Prevention of polyglutamine oligomerization and neurodegeneration by the peptide inhibitor QBP1 in Drosophila

Polyglutamine (polyQ) diseases are a growing class of inherited neurodegenerative diseases including Huntington's disease, which are caused by abnormal expansions of the polyQ stretch in each unrelated disease protein. The expanded polyQ stretch is thought to confer toxic properties on the disease proteins through alteration of their conformation leading to pathogenic protein-protein interactions including oligomerization and/or aggregation. Hypothesizing that molecules with selective binding affinity to the expanded polyQ stretch may interfere with the pathogenic properties, we previously identified Polyglutamine Binding Peptide 1 (QBP1) from combinatorial peptide phage display libraries. We show here that a tandem repeat of the inhibitor peptide QBP1, (QBP1)(2), significantly suppresses polyQ aggregation and polyQ-induced neurodegeneration in the compound eye of Drosophila polyQ disease models, which express the expanded polyQ protein under the eye specific promoter. Most importantly, (QBP1)(2) expression dramatically rescues premature death of flies expressing the expanded polyQ protein in the nervous system, resulting in the dramatic increase of the median life span from 5.5 to 52 days. These results suggest that QBP1 can prevent polyQ-induced neurodegeneration in vivo. We propose that QBP1 prevents polyQ oligomerization and/or aggregation either by altering the toxic conformation of the expanded polyQ stretch, or by simply competing with the expanded polyQ stretches for binding to other expanded polyQ proteins. The peptide inhibitor QBP1 is a promising candidate with great potential as a therapeutic molecule against the currently untreatable polyQ diseases.     

Soluble polyglutamine oligomers formed prior to inclusion body formation are cytotoxic

Expanded polyglutamine (polyQ) repeats cause neurodegenerative disorders, but their cytotoxic structures remain to be elucidated. Although soluble polyQ oligomers have been proposed as a cytotoxic structure, the cytotoxicity of soluble polyQ oligomers, not inclusion bodies (IBs), has not been proven in living cells. To clarify the cytotoxicity of soluble polyQ oligomers, we carried our fluorescence resonance energy transfer (FRET) confocal microscopy and distinguished oligomers from monomers and IBs in a single living cell. FRET signals were detected when donor and acceptor fluorescent proteins were attached to the same side, not the opposite side, of polyQ repeats, which agrees with a parallel beta-sheet or a head-to-tail cylindrical beta-sheet model. These FRET signals disappeared in semi-intact cells, indicating that these polyQ oligomers are soluble. PolyQ monomers assembled into soluble oligomers in a length-dependent manner, which was followed by the formation of IBs. Notably, survival assay of neuronally differentiated cells revealed that cells with soluble oligomers died faster than those with IBs or monomers. These results indicate that a length-dependent formation of oligomers is an essential mechanism underlying neurodegeneration in polyQ-mediated disorders.     

Cytoskeleton proteins are modulators of mutant tau-induced neurodegeneration in Drosophila

Tauopathies, including Alzheimer's disease and fronto-temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), are a group of neurodegenerative disorders characterized by the presence of intraneuronal filamentous inclusions of aberrantly phosphorylated-tau. Tau is a neuronal microtubule-associated protein involved in microtubule assembly and stabilization. Currently, the molecular mechanisms underlying tau-mediated cellular toxicity remain elusive. To address the determinants of tau neurotoxicity, we first characterized the cellular alterations resulting from the over-expression of a mutant form of human tau associated with FTDP-17 (tau V337M) in Drosophila. We found that the over-expression of tau V337M, in Drosophila larval motor neurons, induced disruption of the microtubular network at presynaptic nerve terminals and changes in neuromuscular junctions morphological features. Secondly, we performed a misexpression screen to identify genetic modifiers of the tau V337M-mediated rough eye phenotype. The screening of 1250 mutant Drosophila lines allowed us to identify several components of the cytoskeleton, and particularly from the actin network, as specific modifiers of tau V337M-induced neurodegeneration. Furthermore, we found that numerous tau modulators identified in our screen were involved in the maintenance of synaptic function. Taken together, these findings suggest that disruption of the microtubule network in presynaptic nerve terminals could constitute early events in the pathological process leading to synaptic dysfunction in tau V337M pathology.     

