Htt Exon1 Research Articles

Pathogenic TDP-43 accelerates the generation of toxic exon1 HTT in Huntington's disease knock-in mice.

Huntington's disease (HD) is caused by a CAG repeat expansion in exon1 of the HTT gene that encodes a polyglutamine tract in huntingtin protein. The formation of HTT exon1 fragments with an expanded polyglutamine repeat has been implicated as a key step in the pathogenesis of HD. It was reported that the CAG repeat length-dependent aberrant splicing of exon1 HTT results in a short polyadenylated mRNA that is translated into an exon1 HTT protein. Under normal conditions, TDP-43 is predominantly found in the nucleus, where it regulates gene expression. However, in various pathological conditions, TDP-43 is mislocalized in the cytoplasm. By investigating HD knock-in mice, we explore whether the pathogenic TDP-43 in the cytoplasm contributes to HD pathogenesis, through expressing the cytoplasmic TDP-43 without nuclear localization signal. We found that the cytoplasmic TDP-43 is increased in the HD mouse brain and that its mislocalization could deteriorate the motor and gait behavior. Importantly, the cytoplasmic TDP-43, via its binding to the intron1 sequence (GU/UG)n of the mouse Htt pre-mRNA, promotes the transport of exon1-intron1 Htt onto ribosome, resulting in the aberrant generation of exon1 Htt. Our findings suggest that cytoplasmic TDP-43 contributes to HD pathogenesis via its binding to and transport of nuclear un-spliced mRNA to the ribosome for the generation of a toxic protein product.

Exon 1-targeting miRNA reduces the pathogenic exon 1 HTT protein in Huntington disease models.

Huntington disease (HD) is a fatal neurodegenerative disease caused by a trinucleotide repeat expansion in exon 1 of the huntingtin gene (HTT) resulting in toxic gain-of-function and cell death. Despite its monogenic cause, the pathogenesis of HD is highly complex and increasing evidence indicates that, in addition to the full-length (FL) mutant HTT protein, the expanded exon 1 HTT (HTTexon1) protein that is translated from the HTT1a transcript generated by aberrant splicing is prone to aggregate and may contribute to HD pathology. This finding suggests that reducing the expression of HTT1a may achieve a greater therapeutic benefit than targeting only FL mutant HTT. Conversely, strategies that exclusively target FL HTT may not fully prevent the pathogenesis of HD. We have developed an engineered microRNA targeting the HTT exon 1 sequence (miHTT), delivered via adeno-associated virus serotype 5 (AAV5). The target sequence of miHTT is present in both FL HTT and HTT1a transcripts. Preclinical studies with AAV5-miHTT have demonstrated efficacy in several rodent and large animal models by reducing FL HTT mRNA and protein and rescuing HD-like phenotypes, and have been the rationale for phase I/II clinical studies now ongoing in the US and Europe. In the present study, we evaluated the ability of AAV5-miHTT to reduce the levels of aberrantly spliced HTT1a mRNA and the HTTexon1 protein in the brain of two mouse models of HD (heterozygous zQ175 knock-in mice and humanized Hu128/21 mice). Polyadenylated HTT1a mRNA and HTTexon1 protein were detected in the striatum and cortex of heterozygous zQ175 knock-in mice, but not in wild-type, littermate control mice. Intrastriatal administration of AAV5-miHTT resulted in dose-dependent expression of mature miHTT microRNA in cortical brain regions, accompanied by significant lowering of both FL HTT and HTT1a mRNA expression at two months post-injection. Mutant HTT and HTTexon1 protein levels were also significantly reduced in the striatum and cortex of heterozygous zQ175 knock-in at 2 months after AAV5-miHTT treatment and in humanized Hu128/21 mice 7 months post-treatment. The effects were confirmed in primary Hu128/21 neuronal cultures. These results demonstrate that AAV5-miHTT gene therapy is an effective approach to lower both FL HTT and the pathogenic HTTexon1 levels, which could potentially have an additive therapeutic benefit compared to other HTT-targeting modalities.

