Htt Aggregation Research Articles

Dysfunction of the CNS-heart axis in mouse models of Huntington's disease.

Cardiac remodelling and contractile dysfunction occur during both acute and chronic disease processes including the accumulation of insoluble aggregates of misfolded amyloid proteins that are typical features of Alzheimer's, Parkinson's and Huntington's disease (HD). While HD has been described mainly as a neurological disease, multiple epidemiological studies have shown that HD patients exhibit a high incidence of cardiovascular events leading to heart failure, and that this is the second highest cause of death. Given that huntingtin is ubiquitously expressed, cardiomyocytes may be at risk of an HD-related dysfunction. In mice, the forced expression of an expanded polyQ repeat under the control of a cardiac specific promoter led to severe heart failure followed by reduced lifespan. However the mechanism leading to cardiac dysfunction in the clinical and pre-clinical HD settings remains unknown. To unravel this mechanism, we employed the R6/2 transgenic and HdhQ150 knock-in mouse models of HD. We found that pre-symptomatic animals developed connexin-43 relocation and a significant deregulation of hypertrophic markers and Bdnf transcripts. In the symptomatic animals, pronounced functional changes were visualised by cardiac MRI revealing a contractile dysfunction, which might be a part of dilatated cardiomyopathy (DCM). This was accompanied by the re-expression of foetal genes, apoptotic cardiomyocyte loss and a moderate degree of interstitial fibrosis. To our surprise, we could identify neither mutant HTT aggregates in cardiac tissue nor a HD-specific transcriptional dysregulation, even at the end stage of disease. We postulate that the HD-related cardiomyopathy is caused by altered central autonomic pathways although the pathogenic effects of mutant HTT acting intrinsically in the heart may also be a contributing factor.

Ubiquitin-specific protease-14 reduces cellular aggregates and protects against mutant huntingtin-induced cell degeneration: involvement of the proteasome and ER stress-activated kinase IRE1α.

Huntington's disease (HD) is an autosomal inherited neurological disease caused by a CAG-repeat expansion in the first exon of huntingtin gene encoding for the huntingtin protein (Htt). In HD, there is an accumulation of intracellular aggregates of mutant Htt that negatively influence cellular functions. The aggregates contain ubiquitin, and part of the HD pathophysiology could result from an imbalance in cellular ubiquitin levels. Deubiquitinating enzymes are important for replenishing the ubiquitin pool, but less is known about their roles in brain diseases. We show here that overexpression of the ubiquitin-specific protease-14 (Usp14) reduces cellular aggregates in mutant Htt-expressing cells mainly via the ubiquitin proteasome system. We also observed that the serine-threonine kinase IRE1 involved in endoplasmic reticulum (ER) stress responses is activated in mutant Htt-expressing cells in culture as well as in the striatum of mutant Htt transgenic (BACHD) mice. Usp14 interacted with IRE1 in control cells but less in mutant Htt-expressing cells. Overexpression of Usp14 in turn was able to inhibit phosphorylation of IRE1α in mutant Htt-overexpressing cells and to protect against cell degeneration and caspase-3 activation. These results show that ER stress-mediated IRE1 activation is part of mutant Htt toxicity and that this is counteracted by Usp14 expression. Usp14 effectively reduced cellular aggregates and counteracted cell degeneration indicating an important role of this protein in mutant Htt-induced cell toxicity.

Mglur5: a Potential Target for the Treatment of Huntington's Disease

ISSN 1479-6708 Future Neurology (2014) 9(3), 289–293 Huntington’s disease Huntington’s disease (HD) is a progressive neurodegenerative disorder with an age of onset between 35 and 50 years. At first, HD patients experience chorea and psychiatric alterations, associated with intellectual decline and inevitable death within 15–20 years [1,2]. HD is caused by a polyglutamine expansion in the amino terminus of the htt protein [3]. The length of the polyglutamine repeat inversely correlates with the age of disease onset and directly correlates with the severity of symptoms [4]. Pathological analyses of HD-affected brains has revealed neuronal loss of the GABAergic medium-sized spiny neurons (MSN) present in the striatum [5]. Although it is well established that the htt polyglutamine expansion is the cause of HD, it is unknown why this ubiquitously expressed mutant protein selectively results in the loss of MSNs. Moreover, it remains unclear whether HD occurs as the consequence of either the loss of normal htt protein or as a toxic gain of function due to the polyglutamine expanded form of the protein. Wild-type htt protein is both antiapoptotic [6] and essential for normal embryonic development [7,8]. Nevertheless, the resulting polyglutamine stretch confers neurotoxic functions of the htt protein, resulting in the activation of cell signaling pathways involved in neuronal dysfunction as a consequence of increased oxidative stress, gene transcriptional alterations, glutamate excitotoxicity, apoptosis, mitochondrial dysregulation and energy depletion [9–11]. Cleavage of the mutant htt amino-terminal region results in the generation of insoluble nuclear aggregates, which are suggested to play a key role in HD-related neuronal cell death. Notably, HD symptom severity is strongly correlated with htt aggregate formation and loss of striatal neurons [12]. Htt aggregates and inclusions are found first in the striatum and, over time, in the rest of the basal ganglia, followed by progressive localization throughout the substantia nigra and cortex. It is not known whether htt aggregates and inclusions are toxic as other evidences suggest that htt nuclear inclusions may be neuroprotective for MSNs [13,14].