Aggregated polyglutamine peptides delivered to nuclei are toxic to mammalian cells

A number of observations point to the aggregation of expanded polyglutamine [poly(Q)]-containing proteins as playing a central role in the etiology of Huntington's disease (HD) and other expanded CAG-repeat diseases. Transfected cell and transgenic animal models provide some of this support, but irrefutable data on the cytotoxicity of poly(Q) aggregates is lacking. This may be due in part to difficulties in observing all aggregated states in these models, and in part to the inability to conclusively rule out the role of monomeric states of the poly(Q) protein. To address these questions, we produced aggregates of simple poly(Q) peptides in vitro and introduced them to mammalian cells in culture. We find that Cos-7 and PC-12 cells in culture readily take up aggregates of chemically synthesized poly(Q) peptides. Simple poly(Q) aggregates are localized to the cytoplasm and have little impact on cell viability. Aggregates of poly(Q) peptides containing a nuclear localization signal, however, are localized to nuclei and lead to dramatic cell death. Amyloid fibrils of a non-poly(Q) peptide are non-toxic, whether localized to the cytoplasm or nucleus. Nuclear localization of an aggregate of a short, Q(20), poly(Q) peptide is just as toxic as that of a long poly(Q) peptide, supporting the notion that the influence of poly(Q) repeat length on disease risk and age of onset is at the level of aggregation efficiency. The results support a direct role for poly(Q) aggregates in HD-related neurotoxicity.     

Expanded ATXN3 frameshifting events are toxic in Drosophila and mammalian neuron models

Spinocerebellar ataxia type 3 is caused by the expansion of the coding CAG repeat in the ATXN3 gene. Interestingly, a -1 bp frameshift occurring within an (exp)CAG repeat would henceforth lead to translation from a GCA frame, generating polyalanine stretches instead of polyglutamine. Our results show that transgenic expression of (exp)CAG ATXN3 led to -1 frameshifting events, which have deleterious effects in Drosophila and mammalian neurons. Conversely, transgenic expression of polyglutamine-encoding (exp)CAA ATXN3 was not toxic. Furthermore, (exp)CAG ATXN3 mRNA does not contribute per se to the toxicity observed in our models. Our observations indicate that expanded polyglutamine tracts in Drosophila and mouse neurons are insufficient for the development of a phenotype. Hence, we propose that -1 ribosomal frameshifting contributes to the toxicity associated with (exp)CAG repeats.     

ubiquilin antagonizes presenilin and promotes neurodegeneration in Drosophila

The majority of familial Alzheimer's disease (AD) cases are caused by mutations in presenilins, therefore, identifying regulators of presenilins is crucial for understanding AD pathogenesis. Ubiquilin 1 (UBQLN1) binds Presenilins in mammalian cells; however, the functional significance of this interaction in vivo remains unclear. Moreover, while genetic variants in UBQLN1 have recently been reported to associate with an increased risk for AD, whether these variants have altered function is unknown. Here, we show that Drosophila Ubiquilin (Ubqn) binds to Drosophila Presenilin (Psn), and that loss of ubqn function suppresses phenotypes that arise from loss of psn function in vivo. In addition, overexpression of ubqn in the eye results in adult-onset, age-dependent retinal degeneration, which is at least partially apoptotic in nature. The degeneration associated with ubqn overexpression can also be suppressed by psn overexpression and enhanced by expression of a dominant negative version of Psn. Remarkably, expression of the human AD-associated variant of UBQLN1 leads to more severe degeneration than does comparable expression of the human wildtype UBQLN1. Together, these data identify Ubqn as a regulator of Psn, support an important role for UBQLN1 in AD pathogenesis, and suggest the possibility that expression of a human AD-associated variant can cause neurodegeneration independent of amyloid production.     

Inactivation of Drosophila Apaf-1 related killer suppresses formation of polyglutamine aggregates and blocks polyglutamine pathogenesis

Huntington's disease (HD) is caused by expansion of a polyglutamine tract near the N-terminal of huntingtin. Mutant huntingtin forms aggregates in striatum and cortex, where extensive cell death occurs. We used a Drosophila polyglutamine peptide model to assess the role of specific cell death regulators in polyglutamine-induced cell death. Here, we report that polyglutamine-induced cell death was dramatically suppressed in flies lacking Dark, the fly homolog of human Apaf-1, a key regulator of apoptosis. Dark appeared to play a role in the accumulation of polyglutamine-containing aggregates. Suppression of cell death, caspase activation and aggregate formation were also observed when mutant huntingtin exon 1 was expressed in homozygous dark mutant animals. Expanded polyglutamine induced a marked increase in expression of Dark, and Dark was observed to colocalize with ubiquitinated protein aggregates. Apaf-1 also was found to colocalize with huntingtin-containing aggregates in a murine model and HD brain, suggesting a common role for Dark/Apaf-1 in polyglutamine pathogenesis in invertebrates, mice and man. These findings suggest that limiting Apaf-1 activity may alleviate both pathological protein aggregation and neuronal cell death in HD.     

