aggregates, but their relation to pathogenesis is unclear. We show that expression of PML isoform IV leads to the formation of distinct nuclear bodies enriched in components of the ubiquitin-proteasome system. These bodies recruit soluble mutant ataxin-7 and promote its degradation by proteasome-dependent proteolysis, thus preventing the aggregate formation. Inversely, disruption of the endogenous nuclear bodies with cadmium increases the nuclear accumulation and aggregation of mutant ataxin-7, demonstrating their role in ataxin-7 turnover. Interestingly, beta-interferon treatment, which induces the expression of endogenous PML IV, prevents the accumulation of transiently expressed mutant ataxin-7 without affecting the level of the endogenous wild-type protein. Therefore, clastosomes represent a potential therapeutic target for preventing polyQ disorders.     

Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila

We tested whether proteins implicated in Huntington's and other polyglutamine (polyQ) expansion diseases can cause axonal transport defects. Reduction of Drosophila huntingtin and expression of proteins containing pathogenic polyQ repeats disrupt axonal transport. Pathogenic polyQ proteins accumulate in axonal and nuclear inclusions, titrate soluble motor proteins, and cause neuronal apoptosis and organismal death. Expression of a cytoplasmic polyQ repeat protein causes adult retinal degeneration, axonal blockages in larval neurons, and larval lethality, but not neuronal apoptosis or nuclear inclusions. A nuclear polyQ repeat protein induces neuronal apoptosis and larval lethality but no axonal blockages. We suggest that pathogenic polyQ proteins cause neuronal dysfunction and organismal death by two non-mutually exclusive mechanisms. One mechanism requires nuclear accumulation and induces apoptosis; the other interferes with axonal transport. Thus, disruption of axonal transport by pathogenic polyQ proteins could contribute to early neuropathology in Huntington's and other polyQ expansion diseases.     

Solution structure of polyglutamine tracts in GST-polyglutamine fusion proteins

Aggregation of expanded polyglutamine (polyQ) seems to be the cause of various genetic neurodegenerative diseases. Relatively little is known as yet about the polyQ structure and the mechanism that induces aggregation. We have characterised the solution structure of polyQ in a proteic context using a model system based on glutathione S-transferase fusion proteins. A wide range of biophysical techniques was applied. For the first time, nuclear magnetic resonance was used to observe directly and selectively the conformation of polyQ in the pathological range. We demonstrate that, in solution, polyQs are in a random coil conformation. However, under destabilising conditions, their aggregation behaviour is determined by the polyQ length.     

Nature of the nuclear inclusions formed by PQBP1, a protein linked to neurodegenerative polyglutamine diseases

PQBP1, for polyglutamine tract-binding protein-1, has been linked to progressive neurodegenerative diseases, such as spinocerebellar ataxia, that are caused by the expansion of a polyglutamine repeat in a key regulatory protein. The overexpression of PQBP1 results in the formation of nuclear inclusions, reminiscent of the protein aggregates that are detected in polyglutamine diseases. We show here that the occurrence of PQBP1-induced nuclear inclusions is dramatically increased by the co-expression of the pre-mRNA splicing factor SIPP1, a protein ligand of PQBP1. These nuclear inclusions did not co-localise with nuclear structures such as nucleoli, coiled bodies, PML bodies, speckles and stress bodies, and were not associated with (in)active chromatin or with nucleic acids. Site-directed mutagenesis showed that the facilitation in the formation of the nuclear inclusions required multiple independent interaction sites between SIPP1 and PQBP1. Moreover, the nuclear inclusions were highly dynamic and their formation did not require energy. Our data suggest that the SIPP1-PQBP1-induced nuclear inclusions are distinct from the protein aggregates that are associated with polyglutamine diseases and represent dynamic nucleoplasmic heteropolymers of SIPP1 and PQBP1.     

Leuprorelin rescues polyglutamine-dependent phenotypes in a transgenic mouse model of spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) is an adult-onset motor neuron disease that affects males. It is caused by the expansion of a polyglutamine (polyQ) tract in androgen receptors. Female carriers are usually asymptomatic. No specific treatment has been established. Our transgenic mouse model carrying a full-length human androgen receptor with expanded polyQ has considerable gender-related motor impairment. This phenotype was abrogated by castration, which prevented nuclear translocation of mutant androgen receptors. We examined the effect of androgen-blockade drugs on our mouse model. Leuprorelin, a lutenizing hormone-releasing hormone (LHRH) agonist that reduces testosterone release from the testis, rescued motor dysfunction and nuclear accumulation of mutant androgen receptors in male transgenic mice. Moreover, leuprorelin treatment reversed the behavioral and histopathological phenotypes that were once caused by transient increases in serum testosterone. Flutamide, an androgen antagonist promoting nuclear translocation of androgen receptors, yielded no therapeutic effect. Leuprorelin thus seems to be a promising candidate for the treatment of SBMA.     

