Polyglutamine- and Temperature-Dependent Conformational Rigidity in Mutant Huntingtin Revealed by Immunoassays and Circular Dichroism Spectroscopy

Background

In Huntington''s disease, expansion of a CAG triplet repeat occurs in exon 1 of the huntingtin gene (HTT), resulting in a protein bearing>35 polyglutamine residues whose N-terminal fragments display a high propensity to misfold and aggregate. Recent data demonstrate that polyglutamine expansion results in conformational changes in the huntingtin protein (HTT), which likely influence its biological and biophysical properties. Developing assays to characterize and measure these conformational changes in isolated proteins and biological samples would advance the testing of novel therapeutic approaches aimed at correcting mutant HTT misfolding. Time-resolved Förster energy transfer (TR-FRET)-based assays represent high-throughput, homogeneous, sensitive immunoassays widely employed for the quantification of proteins of interest. TR-FRET is extremely sensitive to small distances and can therefore provide conformational information based on detection of exposure and relative position of epitopes present on the target protein as recognized by selective antibodies. We have previously reported TR-FRET assays to quantify HTT proteins based on the use of antibodies specific for different amino-terminal HTT epitopes. Here, we investigate the possibility of interrogating HTT protein conformation using these assays.

Methodology/Principal Findings

By performing TR-FRET measurements on the same samples (purified recombinant proteins or lysates from cells expressing HTT fragments or full length protein) at different temperatures, we have discovered a temperature-dependent, reversible, polyglutamine-dependent conformational change of wild type and expanded mutant HTT proteins. Circular dichroism spectroscopy confirms the temperature and polyglutamine-dependent change in HTT structure, revealing an effect of polyglutamine length and of temperature on the alpha-helical content of the protein.

Conclusions/Significance

The temperature- and polyglutamine-dependent effects observed with TR-FRET on HTT proteins represent a simple, scalable, quantitative and sensitive assay to identify genetic and pharmacological modulators of mutant HTT conformation, and potentially to assess the relevance of conformational changes during onset and progression of Huntington''s disease.     

Huntingtin cleavage product A forms in neurons and is reduced by gamma-secretase inhibitors

Background

The mutation in Huntington's disease is a polyglutamine expansion near the N-terminus of huntingtin. Huntingtin expressed in immortalized neurons is cleaved near the N-terminus to form N-terminal polypeptides known as cleavage products A and B (cpA and cpB). CpA and cpB with polyglutamine expansion form inclusions in the nucleus and cytoplasm, respectively. The formation of cpA and cpB in primary neurons has not been established and the proteases involved in the formation of these fragments are unknown.

Results

Delivery of htt cDNA into the mouse striatum using adeno-associated virus or into primary cortical neurons using lentivirus generated cpA and cpB, indicating that neurons in brain and in vitro can form these fragments. A screen of small molecule protease inhibitors introduced to clonal striatal X57 cells and HeLa cells identified compounds that reduced levels of cpA and are inhibitors of the aspartyl proteases cathepsin D and cathepsin E. The most effective compound, P1-N031, is a transition state mimetic for aspartyl proteases. By western blot analysis, cathepsin D was easily detected in clonal striatal X57 cells, mouse brain and primary neurons, whereas cathepsin E was only detectible in clonal striatal X57 cells. In primary neurons, levels of cleavage product A were not changed by the same compounds that were effective in clonal striatal cells or by mRNA silencing to partially reduce levels of cathepsin D. Instead, treating primary neurons with compounds that are known to inhibit gamma secretase activity either indirectly (Imatinib mesylate, Gleevec) or selectively (LY-411,575 or DAPT) reduced levels of cpA. LY-411,575 or DAPT also increased survival of primary neurons expressing endogenous full-length mutant huntingtin.