Aberrant splicing of mutant huntingtin in Huntington's disease knock-in pigs

Huntington's disease (HD) is an autosomal-dominant inherited neurodegenerative disease caused by a CAG repeat expansion in exon1 of the huntingtin gene (HTT). This expansion leads to the production of N-terminal mutant huntingtin protein (mHtt) that contains an expanded polyglutamine tract, which is toxic to neurons and causes neurodegeneration. While the production of N-terminal mHtt can be mediated by proteolytic cleavage of full-length mHtt, abnormal splicing of exon1-intron1 of mHtt has also been identified in the brains of HD mice and patients. However, the proportion of aberrantly spliced exon1 mHTT in relation to normal mHTT exon remains to be defined. In this study, HTT exon1 production was examined in the HD knock-in (KI) pig model, which more closely recapitulates neuropathology seen in HD patient brains than HD mouse models. The study revealed that aberrant spliced HTT exon1 is also present in the brains of HD pigs, but it is expressed at a much lower level than the normally spliced HTT exon products. These findings suggest that careful consideration is needed when assessing the contribution of aberrantly spliced mHTT exon1 to HD pathogenesis, and further rigorous investigation is required.

Huntingtin exon 1 deletion does not alter the subcellular distribution of huntingtin and gene transcription in mice.

Huntington disease (HD) is caused by the expansion of CAG triplet repeats in exon 1 of the huntingtin (HTT) gene, which also encodes the first 17 amino acids (N-17) that can modulate the toxicity of the expanded polyQ repeat. N-17 are conserved in a wide range of species and are found to influence the subcellular distribution of mutant Htt. Moreover, N-17 is subject to many posttranslational modifications that may regulate the function, stability, and distribution of HTT. However, the function of Htt exon 1 and its influence on the normal Htt remains to be fully investigated. By investigating a knock-in mouse model that lacks Htt exon1, we found that deletion of Htt exon1 does not affect the survival of mice and differentiation of cultured mouse neurons. Furthermore, the lack of Htt exon 1 does not alter the subcellular distribution of Htt, autophagy protein expression, and global gene transcription in the mouse brain. These results suggest that removing the entire exon 1 of Htt could be a therapeutic approach to eliminate expanded polyQ toxicity.

A plant-based mutant huntingtin model-driven discovery of impaired expression of GTPCH and DHFR

Pathophysiology associated with Huntington’s disease (HD) has been studied extensively in various cell and animal models since the 1993 discovery of the mutant huntingtin (mHtt) with abnormally expanded polyglutamine (polyQ) tracts as the causative factor. However, the sequence of early pathophysiological events leading to HD still remains elusive. To gain new insights into the early polyQ-induced pathogenic events, we expressed Htt exon1 (Httex1) with a normal (21), or an extended (42 or 63) number of polyQ in tobacco plants. Here, we show that transgenic plants accumulated Httex1 proteins with corresponding polyQ tracts, and mHttex1 induced protein aggregation and affected plant growth, especially root and root hair development, in a polyQ length-dependent manner. Quantitative proteomic analysis of young roots from severely affected Httex1Q63 and unaffected Httex1Q21 plants showed that the most reduced protein by polyQ63 is a GTP cyclohydrolase I (GTPCH) along with many of its related one-carbon (C1) metabolic pathway enzymes. GTPCH is a key enzyme involved in folate biosynthesis in plants and tetrahydrobiopterin (BH4) biosynthesis in mammals. Validating studies in 4-week-old R6/2 HD mice expressing a mHttex1 showed reduced levels of GTPCH and dihydrofolate reductase (DHFR, a key folate utilization/alternate BH4 biosynthesis enzyme), and impaired C1 and BH4 metabolism. Our findings from mHttex1 plants and mice reveal impaired expressions of GTPCH and DHFR and may contribute to a better understanding of mHtt-altered C1 and BH4 metabolism, and their roles in the pathogenesis of HD.

Myelinosome Organelles in the Retina of R6/1 Huntington Disease (HD) Mice: Ubiquitous Distribution and Possible Role in Disease Spreading