RNA-binding protein TLS is a major nuclear aggregate-interacting protein in huntingtin exon 1 with expanded polyglutamine-expressing cells

Formation of intracellular aggregates is the hallmark of polyglutamine (polyQ) diseases. We analyzed the components of purified nuclear polyQ aggregates by mass spectrometry. As a result, we found that the RNA-binding protein translocated in liposarcoma (TLS) was one of the major components of nuclear polyQ aggregate-interacting proteins in a Huntington disease cell model and was also associated with neuronal intranuclear inclusions of R6/2 mice. In vitro study revealed that TLS could directly bind to truncated N-terminal huntingtin (tNhtt) aggregates but could not bind to monomer GST-tNhtt with 18, 42, or 62Q, indicating that the tNhtt protein acquired the ability to sequester TLS after forming aggregates. Thioflavin T assay and electron microscopic study further supported the idea that TLS bound to tNhtt-42Q aggregates at the early stage of tNhtt-42Q amyloid formation. Immunohistochemistry showed that TLS was associated with neuronal intranuclear inclusions of Huntington disease human brain. Because TLS has a variety of functional roles, the sequestration of TLS to polyQ aggregates may play a role in diverse pathological changes in the brains of patients with polyQ diseases.     

Entrapment of viral capsids in nuclear PML cages is an intrinsic antiviral host defense against varicella-zoster virus

The herpesviruses, like most other DNA viruses, replicate in the host cell nucleus. Subnuclear domains known as promyelocytic leukemia protein nuclear bodies (PML-NBs), or ND10 bodies, have been implicated in restricting early herpesviral gene expression. These viruses have evolved countermeasures to disperse PML-NBs, as shown in cells infected in vitro, but information about the fate of PML-NBs and their functions in herpesvirus infected cells in vivo is limited. Varicella-zoster virus (VZV) is an alphaherpesvirus with tropism for skin, lymphocytes and sensory ganglia, where it establishes latency. Here, we identify large PML-NBs that sequester newly assembled nucleocapsids (NC) in neurons and satellite cells of human dorsal root ganglia (DRG) and skin cells infected with VZV in vivo. Quantitative immuno-electron microscopy revealed that these distinctive nuclear bodies consisted of PML fibers forming spherical cages that enclosed mature and immature VZV NCs. Of six PML isoforms, only PML IV promoted the sequestration of NCs. PML IV significantly inhibited viral infection and interacted with the ORF23 capsid surface protein, which was identified as a target for PML-mediated NC sequestration. The unique PML IV C-terminal domain was required for both capsid entrapment and antiviral activity. Similar large PML-NBs, termed clastosomes, sequester aberrant polyglutamine (polyQ) proteins, such as Huntingtin (Htt), in several neurodegenerative disorders. We found that PML IV cages co-sequester HttQ72 and ORF23 protein in VZV infected cells. Our data show that PML cages contribute to the intrinsic antiviral defense by sensing and entrapping VZV nucleocapsids, thereby preventing their nuclear egress and inhibiting formation of infectious virus particles. The efficient sequestration of virion capsids in PML cages appears to be the outcome of a basic cytoprotective function of this distinctive category of PML-NBs in sensing and safely containing nuclear aggregates of aberrant proteins.     

Wild type huntingtin toxicity in yeast: Implications for the role of amyloid cross-seeding in polyQ diseases

Proteins with expanded polyglutamine (polyQ) regions are prone to form amyloids, which can cause diseases in humans and toxicity in yeast. Recently, we showed that in yeast non-toxic amyloids of Q-rich proteins can induce aggregation and toxicity of wild type huntingtin (Htt) with a short non-pathogenic polyglutamine tract. Similarly to mutant Htt with an elongated N-terminal polyQ sequence, toxicity of its wild type counterpart was mediated by induced aggregation of the essential Sup35 protein, which contains a Q-rich region. Notably, polymerization of Sup35 was not caused by the initial benign amyloids and, therefore, aggregates of wild type Htt acted as intermediaries in seeding Sup35 polymerization. This exemplifies a protein polymerization cascade which can generate a network of interdependent polymers. Here we discuss cross-seeded protein polymerization as a possible mechanism underlying known interrelations between different polyQ diseases. We hypothesize that similar mechanisms may enable proteins, which possess expanded Q-rich tracts but are not associated with diseases, to promote the development of polyQ diseases.     