Conclusion

We show that cpA and cpB are produced from a larger huntingtin fragment in vivo in mouse brain and in primary neuron cultures. The aspartyl protease involved in forming cpA has cathepsin-D like properties in immortalized neurons and gamma secretase-like properties in primary neurons, suggesting that cell type may be a critical factor that specifies the aspartyl protease responsible for cpA. Since gamma secretase inhibitors were also protective in primary neurons, further study of the role of gamma-secretase activity in HD neurons is justified.     

The mTOR kinase inhibitor Everolimus decreases S6 kinase phosphorylation but fails to reduce mutant huntingtin levels in brain and is not neuroprotective in the R6/2 mouse model of Huntington's disease

Background

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion within the huntingtin gene. Mutant huntingtin protein misfolds and accumulates within neurons where it mediates its toxic effects. Promoting mutant huntingtin clearance by activating macroautophagy is one approach for treating Huntington's disease (HD). In this study, we evaluated the mTOR kinase inhibitor and macroautophagy promoting drug everolimus in the R6/2 mouse model of HD.

Results

Everolimus decreased phosphorylation of the mTOR target protein S6 kinase indicating brain penetration. However, everolimus did not activate brain macroautophagy as measured by LC3B Western blot analysis. Everolimus protected against early declines in motor performance; however, we found no evidence for neuroprotection as determined by brain pathology. In muscle but not brain, everolimus significantly decreased soluble mutant huntingtin levels.

Conclusions

Our data suggests that beneficial behavioral effects of everolimus in R6/2 mice result primarily from effects on muscle. Even though everolimus significantly modulated its target brain S6 kinase, this did not decrease mutant huntingtin levels or provide neuroprotection.

     

Mitochondrial permeability transition pore induces mitochondria injury in Huntington disease

Background

Mitochondrial impairment has been implicated in the pathogenesis of Huntington’s disease (HD). However, how mutant huntingtin impairs mitochondrial function and thus contributes to HD has not been fully elucidated. In this study, we used striatal cells expressing wild type (STHdh^Q7/Q7) or mutant (STHdh^Q111/Q111) huntingtin protein, and cortical neurons expressing the exon 1 of the huntingtin protein with physiological or pathological polyglutamine domains, to examine the interrelationship among specific mitochondrial functions.

Results

Depolarization induced by KCl resulted in similar changes in calcium levels without compromising mitochondrial function, both in wild type and mutant cells. However, treatment of mutant cells with thapsigargin (a SERCA antagonist that raises cytosolic calcium levels), resulted in a pronounced decrease in mitochondrial calcium uptake, increased production of reactive oxygen species (ROS), mitochondrial depolarization and fragmentation, and cell viability loss. The mitochondrial dysfunction in mutant cells was also observed in cortical neurons expressing exon 1 of the huntingtin protein with 104 Gln residues (Q104-GFP) when they were exposed to calcium stress. In addition, calcium overload induced opening of the mitochondrial permeability transition pore (mPTP) in mutant striatal cells. The mitochondrial impairment observed in mutant cells and cortical neurons expressing Q104-GFP was prevented by pre-treatment with cyclosporine A (CsA) but not by FK506 (an inhibitor of calcineurin), indicating a potential role for mPTP opening in the mitochondrial dysfunction induced by calcium stress in mutant huntingtin cells.

Conclusions

Expression of mutant huntingtin alters mitochondrial and cell viability through mPTP opening in striatal cells and cortical neurons.

     

Are there multiple pathways in the pathogenesis of Huntington's disease?

Studies of huntingtin localization in human post-mortem brain offer insights and a framework for basic experiments in the pathogenesis of Huntington''s disease. In neurons of cortex and striatum, we identified changes in the cytoplasmic localization of huntingtin including a marked perinuclear accumulation of huntingtin and formation of multivesicular bodies, changes conceivably pointing to an altered handling of huntingtin in neurons. In Huntington''s disease, huntingtin also accumulates in aberrant subcellular compartments such as nuclear and neuritic aggregates co-localized with ubiquitin. The site of protein aggregation is polyglutamine-dependent, both in juvenile-onset patients having more aggregates in the nucleus and in adult-onset patients presenting more neuritic aggregates. Studies in vitro reveal that the genesis of these aggregates and cell death are tied to cleavage of mutant huntingtin. However, we found that the aggregation of mutant huntingtin can be dissociated from the extent of cell death. Thus properties of mutant huntingtin more subtle than its aggregation, such as its proteolysis and protein interactions that affect vesicle trafficking and nuclear transport, might suffice to cause neurodegeneration in the striatum and cortex. We propose that mutant huntingtin engages multiple pathogenic pathways leading to neuronal death.     