Visual deficit is one of the complications of Huntington disease (HD), a fatal neurological disorder caused by CAG trinucleotide expansions in the Huntingtin gene, leading to the production of mutant Huntingtin (mHTT) protein. Transgenic HD R6/1 mice expressing human HTT exon1 with 115 CAG repeats recapitulate major features of the human pathology and exhibit a degeneration of the retina. Our aim was to gain insight into the ultrastructure of the pathological HD R6/1 retina by electron microscopy (EM). We show that the HD R6/1 retina is enriched with unusual organelles myelinosomes, produced by retinal neurons and glia. Myelinosomes are present in all nuclear and plexiform layers, in the synaptic terminals of photoreceptors, in the processes of retinal neurons and glial cells, and in the subretinal space. In vitro study shows that myelinosomes secreted by human retinal glial Müller MIO-M1 cells transfected with EGFP-mHTT-exon1 carry EGFP-mHTT-exon1 protein, as revealed by immuno-EM and Western-blotting. Myelinosomes loaded with mHTT-exon1 are incorporated by naive neuronal/neuroblastoma SH-SY5Y cells. This results in the emergence of mHTT-exon1 in recipient cells. This process is blocked by membrane fusion inhibitor MDL 28170. Conclusion: Incorporation of myelinosomes carrying mHTT-exon1 in recipient cells may contribute to HD spreading in the retina. Exploring ocular fluids for myelinosome presence could bring an additional biomarker for HD diagnostics.

An Expanded Polyproline Domain Maintains Mutant Huntingtin Soluble in vivo and During Aging.

Huntington’s disease is a dominantly inherited neurodegenerative disorder caused by the expansion of a CAG repeat, encoding for the amino acid glutamine (Q), present in the first exon of the protein huntingtin. Over the threshold of Q39 HTT exon 1 (HTTEx1) tends to misfold and aggregate into large intracellular structures, but whether these end-stage aggregates or their on-pathway intermediates are responsible for cytotoxicity is still debated. HTTEx1 can be separated into three domains: an N-terminal 17 amino acid region, the polyglutamine (polyQ) expansion and a C-terminal proline rich domain (PRD). Alongside the expanded polyQ, these flanking domains influence the aggregation propensity of HTTEx1: with the N17 initiating and promoting aggregation, and the PRD modulating it. In this study we focus on the first 11 amino acids of the PRD, a stretch of pure prolines, which are an evolutionary recent addition to the expanding polyQ region. We hypothesize that this proline region is expanding alongside the polyQ to counteract its ability to misfold and cause toxicity, and that expanding this proline region would be overall beneficial. We generated HTTEx1 mutants lacking both flanking domains singularly, missing the first 11 prolines of the PRD, or with this stretch of prolines expanded. We then followed their aggregation landscape in vitro with a battery of biochemical assays, and in vivo in novel models of C. elegans expressing the HTTEx1 mutants pan-neuronally. Employing fluorescence lifetime imaging we could observe the aggregation propensity of all HTTEx1 mutants during aging and correlate this with toxicity via various phenotypic assays. We found that the presence of an expanded proline stretch is beneficial in maintaining HTTEx1 soluble over time, regardless of polyQ length. However, the expanded prolines were only advantageous in promoting the survival and fitness of an organism carrying a pathogenic stretch of Q48 but were extremely deleterious to the nematode expressing a physiological stretch of Q23. Our results reveal the unique importance of the prolines which have and still are evolving alongside expanding glutamines to promote the function of HTTEx1 and avoid pathology.

Length-Dependent Structural Transformations of Huntingtin PolyQ Domain Upon Binding to 2D-Nanomaterials.

There is a strong negative correlation between the polyglutamine (polyQ) domain length (Q-length) in the intrinsically disordered Huntingtin protein (Htt) exon-1 and the age of onset of Huntington's disease (HD). PolyQ of Q-length longer than 40 has the propensity of forming very compact aggregate structures, leading to HD at full penetrance. Recent advances in nanobiotechnology provided a new platform for the development of novel diagnosis and therapeutics. Here, we explore the possibility of utilizing 2D-nanomaterials to inhibit the formation of supercompact polyQ structures through the so-called “folding-upon-binding” where the protein structure is dependent on the binding substrate. Using molecular dynamics simulations, we characterize two polyQ peptides with Q-length of 22 (Q22, normal length) and 46 (Q46, typical length causing HD) binding to both graphene and molybdenum disulfide (MoS2) nanosheets, which have been applied as antibacterial or anticancer agents. Upon binding, Q22 unfolds and elongates on both grapheme and MoS2 surfaces, regardless of its initial conformation, with graphene showing slightly stronger effect. In contrast, initially collapsed Q46 remains mostly collapsed within our simulation time on both nanosheets even though they do provide some “stretching” to Q46 as well. Further analyses indicate that the hydrophobic nature of graphene/MoS2 promotes the stretching of polyQ on nanosheets. However, there is strong competition with the intra-polyQ interactions (mainly internal hydrogen bonds) leading to the disparate folding/binding behaviors of Q22 and Q46. Our results present distinct Q-length specific behavior of the polyQ domain upon binding to two types of 2D-nanomaterials which holds clinical relevance for Huntington's disease.