Differential ubiquitination and degradation of huntingtin fragments modulated by ubiquitin-protein ligase E3A

Ubiquitination of misfolded proteins, a common feature of many neurodegenerative diseases, is mediated by different lysine (K) residues in ubiquitin and alters the levels of toxic proteins. In Huntington disease, polyglutamine expansion causes N-terminal huntingtin (Htt) to misfold, inducing neurodegeneration. Here we report that shorter N-terminal Htt fragments are more stable than longer fragments and find differential ubiquitination via K63 of ubiquitin. Aging decreases proteasome-mediated Htt degradation, at the same time increasing K63-mediated ubiquitination and subsequent Htt aggregation in HD knock-in mice. The association of Htt with the K48-specific E3 ligase, Ube3a, is decreased in aged mouse brain. Overexpression of Ube3a in HD mouse brain reduces K63-mediated ubiquitination and Htt aggregation, enhancing its degradation via the K48 ubiquitin-proteasome system. Our findings suggest that aging-dependent Ube3a levels result in differential ubiquitination and degradation of Htt fragments, thereby contributing to the age-related neurotoxicity of mutant Htt.

Silencing Mutant Huntingtin by Adeno-Associated Virus-Mediated RNA Interference Ameliorates Disease Manifestations in the YAC128 Mouse Model of Huntington's Disease

Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disease caused by an increase in the number of polyglutamine residues in the huntingtin (Htt) protein. With the identification of the underlying basis of HD, therapies are being developed that reduce expression of the causative mutant Htt. RNA interference (RNAi) that seeks to selectively reduce the expression of such disease-causing agents is emerging as a potential therapeutic strategy for this and similar disorders. This study examines the merits of administering a recombinant adeno-associated viral (AAV) vector designed to deliver small interfering RNA (siRNA) that targets the degradation of the Htt transcript. The aim was to lower Htt levels and to correct the behavioral, biochemical, and neuropathological deficits shown to be associated with the YAC128 mouse model of HD. Our data demonstrate that AAV-mediated RNAi is effective at transducing greater than 80% of the cells in the striatum and partially reducing the levels (~40%) of both wild-type and mutant Htt in this region. Concomitant with these reductions are significant improvements in behavioral deficits, reduction of striatal Htt aggregates, and partial correction of the aberrant striatal transcriptional profile observed in YAC128 mice. Importantly, a partial reduction of both the mutant and wild-type Htt levels is not associated with any notable overt neurotoxicity. Collectively, these results support the continued development of AAV-mediated RNAi as a therapeutic strategy for HD.

Ubiquilin-1 Overexpression Increases the Lifespan and Delays Accumulation of Huntingtin Aggregates in the R6/2 Mouse Model of Huntington's Disease

Huntington's Disease (HD) is a neurodegenerative disorder that is caused by abnormal expansion of a polyglutamine tract in huntingtin (htt) protein. The expansion leads to increased htt aggregation and toxicity. Factors that aid in the clearance of mutant huntingtin proteins should relieve the toxicity. We previously demonstrated that overexpression of ubiqulin-1, which facilitates protein clearance through the proteasome and autophagy pathways, reduces huntingtin aggregates and toxicity in mammalian cell and invertebrate models of HD. Here we tested whether overexpression of ubiquilin-1 delays or prevents neurodegeneration in R6/2 mice, a well-established model of HD. We generated transgenic mice overexpressing human ubiquilin-1 driven by the neuron-specific Thy1.2 promoter. Immunoblotting and immunohistochemistry revealed robust and widespread overexpression of ubiquilin-1 in the brains of the transgenic mice. Similar analysis of R6/2 animals revealed that ubiquilin is localized in huntingtin aggregates and that ubiquilin levels decrease progressively to 30% during the end-stage of disease. We crossed our ubiquilin-1 transgenic line with R6/2 mice to assess whether restoration of ubiquilin levels would delay HD symptoms and pathology. In the double transgenic progeny, ubiquilin levels were fully restored, and this correlated with a 20% increase in lifespan and a reduction in htt inclusions in the hippocampus and cortex. Furthermore, immunoblots indicated that endoplasmic reticulum stress response that is elevated in the hippocampus of R6/2 animals was attenuated by ubiquilin-1 overexpression. However, ubiquilin-1 overexpression neither altered the load of htt aggregates in the striatum nor improved motor impairments in the mice.