Androgen-dependent neurodegeneration by polyglutamine-expanded human androgen receptor in Drosophila

Spinal and bulbar muscular atrophy (SBMA) is an X-linked, adult-onset, neurodegenerative disorder affecting only males and is caused by expanded polyglutamine (polyQ) stretches in the N-terminal A/B domain of human androgen receptor (hAR). Although no overt phenotype was detected in adult fly eye photoreceptor neurons expressing mutant hAR (polyQ 52), ingestion of androgen or its known antagonists caused marked neurodegeneration with nuclear localization and structural alteration of the hAR mutant. Ligand-independent toxicity was detected with a truncated polyQ-expanded A/B domain alone, which was attenuated with cytosolic trapping by coexpression of the unliganded hAR E/F ligand binding domain. Thus, our findings suggest that the full binding of androgen to the polyQ-expanded hAR mutants leads to structural alteration with nuclear translocation that eventually results in the onset of SBMA in male patients.     

Expression of extended polyglutamine sequentially activates initiator and effector caspases

To date, eight neurodegenerative disorders, including Huntington's disease and dentatorubral-pallidoluysian atrophy, have been identified to be caused by expansion of a CAG repeat coding for a polyglutamine (polyQ) stretch. It is, however, unclear how polyQ expansion mediates neuronal cell death observed in these disorders. Here, we have established a tetracycline-regulated expression system producing 19 and 56 repeats of glutamine fused with green fluorescent protein. Induced expression of the 56 polyQ, but not of the 19 polyQ stretch caused marked nuclear aggregation and apoptotic morphological changes of the nucleus. In vitro enzyme assays and Western blotting showed that polyQ56 expression sequentially activated initiator and effector caspases, such as caspase-8 or -9, and caspase-3, respectively. Furthermore, using cell-permeable fluorogenic substrate, the activation of caspase-3-like proteases was demonstrated in intact cells with aggregated polyQ. This is the first direct evidence that the expression of extended polyQ activates caspases and together with the previous findings that some of the products of genes responsible for CAG repeat diseases are substrates of caspase-3 indicates an important role of caspases in the pathogenesis of these diseases.     

Experimental and Computational Analysis of Polyglutamine-Mediated Cytotoxicity

Expanded polyglutamine (polyQ) proteins are known to be the causative agents of a number of human neurodegenerative diseases but the molecular basis of their cytoxicity is still poorly understood. PolyQ tracts may impede the activity of the proteasome, and evidence from single cell imaging suggests that the sequestration of polyQ into inclusion bodies can reduce the proteasomal burden and promote cell survival, at least in the short term. The presence of misfolded protein also leads to activation of stress kinases such as p38MAPK, which can be cytotoxic. The relationships of these systems are not well understood. We have used fluorescent reporter systems imaged in living cells, and stochastic computer modeling to explore the relationships of polyQ, p38MAPK activation, generation of reactive oxygen species (ROS), proteasome inhibition, and inclusion body formation. In cells expressing a polyQ protein inclusion, body formation was preceded by proteasome inhibition but cytotoxicity was greatly reduced by administration of a p38MAPK inhibitor. Computer simulations suggested that without the generation of ROS, the proteasome inhibition and activation of p38MAPK would have significantly reduced toxicity. Our data suggest a vicious cycle of stress kinase activation and proteasome inhibition that is ultimately lethal to cells. There was close agreement between experimental data and the predictions of a stochastic computer model, supporting a central role for proteasome inhibition and p38MAPK activation in inclusion body formation and ROS-mediated cell death.     

Surface plasmon resonance characterization of specific binding of polyglutamine aggregation inhibitors to the expanded polyglutamine stretch

Proteins with an abnormally expanded polyglutamine (polyQ) stretch are prone to change their conformations, leading to their aggregation, and cause inherited neurodegenerative diseases called the polyQ diseases. Although screening for polyQ aggregation inhibitors has been extensively performed, many common false-positive hits have been identified so far. In this study, we employed surface plasmon resonance (SPR) to characterize the binding specificities and affinities of polyQ aggregation inhibitors to the expanded polyQ stretch. SPR successfully detected specific binding of polyQ binding peptide 1 (QBP1) to the expanded polyQ stretch (K_d = 5.7 μM), and non-specific binding of Congo red to polyQ proteins independent of their polyQ-length. Binding affinities of polyQ aggregation inhibitors to the expanded polyQ stretch were correlated with their inhibitory effects on polyQ aggregation. We therefore conclude that SPR is a useful technique for screening for specific polyQ aggregation inhibitors as promising therapeutic candidates for the currently untreatable polyQ diseases.     