Longitudinal Biochemical Assay Analysis of Mutant Huntingtin Exon 1 Protein in R6/2 Mice.

Biochemical analysis of mutant huntingtin (mHTT) aggregation species in HD mice is a common measure to track disease. A longitudinal and systematic study of how tissue processing affects detection of conformers has not yet been reported. Understanding the homeostatic flux of mHTT over time and under different processing conditions would aid in interpretation of pre-clinical assessments of disease interventions. Provide a systematic evaluation of tissue lysis methods and molecular and biochemical assays in parallel with behavioral readouts in R6/2 mice to establish a baseline for HTT exon1 protein accumulation. Established biochemical methods were used to process tissue from R6/2 mice of specific ages following behavior tasks. Aggregation states and accumulation of mHTT exon 1 protein were evaluated using multiple break and assay methods to determine potential conformational flux assay specificity in detection of mHTT species, and tissue specificity of conformers. Detection of mHTT exon 1 protein species varied based on biochemical processing and analysis providing a baseline for subsequent studies in R6/2 mice. Insoluble, high molecular weight species of mHTT exon 1 protein increased and tracked with onset of behavioral impairments in R6/2 mice using multiple assay methods. Conformational flux from soluble monomer to high molecular weight, insoluble species of mHTT exon 1 protein was generally consistent for multiple assay methods throughout R6/2 disease progression; however, the results support the use of multiple biochemical techniques to detect mHTT exon 1 protein species for preclinical assessments in HD mouse models expressing mHTT exon 1 protein.

Mutational analysis implicates the amyloid fibril as the toxic entity in Huntington's disease

In Huntington disease (HD), an expanded polyglutamine (polyQ > 37) sequence within huntingtin (htt) exon1 leads to enhanced disease risk. It has proved difficult, however, to determine whether the toxic form generated by polyQ expansion is a misfolded or avid-binding monomer, an α-helix-rich oligomer, or a β-sheet-rich amyloid fibril. Here we describe an engineered htt exon1 analog featuring a short polyQ sequence that nonetheless quickly forms amyloid fibrils and causes HD-like toxicity in rat neurons and Drosophila. Additional modifications within the polyQ segment produce htt exon1 analogs that populate only spherical oligomers and are non-toxic in cells and flies. Furthermore, in mixture with expanded-polyQ htt exon1, the latter analogs in vitro suppress amyloid formation and promote oligomer formation, and in vivo rescue neurons and flies expressing mhtt exon1 from dysfunction and death. Thus, in our experiments, while htt exon1 toxicity tracks with aggregation propensity, it does so in spite of the toxic construct's possessing polyQ tracts well below those normally considered to be disease-associated. That is, aggregation propensity proves to be a more accurate surrogate for toxicity than is polyQ repeat length itself, strongly supporting a major toxic role for htt exon1 aggregation in HD. In addition, the results suggest that the aggregates that are most toxic in these model systems are amyloid-related. These engineered analogs are novel tools for mapping properties of polyQ self-assembly intermediates and products that should similarly be useful in the analysis of other expanded polyQ diseases. Small molecules with similar amyloid inhibitory properties might be developed into effective therapeutic agents.

The 17-residue-long N terminus in huntingtin controls stepwise aggregation in solution and on membranes via different mechanisms

Aggregation of huntingtin protein arising from expanded polyglutamine (polyQ) sequences in the exon-1 region of mutant huntingtin plays a central role in the pathogenesis of Huntington's disease. The huntingtin aggregation pathways are of therapeutic and diagnostic interest, but obtaining critical information from the physiologically relevant htt exon-1 (Httex1) protein has been challenging. Using biophysical techniques and an expression and purification protocol that generates clean, monomeric Httex1, we identified and mapped three distinct aggregation pathways: 1) unseeded in solution; 2) seeded in solution; and 3) membrane-mediated. In solution, aggregation proceeded in a highly stepwise manner, in which the individual domains (N terminus containing 17 amino acids (N17), polyQ, and proline-rich domain (PRD)) become ordered at very different rates. The aggregation was initiated by an early oligomer requiring a pathogenic, expanded Gln length and N17 α-helix formation. In the second phase, β-sheet forms in the polyQ. The slowest step is the final structural maturation of the PRD. This stepwise mechanism could be bypassed by seeding, which potently accelerated aggregation and was a prerequisite for prion-like spreading in vivo Remarkably, membranes could catalyze aggregation even more potently than seeds, in a process that caused significant membrane damage. The N17 governed membrane-mediated aggregation by anchoring Httex1 to the membrane, enhancing local concentration and promoting collision via two-dimensional diffusion. Considering its central roles in solution and in membrane-mediated aggregation, the N17 represents an attractive target for inhibiting multiple pathways. Our approach should help evaluate such inhibitors and identify diagnostic markers for the misfolded forms identified here.