Genetic modulation of polyglutamine toxicity by protein conjugation pathways in Drosophila

Spinal and bulbar muscular atrophy (SBMA) is a heritable neurodegenerative disease caused by the expansion of a polyglutamine [poly(Q)] repeat within the androgen receptor (AR) protein. We studied SBMA in Drosophila using an N-terminal fragment of the human AR protein. Expression of a pathogenic AR protein with an expanded poly(Q) repeat in Drosophila results in nuclear and cytoplasmic inclusion formation, and cellular degeneration, preferentially in neuronal tissues. We have studied the influence of ubiquitin-dependent modification and the proteasome pathway on neural degeneration and AR protein fragment solubility. Compromising the ubiquitin/proteasome pathway enhances degeneration and decreases poly(Q) protein solubility. Our data further suggest that Hsp70 and the proteasome act in an additive manner to modulate neurodegeneration. Through the over-expression of a mutant of the SUMO-1 activating enzyme Uba2, we further show that poly(Q)-induced degeneration is intensified when the cellular SUMO-1 protein conjugation pathway is altered. These data suggest that post-translational protein modification, including the ubiquitin/proteasome and the SUMO-1 pathways, modulate poly(Q) pathogenesis.     

Influenza basic polymerase 2 peptides are recognized by influenza nucleoprotein-specific cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTL) play an important role in limiting viral infections and in eradicating virus from host tissues. Recent progress in understanding the processing and presentation of viral antigens to CTL indicates that the CTL antigen receptor recognizes peptides derived from viral proteins that are bound to an antigen binding groove present in class I major histocompatibility complex (MHC) molecules. In understanding CTL anti-viral responses and in creating vaccines designed to elicit CTL responses, it is critical to identify the portions of viral proteins that bind class I molecules and are recognized by T cell receptors. Previous findings have indicated that a significant portion of the CTL response of H-2d mice to influenza virus is specific for one of the viral polymerases (PB2). To identify the region of PB2 naturally processed and presented by influenza virus-infected mouse cells to CTL, 31 PB2 peptides of 9-16 residues in length were chosen and chemically synthesized. Two peptides, PB2, residues 146-159 and 187-195, were found to sensitize histocompatible target cells for recognition by influenza virus-specific CTL. When CTL were generated to individual viral proteins using influenza-vaccinia recombinant viruses, we found, to our surprise, that PB2-specific CTL failed to recognize cells sensitized with PB2 peptides 146-159 and 187-195. Further analysis showed that these PB2 peptides were, in fact, recognized by nucleoprotein (NP)-specific CTL generated by NP-vac virus priming and influenza A virus stimulation, or NP peptide stimulation in vitro of NP-vac or influenza A-primed CTL. These results demonstrate that while screening peptide libraries one cannot assume that positive peptides necessarily identify the viral protein to which the CTL response is directed.     

Both avian and mammalian embryonic myoblasts are intrinsically heterogeneous

Adult skeletal muscles are composed of different fibre types. What initiates the distinctive muscle fibre type-specific specialization in a developing embryo is still controversial. In vitro studies of avian muscles have shown the expression of one of the slow myosin heavy chains, SM2, in only some myotubes. In this report we demonstrate the expression of another slow myosin heavy chain, SM1, restricted to only some chicken myotubes (presumptive slow) in vitro. We also demonstrate that as is the case for avian species, distinct fast and slow myogenic cells are detectable in mammalian species, human and rat, during in vitro development in the absence of innervation. While antibodies to fast myosin heavy chains stained all myotubes dark in these muscle cell cultures, antibodies to slow myosin heavy chains stained only a proportion of the myotubes (presumptive slow). The other myotubes were either unstained or only weakly stained with slow myosin heavy c hain antibodies. The muscle cell cultures prepared from different developmental stages of rat skeletal muscles showed a reduction in the number of slow myosin heavy chain-positive myotubes with advancing foetal growth. It is concluded that embryonic myogenic cells that are likely to form distinct fast or slow muscle fibre types are intrinsically heterogeneous, not only in avian but also in mammalian species, although extrinsic factors reinforce and modify such commitment throughout subsequent development. © Kluwer Academic Publishers.     