Polyglutamine Flanking Regions in Huntingtin Highlight Key Structural Intermediates Relevant for Molecular Chaperone Interaction and Huntington's Disease Pathogenesis

Huntington's Disease (HD) is a neurodegenerative disorder caused by an expansion in the polyglutamine (polyQ) tract of the Huntingtin (Htt) protein. The clinical hallmark of this mutation is the accumulation of amyloid Htt aggregates in neurons. While Htt aggregation is highly correlated with the length of this polyQ tract both in vitro and in vivo, recent studies suggest that the regions flanking the polyQ tract can influence Htt aggregation and toxicity independent of polyQ length. In addition, it is well established that two polyQ flanking regions in exon1 of Htt, the N-terminal first 17 amino acids (N17) and a proline-rich region (PolyPro), influence Htt aggregation propensity. Here, we show that the N17 and PolyPro regions of Htt dramatically influence the rate of the Htt aggregation pathway. We also show that mutations within these polyQ-flanking regions alter Htt toxicity in a brain slice model. The influence of these flanking regions on the heterogeneous distribution of the aggregate species may account for these differences in toxicity. This theory is increasingly relevant in light of recent thought that Htt toxicity may derive from a species other than the amyloid fibril itself. Finally, previous work has shown that the N17 region of Htt interacts with the molecular chaperonin TRiC, and this interaction exerts a protective effect against both Htt aggregation and toxicity. An understanding how these flanking regions influence Htt aggregation will inform how TRiC works through the N17 element. Together, this information can provide the basis to design HD therapeutics that exploit these flanking regions to suppress HD pathogenesis.

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.

Role of Intrinsic Helicity Within N-Terminal Flanking Sequences on Huntingtin Aggregation Mechanisms

Huntington's disease (HD) is associated with CAG repeats within exon 1 that lead to polyglutamine (polyQ) expansions in the protein huntingtin (htt). Aberrant splicing of htt transcripts generates N-terminal fragments in a polyQ length dependent manner that span exon 1 of htt. These N-terminal fragments can be trafficked into the nucleus and form neuronal nuclear inclusions. Within exon 1 of htt the polyQ region is flanked by a N-terminal 17-residue amphipathic stretch (N17) and a C-terminal proline-rich stretch. Modulation of these flanking sequences has profound effects on HD progression. Specifically, post-translational modifications and single / double point mutations within N17 can modulate the intrinsic helicity of N17, as well as aggregation in vitro and HD phenotypes in animal models. However, the role of helicity within N17 on the modulation of polyQ aggregation remains unresolved. Here, we focus on understanding the synergy between the intrinsic helicity within N17 and polyQ length on htt aggregation mechanisms. Through the use of atomistic Monte Carlo simulations utilizing the ABSINTH implicit solvation model, we examine the monomeric properties of N17 permutants with varying degrees of intrinsic helicity as a function of polyQ length. Results suggest that the degree of coupling between N17 constructs and polyQ is dependent on the intrinsic helicity within N17 constructs. A comparative analysis of N17 constructs in cis with a range of polyQ lengths will be combined with biophysical experiments to understand how monomeric properties of N17 modulate the solubility and mechanism of polyQ aggregation.