Differential effects of soluble and aggregating polyQ proteins on cytotoxicity and type-1 myosin-dependent endocytosis in yeast

Huntington’s disease develops when the polyglutamine (polyQ) repeat in the Huntingtin (Htt) protein is expanded to over 35 glutamines rendering it aggregation-prone. Here, using Htt exon-1 as a polyQ model protein in a genome-wide screen in yeast, we show that the normal and soluble Htt exon-1 is toxic in cells with defects in type-1 myosin-dependent endocytosis. The toxicity of Htt is linked to physical interactions with type-1 myosins, which occur via the Htt proline-rich region, leading to a reduction in actin patch polarization and clathrin-dependent endocytosis. An expansion of the polyQ stretch from 25 to 103 glutamines, which causes Htt aggregation, alleviated Htt toxicity in cells lacking Myo5 or other components involved in early endocytosis. The data suggest that the proline-rich stretch of Htt interacts with type-1 myosin/clathrin-dependent processes and demonstrate that a reduction in the activity of such processes may result in a positive selection for polyQ expansions.

Fibril polymorphism affects immobilized non-amyloid flanking domains of huntingtin exon1 rather than its polyglutamine core

Polyglutamine expansion in the huntingtin protein is the primary genetic cause of Huntington’s disease (HD). Fragments coinciding with mutant huntingtin exon1 aggregate in vivo and induce HD-like pathology in mouse models. The resulting aggregates can have different structures that affect their biochemical behaviour and cytotoxic activity. Here we report our studies of the structure and functional characteristics of multiple mutant htt exon1 fibrils by complementary techniques, including infrared and solid-state NMR spectroscopies. Magic-angle-spinning NMR reveals that fibrillar exon1 has a partly mobile α-helix in its aggregation-accelerating N terminus, and semi-rigid polyproline II helices in the proline-rich flanking domain (PRD). The polyglutamine-proximal portions of these domains are immobilized and clustered, limiting access to aggregation-modulating antibodies. The polymorphic fibrils differ in their flanking domains rather than the polyglutamine amyloid structure. They are effective at seeding polyglutamine aggregation and exhibit cytotoxic effects when applied to neuronal cells.

Distinct cellular toxicity of two mutant huntingtin mRNA variants due to translation regulation.

Huntington’s disease (HD) is a neurodegenerative disorder caused by CAG repeat expansion within exon1 of the HTT gene. The gene generates two mRNA variants that carry either a short or long 3′ untranslated region (3′UTR) while encoding the same protein. It remains unknown whether the two mRNA variants play distinct roles in HD pathogenesis. We found that the long HTT 3′UTR was capable of guiding mRNA to neuronal dendrites, suggesting that some long-form HTT mRNA is transported to dendrites for local protein synthesis. To assay roles of two HTT mRNA variants in cell bodies, we expressed mRNA harboring HTT exon1 containing 23x or 145x CAGs with the short or long 3′UTR. We found that mutant mRNA containing the short 3′UTR produced more protein aggregates and caused more apoptosis in both cultured neurons and HEK293 cells, compared with mutant mRNA containing the long 3′UTR. Although the two 3′UTRs did not affect mRNA stability, we detected higher levels of protein synthesis from mRNA containing the short 3′UTR than from mRNA containing the long 3′UTR. These results indicate that the long HTT 3′UTR suppresses translation. Thus, short-form mutant HTT mRNA will be more efficient in producing toxic protein than its long-form counterpart.

The phasor-FLIM fingerprints reveal shifts from OXPHOS to enhanced glycolysis in Huntington Disease.