Melanocortin 1 Receptor-derived peptides are efficiently recognized by cytotoxic T lymphocytes from melanoma patients

Background

Melanocortin 1 Receptor (MC1R) is expressed in a majority of melanoma biopsies and cell lines. We previously demonstrated that three hydrophobic low-affinity HLA-A2-restricted MC1R-derived peptides: MC1R_291–298, MC1R_244–252 and MC1R_283–291 can elicit cytotoxic T-lymphocytes (CTL) responses from normal donor peripheral blood lymphocytes (PBL). Moreover, peptide-specific CTL recognized a panel of MHC-matched melanomas, demonstrating that human melanoma cell lines naturally present MC1R epitopes. However, the natural presence of MC1R-specific T cells in melanoma patient's tumour and blood remains unknown.

Methods

The presence of anti-MC1R specific CD8⁺ T cells was established in a population of melanoma-specific T cells derived from peripheral blood mononuclear cells (PBMC) and tumour-infiltrating lymphocytes (TIL) from HLA-A2⁺ melanoma patients.

Results

CTLs specific for the three MC1R-derived peptides that lysed allogeneic HLA-A2⁺MC1R⁺ melanomas were elicited from PBMC, demonstrating the existence of an anti-MC1R T cell repertoire in melanoma patients. Moreover, TILs also recognized MC1R epitopes and HLA-A2⁺ melanoma cell lines. Finally, HLA-A2/MC1R₂₄₄-specific CD8⁺ T cell clones derived from TILs and a subset of MC1R₂₉₁ specific TILs were identified using HLA-A2/MC1R tetramers.

Conclusion

Our results demonstrate that MC1R-derived peptides are common immunogenic epitopes for melanoma-specific CTLs and TILs, and may thus be useful for the development of anti-melanoma immunotherapy.     

Soluble HLA-A2.1 restricted peptides that are recognized by influenza virus specific cytotoxic T lymphocytes

The influenza A virus matrix protein derived peptide with amino acids 57-68 (Lys-Gly-Ileu-Leu-Gly-Phe-Val-Phe-Thr-Leu-Thr-Val) is recognized by influenza virus HLA-A2 restricted CTL. Because of the large number of hydrophobic residues this peptide is very insoluble. Substitution with a number of polar amino acids resulted in a soluble peptide (Lys-Lys-Ala-Leu-Gly-Phe-Val-Phe-Thr-Leu-Asp-Lys) that was very effective in sensitizing HLA-A2 positive target cells. Further substitution of threonine in position 65 with lysine resulted in a soluble antagonist peptide that inhibited sensitization. Both agonist and antagonist peptides retained 20% of their biological activity when tyrosine was added at the N terminus. Soluble radio-iodinated peptides can now be prepared that will be useful reagents to study the interaction of peptides and class I molecules.     

Impaired ERAD and ER stress are early and specific events in polyglutamine toxicity

Protein misfolding, whether caused by aging, environmental factors, or genetic mutations, is a common basis for neurodegenerative diseases. The misfolding of proteins with abnormally long polyglutamine (polyQ) expansions causes several neurodegenerative disorders, such as Huntington’s disease (HD). Although many cellular pathways have been documented to be impaired in HD, the primary triggers of polyQ toxicity remain elusive. We report that yeast cells and neuron-like PC12 cells expressing polyQ-expanded huntingtin (htt) fragments display a surprisingly specific, immediate, and drastic defect in endoplasmic reticulum (ER)-associated degradation (ERAD). We further decipher the mechanistic basis for this defect in ERAD: the entrapment of the essential ERAD proteins Npl4, Ufd1, and p97 by polyQ-expanded htt fragments. In both yeast and mammalian neuron-like cells, overexpression of Npl4 and Ufd1 ameliorates polyQ toxicity. Our results establish that impaired ER protein homeostasis is a broad and highly conserved contributor to polyQ toxicity in yeast, in PC12 cells, and, importantly, in striatal cells expressing full-length polyQ-expanded huntingtin.     