A mitochondrial basis for Huntington’s disease: therapeutic prospects

Huntington's disease (HD) is an autosomal dominant disease, with overt movement dysfunctions. Despite focused research on the basis of neurodegeneration in HD for last few decades, the mechanism for the site-specific lesion of neurons in the brain is not clear. All the explanations that partially clarify the phenomenon of neurodegeneration leads to one organelle, mitochondrion, which is severely affected in HD at the level of electron transport chain, Ca(2+) buffering efficiency and morphology. But, with the existing knowledge, it is not clear whether the cell death processes in HD initiate from mitochondria, though the Huntingtin (Htt) aggregates show close proximity to this organelle, or do some extracellular stimuli like TNFα or FasL trigger the process. Mainly because of the disparity in the different available experimental models, the results are quite confusing or at least inconsistent to a great extent. The fact remains that the mutant Htt protein was seen to be associated with mitochondria directly, and as the striatum is highly enriched with dopamine and glutamate, it may make the striatal mitochondria more vulnerable because of the presence of dopa-quinones, and due to an imbalance in Ca(2+). The current therapeutic strategies are based on symptomatic relief, and, therefore, mainly target neurotransmitter(s) and their receptors to modulate behavioral outputs, but none of them targets mitochondria or try to address the basic molecular events that cause neurons to die in discrete regions of the brain, which could probably be resulting from grave mitochondrial dysfunctions. Therefore, targeting mitochondria for their protection, while addressing symptomatic recovery, holds a great potential to tone down the progression of the disease, and to provide better relief to the patients and caretakers.

Grb2 Is Regulated by Foxd3 and Has Roles in Preventing Accumulation and Aggregation of Mutant Huntingtin

Growth factor receptor protein binding protein 2 (Grb2) is known to be associated with intracellular growth and proliferation related signaling cascades. Huntingtin (Htt), a ubiquitously expressed protein, when mutated, forms toxic intracellular aggregates - the hallmark of Huntington’s disease (HD). We observed an elevated expression of Grb2 in neuronal cells in animal and cell models of HD. Grb2 overexpression was predominantly regulated by the transcription factor Forkhead Box D3 (Foxd3). Exogenous expression of Grb2 also reduced aggregation of mutant Htt in Neuro2A cells. Grb2 is also known to interact with Htt, depending on epidermal growth factor receptor (EGFR) activation. Grb2- mutant Htt interaction in the contrary, took place in vesicular structures, independent of EGFR activation that eventually merged with autophagosomes and activated the autophagy machinery helping in autophagosome and lysosome fusion. Grb2, with its emerging dual role, holds promise for a survival mechanism for HD.

Expression of Huntingtons disease markers in the cornea of minipig transgenic for the human mutated huntingtin

AbstractPurpose Huntingtons disease (HD) is a neurodegenerative disorder that is caused by an expansion of a polyglutamine (polyQ) domain in the protein of huntingtin (htt). PolyQ expansion above 35–40 results in disease associated with htt aggregation into inclusion bodies. The recent studies have shown that the illness arises not only in neurons, but also in non‐neuronal cells. Purpose of this study was to investigate the expression of selected HD markers (on htt and their aggregates, ferritin and ubiquitin) in corneas of minipig transgenic for the human mutated huntingtin and compare them with corneas of wild type (WT) minipig.Methods In this study we used WT minipigs and minipigs transgenic for N‐terminal part of the human mutated huntingtin (both F2 generation, age 24 months). HD markers were examined on cryostat sections immunohistochemically by using the following primary antibodies: anti‐polyglutamines, anti‐polyglutamine‐expansion diseases marker, MW8 marker, anti‐ferritin and anti‐ubiguitin.Results Immunohistochemical examination on huntingtin demonstrated the increased expression of this protein in corneal epithelium and keratocytes of transgenic minipig in comparison with wild type cornea. The expression of ferritin detected in epithelial cells was very low in both studied corneas without significant difference between them. No nuclear inclusions or ubiquitous proteins were found in the cornea of transgenic minipig.Conclusion This study showed that preclinical (silent) stages of HD are characterized by an elevated level of mutagenic huntingtin without formation of nuclear inclusions and ubiquitination of proteins. Supported by CHDI Foundation (A‐5378 and A‐5379), TA01011466, CZ.1.05./2.1.00/03.0124 and RVO: 67985904.

MiR-196a Ameliorates Phenotypes of Huntington Disease in Cell, Transgenic Mouse, and Induced Pluripotent Stem Cell Models