Huntington disease (HD) is an autosomal neurodegenerative disorder caused by the expansion of Polyglutamine (polyQ) in exon 1 of the Huntingtin protein. Glutamine repeats below 36 are considered normal while repeats above 40 lead to HD. Impairment in energy metabolism is a common trend in Huntington pathogenesis; however, this effect is not fully understood. Here, we used the phasor approach and Fluorescence Lifetime Imaging Microscopy (FLIM) to measure changes between free and bound fractions of NADH as a indirect measure of metabolic alteration in living cells. Using Phasor-FLIM, pixel maps of metabolic alteration in HEK293 cell lines and in transgenic Drosophila expressing expanded and unexpanded polyQ HTT exon1 in the eye disc were developed. We found a significant shift towards increased free NADH, indicating an increased glycolytic state for cells and tissues expressing the expanded polyQ compared to unexpanded control. In the nucleus, a further lifetime shift occurs towards higher free NADH suggesting a possible synergism between metabolic dysfunction and transcriptional regulation. Our results indicate that metabolic dysfunction in HD shifts to increased glycolysis leading to oxidative stress and cell death. This powerful label free method can be used to screen native HD tissue samples and for potential drug screening.

Myelinosomes act as natural secretory organelles in Sertoli cells to prevent accumulation of aggregate-prone mutant Huntingtin and CFTR

Inappropriate deposition of insoluble aggregates of proteins with abnormal structures is a hallmark of affected organs in protein aggregation disease. Very rare, affected organs avoid aggregation naturally. This concerns atrophic testis in Huntington disease (HD). We aimed to understand how HD testis avoids aggregation. Using HD model R6/1 mice, we demonstrate that affected testis contain rare organelles myelinosomes. Myelinosomes secreted from testis somatic TM4 Sertoli cells provide the release of aggregate-prone mutant, but not normal Huntingtin (Htt) exon1. Myelinosomes also support the release of other aggregate-prone mutant protein responsible for cystic fibrosis (CF), F508delCFTR. The traffic and discharge of myelinosomes is facilitated by multivesicular bodies (MVB)s. Inhibition of MVB excretion induced reversible retention of both misfolded proteins inside TM4 Sertoli cells. We propose that myelinosome-mediated elimination of mutant proteins is an unusual secretory process allowing Sertoli cells getting rid of misfolded proteins to avoid aggregation and to maintain cell proteostasis.

Factors Modulating the Interaction of Huntingtin with Lipid Membranes: Implications for Huntington's Disease

Huntington disease (HD) is a genetic neurodegenerative disease caused by an expanded polyglutamine (polyQ) domain in the first exon of the huntingtin (Htt) protein. This polyQ expansions leads directly to the formation of proteinaceous aggregates including oligomers and fibrils. Htt interacts with a variety of membraneous structures within the cell, and the first seventeen amino acids (Nt17) of Htt, which directly flanks the polyQ domain, is an amphiphathic α-helix (AH) lipid-binding domain. Here, the role that lipid/htt interactions play in aggregation was investigated with a specific emphasis on the role of Nt17 acetylation and membrane cholesterol content. N-terminal acetylation of htt has been implicated in the etiology of HD, and cholesterol homeostasis is altered in HD. A variety of aggregation assays, lipid binding assays, mass spectrometry, and atomic force microscopy were used. The model membrane systems were vesicles and supported bilayers comprised of total brain lipid extract (TBLE), and these were exposed to a variety of purified or synthetic peptides modeled after htt exon1. Acetylation of htt was achieved using a selective covalent label sulfo-N-hydroxysuccinimide (NHSA). Acetylation retarded fibril formation, promoted the formation of larger globular aggregates, and altered htt's ability to bind lipid membranes. Exogenous cholesterol was incorporated into our model TBLE membranes. As cholesterol content increased, morphological and mechanical changes in the bilayer associated with exposure to Htt were drastically altered. Collectively, these observations suggest that the acetylation and cholesterol content of bilayers play a critical role in htt binding to and aggregation on lipid membranes.