Expanded polyglutamines induce neurodegeneration and trans-neuronal alterations in cerebellum and retina of SCA7 transgenic mice

Among the eight progressive neurodegenerative diseases caused by polyglutamine expansions, spinocerebellar ataxia type 7 (SCA7) is the only one to display degeneration in both brain and retina. We show here that mice overexpressing full-length mutant ataxin-7[Q90] either in Purkinje cells or in rod photoreceptors have deficiencies in motor coordination and vision, respectively. In both models, although with different time courses, an N-terminal fragment of mutant ataxin-7 accumulates into ubiquitinated nuclear inclusions that recruit a distinct set of chaperone/proteasome subunits. A severe degeneration is caused by overexpression of ataxin-7[Q90] in rods, whereas a similar overexpression of normal ataxin-7[Q10] has no obvious effect. The degenerative process is not limited to photoreceptors, showing secondary alterations of post-synaptic neurons. These findings suggest that proteolytic cleavage of mutant ataxin-7 and trans-neuronal responses are implicated in the pathogenesis of SCA7.     

Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy

Protein conformational disorders (PCDs), such as Alzheimer's disease, Huntington's disease (HD), Parkinson's disease and oculopharyngeal muscular dystrophy, are associated with proteins that misfold and aggregate. Here we have used exon 1 of the HD gene with expanded polyglutamine [poly(Q)] repeats and enhanced green fluorescent protein tagged to 19 alanines as models for aggregate-prone proteins, to investigate the pathways mediating their degradation. Autophagy is involved in the degradation of these model proteins, since they accumulated when cells were treated with different inhibitors acting at distinct stages of the autophagy-lysosome pathway, in two different cell lines. Furthermore, rapamycin, which stimulates autophagy, enhanced the clearance of our aggregate-prone proteins. Rapamycin also reduced the appearance of aggregates and the cell death associated with the poly(Q) and polyalanine [poly(A)] expansions. Since rapamycin is used clinically, this drug or related analogues may be suitable candidates for therapeutic investigation in HD and related diseases. We have also re-examined the role of the proteasome, since previous studies in poly(Q) diseases have used lactacystin as an inhibitor--recent studies have shown that lactacystin may also affect lysosomal function. Both lactacystin and the specific proteasomal inhibitor epoxomicin increased soluble protein levels of the poly(Q) constructs, suggesting that these are also cleared by the proteasome. However, while poly(Q) aggregation was enhanced by lactacystin in our inducible PC12 cell model, aggregation was reduced by epoxomicin, suggesting that some other protein(s) induced by epoxomicin may regulate poly(Q) aggregation.     

Comparative toxicity of polyglutamine, polyalanine and polyleucine tracts in Drosophila models of expanded repeat disease

Homopolymeric amino acid repeat sequences in proteins are of particular interest due to the discovery that expanded copy numbers of these repeats are the molecular basis for a growing list of human genetic diseases. Repeat copy numbers above a typical normal range of polyglutamine repeats have been found to be the principal pathogenic agents in a number of these diseases, including Huntington's disease. There is emerging evidence that expansions of amino acids encoded by other reading frames of CAG/CUG repeats, including polyalanine and polyleucine, could contribute to toxicity in the 'polyglutamine' diseases. We have therefore used the Drosophila model system to investigate effects of ectopic expression of polyglutamine, polyleucine and polyalanine repeats in vivo to assess their relative toxicities and the common and distinct characteristics of the pathogenesis that they cause. We find that these homopolymeric sequences all exhibit toxicity and are able to form aggregates in Drosophila, although there are marked differences in the degree of toxicity dependent upon the tissue in which they are expressed.     

A molecular pathway of neurodegeneration linking alpha-synuclein to ApoE and Abeta peptides

Pathogenic aggregates of alpha-synuclein are thought to contribute to the development of Parkinson's disease. Inclusion bodies containing alpha-synuclein are present in Parkinson's disease and other neurodegenerative diseases, including Alzheimer's disease. Moreover, alpha-synuclein mutations are found in cases of familial Parkinson's disease, and transgenic overexpression of alpha-synuclein causes neurodegeneration in mice. The molecular mechanisms involved, however, remain incompletely understood. Here we show that, in transgenic mice, alpha-synuclein induced neurodegeneration involves activation of the ubiquitin/proteasome system, a massive increase in apolipoprotein E (ApoE) levels and accumulation of insoluble mouse Abeta. ApoE was not protective, but was injurious, as deletion of ApoE delayed the neurodegeneration caused by alpha-synuclein and suppressed the accumulation of Abeta. Our data reveal a molecular link between central pathogenic mechanisms implicated in Parkinson's disease and Alzheimer's disease and suggest that intracellular alpha-synuclein is pathogenic, at least in part, by activation of extracellular signaling pathways involving ApoE.