Huntington disease (HD) is a dominantly inherited neurodegenerative disorder characterized by dysregulation of various genes. Recently, microRNAs (miRNAs) have been reported to be involved in this dysregulation, suggesting that manipulation of appropriate miRNA regulation may have a therapeutic benefit. Here, we report the beneficial effects of miR-196a (miR196a) on HD in cell, transgenic mouse models, and human induced pluripotent stem cells derived from one individual with HD (HD-iPSCs). In the in vitro results, a reduction of mutant HTT and pathological aggregates, accompanying the overexpression of miR-196a, was observed in HD models of human embryonic kidney cells and mouse neuroblastoma cells. In the in vivo model, HD transgenic mice overexpressing miR-196a revealed the suppression of mutant HTT in the brain and also showed improvements in neuropathological progression, such as decreases of nuclear, intranuclear, and neuropil aggregates and late-stage behavioral phenotypes. Most importantly, miR-196a also decreased HTT expression and pathological aggregates when HD-iPSCs were differentiated into the neuronal stage. Mechanisms of miR-196a in HD might be through the alteration of ubiquitin-proteasome systems, gliosis, cAMP response element-binding protein pathway, and several neuronal regulatory pathways in vivo. Taken together, these results show that manipulating miR-196a provides beneficial effects in HD, suggesting the potential therapeutical role of miR-196a in HD.

Huntingtin disrupts lipid bilayers in a polyQ-length dependent manner

Huntington's Disease (HD) is a neurodegenerative disorder that is defined by the accumulation of nanoscale aggregates comprised of the huntingtin (htt) protein. Aggregation is directly caused by an expanded polyglutamine (polyQ) domain in htt, leading to a diverse population of aggregate species, such as oligomers, fibrils, and annular aggregates. Furthermore, the length of this polyQ domain is directly related to onset and severity of disease. The first 17 N-terminal amino acids of htt have been shown to further modulate aggregation. Additionally, these 17 amino acids appear to have lipid binding properties as htt interacts with a variety of membrane-containing structures present in cells, such as organelles, and interactions with these membrane surfaces may further modulate htt aggregation. To investigate the interaction between htt exon1 and lipid bilayers, in situ atomic force microscopy (AFM) was used to directly monitor the aggregation of htt exon1 constructs with varying Q-lengths (35Q, 46Q, 51Q, and myc-53Q) on supported lipid membranes comprised of total brain lipid extract. The exon1 fragments accumulated on the lipid membranes, causing disruption of the membrane, in a polyQ dependent manner. Furthermore, the addition of an N-terminal myc-tag to the htt exon1 fragments impeded the interaction of htt with the bilayer.

The Interaction of Polyglutamine Peptides with Lipid Membranes Is Regulated by Flanking Sequences Associated with Huntingtin

Huntington disease (HD) is caused by an expanded polyglutamine (poly(Q)) repeat near the N terminus of the huntingtin (htt) protein. Expanded poly(Q) facilitates formation of htt aggregates, eventually leading to deposition of cytoplasmic and intranuclear inclusion bodies containing htt. Flanking sequences directly adjacent to the poly(Q) domain, such as the first 17 amino acids on the N terminus (Nt17) and the polyproline (poly(P)) domain on the C-terminal side of the poly(Q) domain, heavily influence aggregation. Additionally, htt interacts with a variety of membraneous structures within the cell, and Nt17 is implicated in lipid binding. To investigate the interaction between htt exon1 and lipid membranes, a combination of in situ atomic force microscopy, Langmuir trough techniques, and vesicle permeability assays were used to directly monitor the interaction of a variety of synthetic poly(Q) peptides with different combinations of flanking sequences (KK-Q35-KK, KK-Q35-P10-KK, Nt17-Q35-KK, and Nt17-Q35-P10-KK) on model membranes and surfaces. Each peptide aggregated on mica, predominately forming extended, fibrillar aggregates. In contrast, poly(Q) 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, whereas the poly(P) region enhanced this interaction. The aggregation of Nt17-Q35-P10-KK on the lipid bilayer closely resembled that of a htt exon1 construct containing 35 repeat glutamines. Collectively, this 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 poly(P) domain.

The cell biology of prion-like spread of protein aggregates: mechanisms and implication in neurodegeneration

The misfolding and aggregation of specific proteins is a common hallmark of many neurodegenerative disorders, including highly prevalent illnesses such as Alzheimer's and Parkinson's diseases, as well as rarer disorders such as Huntington's and prion diseases. Among these, only prion diseases are 'infectious'. By seeding misfolding of the PrP(C) (normal conformer prion protein) into PrP(Sc) (abnormal disease-specific conformation of prion protein), prions spread from the periphery of the body to the central nervous system and can also be transmitted between individuals of the same or different species. However, recent exciting data suggest that the transmissibility of misfolded proteins within the brain is a property that goes way beyond the rare prion diseases. Evidence indicates that non-prion aggregates [tau, α-syn (α-synuclein), Aβ (amyloid-β) and Htt (huntingtin) aggregates] can also move between cells and seed the misfolding of their normal conformers. These findings have enormous implications. On the one hand they question the therapeutical use of transplants, and on the other they indicate that it may be possible to bring these diseases to an early arrest by preventing cell-to-cell transmission. To better understand the prion-like spread of these protein aggregates it is essential to identify the underlying cellular and molecular factors. In the present review we analyse and discuss the evidence supporting prion-like spreading of amyloidogenic proteins, especially focusing on the cellular and molecular mechanisms and their significance.