Formation of Polyglutamine Inclusions in a Wide Range of Non-CNS Tissues in the HdhQ150 Knock-In Mouse Model of Huntington's Disease

Background

Huntington''s disease (HD) is an inherited progressive neurodegenerative disorder caused by a CAG repeat expansion in the ubiquitously expressed HD gene resulting in an abnormally long polyglutamine repeat in the huntingtin protein. Polyglutamine inclusions are a hallmark of the neuropathology of HD. We have previously shown that inclusion pathology is also present in the peripheral tissues of the R6/2 mouse model of HD which expresses a small N-terminal fragment of mutant huntingtin. To determine whether this peripheral pathology is a consequence of the aberrant expression of this N-terminal fragment, we extend this analysis to the genetically precise knock-in mouse model of HD, HdhQ150, which expresses mutant mouse huntingtin.

Methodology/Principal Findings

We have previously standardized the CAG repeat size and strain background of the R6/2 and HdhQ150 knock-in mouse models and found that they develop a comparable and widespread neuropathology. To determine whether HdhQ150 knock-in mice also develop peripheral inclusion pathology, homozygous Hdh ^Q150/Q150 mice were perfusion fixed at 22 months of age, and tissues were processed for histology and immunohistochemistry with the anti-huntingtin antibody S830. The peripheral inclusion pathology was almost identical to that found in R6/2 mice at 12 weeks of age with minor differences in inclusion abundance.

Conclusions/Significance

The highly comparable peripheral inclusion pathology that is present in both the R6/2 and HdhQ150 knock-in models of HD indicates that the presence of peripheral inclusions in R6/2 mice is not a consequence of the aberrant expression of an N-terminal huntingtin protein. It remains to be determined whether peripheral inclusions are a pathological feature of the human disease. Both mouse models carry CAG repeats that cause childhood disease in humans, and therefore, inclusion pathology may be a feature of the childhood rather than the adult forms of HD. It is important to establish the extent to which peripheral pathology causes the peripheral symptoms of HD from the perspective of a mechanistic understanding and future treatment options.     

A Genome-Scale RNA–Interference Screen Identifies RRAS Signaling as a Pathologic Feature of Huntington's Disease

A genome-scale RNAi screen was performed in a mammalian cell-based assay to identify modifiers of mutant huntingtin toxicity. Ontology analysis of suppressor data identified processes previously implicated in Huntington''s disease, including proteolysis, glutamate excitotoxicity, and mitochondrial dysfunction. In addition to established mechanisms, the screen identified multiple components of the RRAS signaling pathway as loss-of-function suppressors of mutant huntingtin toxicity in human and mouse cell models. Loss-of-function in orthologous RRAS pathway members also suppressed motor dysfunction in a Drosophila model of Huntington''s disease. Abnormal activation of RRAS and a down-stream effector, RAF1, was observed in cellular models and a mouse model of Huntington''s disease. We also observe co-localization of RRAS and mutant huntingtin in cells and in mouse striatum, suggesting that activation of R-Ras may occur through protein interaction. These data indicate that mutant huntingtin exerts a pathogenic effect on this pathway that can be corrected at multiple intervention points including RRAS, FNTA/B, PIN1, and PLK1. Consistent with these results, chemical inhibition of farnesyltransferase can also suppress mutant huntingtin toxicity. These data suggest that pharmacological inhibition of RRAS signaling may confer therapeutic benefit in Huntington''s disease.     