Proteins with Intrinsically Disordered Domains Are Preferentially Recruited to Polyglutamine Aggregates

Intracellular protein aggregation is the hallmark of several neurodegenerative diseases. Aggregates formed by polyglutamine (polyQ)-expanded proteins, such as Huntingtin, adopt amyloid-like structures that are resistant to denaturation. We used a novel purification strategy to isolate aggregates formed by human Huntingtin N-terminal fragments with expanded polyQ tracts from both yeast and mammalian (PC-12) cells. Using mass spectrometry we identified the protein species that are trapped within these polyQ aggregates. We found that proteins with very long intrinsically-disordered (ID) domains (≥100 amino acids) and RNA-binding proteins were disproportionately recruited into aggregates. The removal of the ID domains from selected proteins was sufficient to eliminate their recruitment into polyQ aggregates. We also observed that several neurodegenerative disease-linked proteins were reproducibly trapped within the polyQ aggregates purified from mammalian cells. Many of these proteins have large ID domains and are found in neuronal inclusions in their respective diseases. Our study indicates that neurodegenerative disease-associated proteins are particularly vulnerable to recruitment into polyQ aggregates via their ID domains. Also, the high frequency of ID domains in RNA-binding proteins may explain why RNA-binding proteins are frequently found in pathological inclusions in various neurodegenerative diseases.     

VCP/p97 in abnormal protein aggregates, cytoplasmic vacuoles, and cell death, phenotypes relevant to neurodegeneration

Neuronal cell death, abnormal protein aggregates, and cytoplasmic vacuolization are major pathologies observed in many neurodegenerative disorders such as the polyglutamine (polyQ) diseases, prion disease, Alzheimer disease, and the Lewy body diseases, suggesting common mechanisms underlying neurodegeneration. Here, we have identified VCP/p97, a member of the AAA+ family of ATPase proteins, as a polyQ-interacting protein in vitro and in vivo, and report on its characterization. Endogenous VCP co-localized with expanded polyQ (ex-polyQ) aggregates in cultured cells expressing ex-polyQ, with nuclear inclusions in Huntington disease patient brains, and with Lewy bodies in patient samples. Moreover, the expression of VCP mutants with mutations in the 2nd ATP binding domain created cytoplasmic vacuoles, followed by cell death. Very similar vacuoles were also induced by ex-polyQ expression or proteasome inhibitor treatment. These results suggest that VCP functions not only as a recognition factor for abnormally folded proteins but also as a pathological effector for several neurodegenerative phenotypes. VCP may thus be an ideal molecular target for the treatment of neurodegenerative disorders.     

Overexpression of F0F1-ATP synthase α suppresses mutant huntingtin aggregation and toxicity in vitro

Huntington’s disease (HD) and other polyglutamine (polyQ) neurodegenerative diseases are characterized by neuronal accumulation of the disease protein, suggesting that the cellular ability to handle abnormal proteins is compromised. As a multi-subunit protein localized in the mitochondria of eukaryotic cells, the F₀F₁-ATP synthase α belongs to the family of stress proteins HSP60. Currently, mounting evidences indicate F₀F₁-ATP synthase α may play a role in neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). Recently, ATP synthase α was reported to have protective and therapeutic roles in primary cardiacmyocytes of iron-overloaded rats by lowering ROS production. However, little is understood about the role of ATP synthase α in cell death and neurodegeneration. Here, we demonstrate that overexpression of ATP synthase α suppresses huntingtin (htt) polyQ aggregation and toxicity in transfected SH-SY5Y cell lines. Overexpression of ATP synthase α is able to protect cell death caused by polyglutamine-expanded htt. Transient overexpression of ATP synthase α suppresses the aggregate formation by estimation of polyQ aggregation, Western blot analysis, and filter trap assay (FTA) in transfected SH-SY5Y cells. These results indicated that ATP synthase α has a strong inhibitory effect on polyglutamine aggregate formation and toxicity in vitro, and suggest a novel neuroprotective role of ATP synthase α.