Studies on Domain Specific Aggregation Behavior of Huntingtin Exon1

The pathogenesis of Huntington's disease results from polyQ expansions (>36Q) in the Huntingtin (Htt) protein.1 These polyQ expansions make Htt less soluble and promote the formation of misfolded oligomers, fibrils and aggregates in Huntington's disease.2 Efforts to prevent aggregation will greatly benefit from a detailed molecular understanding of the misfolding process. However, htt misfolding has been difficult to study as the protein is tough to handle and examine spectroscopically. Here we developed a novel purification scheme to generate recombinant, clean and monomeric Htt exon1, an aggregation prone fragment of htt that is highly expressed in disease. The misfolding of spin labeled derivatives of Htt exon1 were then monitored by EPR in order to obtain site-specific folding information for residues in different regions of the protein. Our data indicate that residues in the N-terminal N17, the middle polyQ region and the C-terminal, proline rich region all undergo structural changes on different time scales. Moreover, we have also found that the aggregation kinetics and mechanisms of aggregation significantly depend on Q length. Overall, the data indicate distinct phases in which different regions of the molecule undergo conformational changes. Each of these stages may afford the design of different compounds intended at altering the aggregation process.1. Bates, GP.; Harper, PS.; Jones, L., editors. Huntington's Disease. Oxford University Press; Oxford, U.K.: 2002.2. Bates, GP.; Benn, C. The polyglutamine diseases. In: Bates, GP.; Harper, PS.; Jones, L., editors. Huntington's Disease. Oxford University Press; Oxford, U.K.: 2002. p. 429-472.

The Role of Membrane Context in the Interaction of Polyglutamine Peptides with Lipid Membranes

Huntington's disease is a dominant genetic neurodegenerative disorder associated with motor and cognitive decline, caused by a mutation in the poly-glutamine (polyQ) region near the N-terminus of the huntingtin (htt) protein. Expansion of the polyQ region above 35-40 repeats results in the disease that is characterized by inclusion body aggregates of mutated protein. The polyQ expansion in htt is flanked by a 17 amino acid N-terminal sequence (Nt17) and a proline-rich (polyP) region. To investigate the interaction between htt exon1 and lipid membranes, a combination of Langmuir trough techniques and vesicle permeability assays measuring calcein leakage were used to directly monitor the interaction of a variety of synthetic polyQ peptides with total brain lipid extract (TBLE) model membranes doped with specific membrane components. Our data suggests that the Nt17 domain plays a critical role in htt binding and aggregation on lipid membranes, and this lipid/htt interaction is enhanced by the presence of the polyP domain. The exogenous addition of cholesterol, a primary neuronal plasma membrane component reduced in many Huntington's disease models, to the TBLE monolayer caused a notable condensing effect in the membrane at low surface pressures. This resulted in reduced peptide insertion into lipid monolayers and decreased levels of induced vesicle permeability, though the effect does not scale linearly with cholesterol concentration. Results from parallel studies on htt-membrane interaction with sphingomyelin, a major component of membranes in the nervous system, will also be discussed.

The Role of Protein and Membrane Context in the Interaction of Polyglutamine Peptides with Lipid Membranes

Huntington's disease is a dominant genetic neurodegenerative disorder associated with motor and cognitive decline, caused by a mutation in the poly-glutamine (polyQ) region near the N-terminus of the huntingtin (htt) protein. Expansion of the polyQ region above 35-40 repeats results in the disease that is characterized by inclusion body aggregates of mutated protein. The polyQ expansion in htt flanked by a 17 amino acid N-terminal sequence (Nt17) and a proline-rich (polyP) region. To investigate the interaction between htt exon1 and lipid membranes, a combination of Langmuir trough techniques and vesicle permeability assays measuring calcein leakage were used to directly monitor the interaction of a variety of synthetic polyQ peptides with different combinations of flanking sequences (KK-Q35-KK, KK-Q35-P10-KK, Nt17-Q35-KK, and Nt17-Q35-P10-KK) on total brain lipid extract (TBLE) model membranes. PolyQ peptides that lacked the Nt17 domain did not appreciably aggregate on or insert into lipid membranes. Nt17 facilitated the interaction of peptides with lipid surfaces while the polyP region enhanced this interaction. Our data suggests that the Nt17 domain plays a critical role in htt binding and aggregation on lipid membranes, and this lipid/htt interaction can be further modulated by the presence of the polyP domain. The addition of cholesterol to TBLE model membranes, to determine its role in htt-membrane interactions, resulted in reduced peptide insertion into lipid monolayers and decreased levels of induced vesicle permeability, though the effect does not scale linearly with cholesterol concentration. Results from parallel studies performed with ganglioside GM1 and sphingomyelin to determine their role will also be presented.