Conformational Targeting of Fibrillar Polyglutamine Proteins in Live Cells Escalates Aggregation and Cytotoxicity

Background

Misfolding- and aggregation-prone proteins underlying Parkinson''s, Huntington''s and Machado-Joseph diseases, namely α-synuclein, huntingtin, and ataxin-3 respectively, adopt numerous intracellular conformations during pathogenesis, including globular intermediates and insoluble amyloid-like fibrils. Such conformational diversity has complicated research into amyloid-associated intracellular dysfunction and neurodegeneration. To this end, recombinant single-chain Fv antibodies (scFvs) are compelling molecular tools that can be selected against specific protein conformations, and expressed inside cells as intrabodies, for investigative and therapeutic purposes.

Methodology/Principal Findings

Using atomic force microscopy (AFM) and live-cell fluorescence microscopy, we report that a human scFv selected against the fibrillar form of α-synuclein targets isomorphic conformations of misfolded polyglutamine proteins. When expressed in the cytoplasm of striatal cells, this conformation-specific intrabody co-localizes with intracellular aggregates of misfolded ataxin-3 and a pathological fragment of huntingtin, and enhances the aggregation propensity of both disease-linked polyglutamine proteins. Using this intrabody as a tool for modulating the kinetics of amyloidogenesis, we show that escalating aggregate formation of a pathologic huntingtin fragment is not cytoprotective in striatal cells, but rather heightens oxidative stress and cell death as detected by flow cytometry. Instead, cellular protection is achieved by suppressing aggregation using a previously described intrabody that binds to the amyloidogenic N-terminus of huntingtin. Analogous cytotoxic results are observed following conformational targeting of normal or polyglutamine-expanded human ataxin-3, which partially aggregate through non-polyglutamine domains.

Conclusions/Significance

These findings validate that the rate of aggregation modulates polyglutamine-mediated intracellular dysfunction, and caution that molecules designed to specifically hasten aggregation may be detrimental as therapies for polyglutamine disorders. Moreover, our findings introduce a novel antibody-based tool that, as a consequence of its general specificity for fibrillar conformations and its ability to function intracellularly, offers broad research potential for a variety of human amyloid diseases.     

Curcumin enhances the polyglutamine-expanded truncated N-terminal huntingtin-induced cell death by promoting proteasomal malfunction

Formation of neuronal intranuclear inclusions of the disease proteins that are ubiquitinated and often associated with various proteasome components is the major hallmark of the polyglutamine diseases. Curcumin is a polyphenolic compound having anti-inflammatory, anti-tumor, and anti-oxidative properties. Recently, curcumin has been reported to suppress the amyloid-beta accumulation, oxidative damage, and inflammation in the transgenic mice model of Alzheimer's disease. Here, we found that the treatment of curcumin increases the polyglutamine-expanded truncated N-terminal huntingtin (mutant huntingtin) aggregation and mutant huntingtin-dependent cell death. Curcumin also causes rapid proteasomal malfunction in the mutant huntingtin expressing cells in comparison with normal glutamine repeat expressing cells. Finally, we show that N-acetyl cysteine (NAC), a potent antioxidant, reverted the curcumin-induced mutant huntingtin aggregation and proteasomal malfunction in the mutant huntingtin expressing cells. NAC also protects curcumin-induced cell death. Our result suggests that curcumin promotes mutant huntingtin-induced cell death by mimicking proteasomal dysfunction.     

Downregulation of Genes Involved in Metabolism and Oxidative Stress in the Peripheral Leukocytes of Huntington's Disease Patients

Background

Huntington''s disease (HD) is caused by expanded CAG repeats encoding a polyglutamine tract in the huntingtin (HTT) protein. A number of differentially-expressed protein molecules have been identified in striatum of HD animal models. Here we examined if the expression changes could be visualized in the peripheral leukocytes of HD patients and pre-symptomatic HD (PreHD) carriers.

Methods and findings

The expression levels of 17 candidate genes that differentially expressed in striatum between transgenic HD and wild-type mice in literature were measured in the peripheral leukocytes of 4 PreHD carriers, 16 HD patients and 20 healthy controls. Four genes majorly involved in metabolism and oxidative stress response, including AHCY1, ACO2, OXCT1 and CAP1, demonstrated consistent downregulation in peripheral leukocytes of both PreHD carriers and HD patients, while UCP2 was only down-regulated in HD patients.

Conclusion

These results provide potential peripheral biomarkers to indicate disease onset in preclinical stage, and to monitor the efficacy of early treatment. Further studies of a large series of preHD carriers and symptomatic HD patients will be warranted to verify the findings and examine if these markers correlate with clinical features.     

A compact beta model of huntingtin toxicity

Huntington disease results from an expanded polyglutamine region in the N terminus of the huntingtin protein. HD pathology is characterized by neuronal degeneration and protein inclusions containing N-terminal fragments of mutant huntingtin. Structural information is minimal, though it is believed that mutant huntingtin polyglutamine adopts β structure upon conversion to a toxic form. To this end, we designed mammalian cell expression constructs encoding compact β variants of Htt exon 1 N-terminal fragment and tested their ability to aggregate and induce toxicity in cultured neuronal cells. In parallel, we performed molecular dynamics simulations, which indicate that constructs with expanded polyglutamine β-strands are stabilized by main-chain hydrogen bonding. Finally, we found a correlation between the reactivity to 3B5H10, an expanded polyglutamine antibody that recognizes a compact β rich hairpin structure, and the ability to induce cell toxicity. These data are consistent with an important role for a compact β structure in mutant huntingtin-induced cell toxicity.     

Expanded glutamines and neurodegeneration – a gain of insight

Glutamine repeat expansion has been established as the mutation underlying five inherited neurodegenerative diseases. The mechanism by which this apparently universal mutation, in ubiquitously expressed proteins, causes highly selective neurodegeneration is unknown. The proteins containing the glutamine expansions are otherwise unrelated and likely to have different functions. Two recently published papers^(1,2) provide evidence of a conformational change occurring in polyglutamine expansions, which may allow novel interactions and is consistent with a toxic gain-of-function hypothesis. HAP1, a protein that interacts with huntingtin (Huntington's disease protein), has an expression profile that intriguingly mirrors the selective neurodegeneration seen in Huntington's disease⁽²⁾.     

Reduced Levels of Proteasome Products in a Mouse Striatal Cell Model of Huntington’s Disease

Huntington’s disease is the result of a long polyglutamine tract in the gene encoding huntingtin protein, which in turn causes a large number of cellular changes and ultimately results in neurodegeneration of striatal neurons. Although many theories have been proposed, the precise mechanism by which the polyglutamine expansion causes cellular changes is not certain. Some evidence supports the hypothesis that the long polyglutamine tract inhibits the proteasome, a multiprotein complex involved in protein degradation. However, other studies report normal proteasome function in cells expressing long polyglutamine tracts. The controversy may be due to the methods used to examine proteasome activity in each of the previous studies. In the present study, we measured proteasome function by examining levels of endogenous peptides that are products of proteasome cleavage. Peptide levels were compared among mouse striatal cell lines expressing either 7 glutamines (STHdh ^Q7/Q7) or 111 glutamines in the huntingtin protein, either heterozygous (STHdh ^Q7/Q111) or homozygous (STHdh ^Q111/Q111). Both of the cell lines expressing huntingtin with 111 glutamines showed a large reduction in nearly all of the peptides detected in the cells, relative to levels of these peptides in cells homozygous for 7 glutamines. Treatment of STHdh ^Q7/Q7 cells with proteasome inhibitors epoxomicin or bortezomib also caused a large reduction in most of these peptides, suggesting that they are products of proteasome-mediated cleavage of cellular proteins. Taken together, these results support the hypothesis that proteasome function is impaired by the expression of huntingtin protein containing long polyglutamine tracts.