An N-terminal Nuclear Export Signal Regulates Trafficking and Aggregation of Huntingtin (Htt) Protein Exon 1*

Huntington disease is a dominantly inherited neurodegenerative condition caused by polyglutamine expansion in the N terminus of the huntingtin protein (Htt). The first 17 amino acids (N17) of Htt play a key role in regulating its toxicity and aggregation. Both nuclear export and cytoplasm retention functions have been ascribed to N17. We have determined that N17 acts as a nuclear export sequence (NES) within Htt exon and when fused to yellow fluorescent protein. We have defined amino acids within N17 that constitute the nuclear export sequence (NES). Mutation of any of the conserved residues increases nuclear accumulation of Htt exon 1. Nuclear export of Htt is sensitive to leptomycin B and is reduced by knockdown of exportin 1. In HEK293 cells, NES mutations decrease overall Htt aggregation but increase the fraction of cells with nuclear inclusions. In primary cultured neurons, NES mutations increase nuclear accumulation and increase overall aggregation. This work defines a bona fide nuclear export sequence within N17 and links it to effects on protein aggregation. This may help explain the important role of N17 in controlling Htt toxicity.

Putting Huntingtin “Aggregation” in View with Windows into the Cellular Milieu

Huntington's disease arises from CAG codon-repeat expansions in the Htt gene, which leads to a Htt gene product with an expanded polyglutamine (polyQ) sequence. The length of the polyQ expansion correlates with an increased tendency to form aggregates and clustering into micrometer-plus sized inclusion bodies in neurons and other cell types. Yet after nearly 20 years since the genetic basis for HD was identified, our knowledge of how polyQ-expanded Htt fragment aggregation relates to disease mechanisms remains fragmentary and controversial. Challenges remain in defining the aggregation process at the molecular level and how this process is influenced by, or influences cellular activities. Insight is further confounded by the term "aggregation" being used to describe a composite of distinct processes that may have opposing consequences to cell health and survival. This review discusses these issues in light of a historic summary of Htt aggregation in the cellular milieu and the intrinsic attributes of polyQ-expanded Htt that lead to aggregation. Finally, discussion centers on strategies forward to improve our knowledge for how aggregation relates to cellular dysfunction.

Intravitreal Administration of HA-1077, a ROCK Inhibitor, Improves Retinal Function in a Mouse Model of Huntington Disease

Huntington disease (HD) is an inherited neurodegenerative disease that affects multiple brain regions. It is caused by an expanded polyglutamine tract in huntingtin (Htt). The development of therapies for HD and other neurodegenerative diseases has been hampered by multiple factors, including the lack of clear therapeutic targets, and the cost and complexity of testing lead compounds in vivo. The R6/2 HD mouse model is widely used for pre-clinical trials because of its progressive and robust neural dysfunction, which includes retinal degeneration. Profilin-1 is a Htt binding protein that inhibits Htt aggregation. Its binding to Htt is regulated by the rho-associated kinase (ROCK), which phosphorylates profilin at Ser-137. ROCK is thus a therapeutic target in HD. The ROCK inhibitor Y-27632 reduces Htt toxicity in fly and mouse models. Here we characterized the progressive retinopathy of R6/2 mice between 6–19 weeks of age to determine an optimal treatment window. We then tested a clinically approved ROCK inhibitor, HA-1077, administered intravitreally via liposome-mediated drug delivery. HA-1077 increased photopic and flicker ERG response amplitudes in R6/2 mice, but not in wild-type littermate controls. By targeting ROCK with a new inhibitor, and testing its effects in a novel in vivo model, these results validate the in vivo efficacy of a therapeutic candidate, and establish the feasibility of using the retina as a readout for CNS function in models of neurodegenerative disease.

The Interactin of Huntingtin Exon1 with Lipid Bilayers is Regulated by polyQ Length and polyQ Flanking Sequences

Huntington's Disease (HD) is a neurodegenerative disorder that is defined by the accumulation of nanoscale aggregates comprised of the huntingtin (htt) protein. Aggregation is directly caused by an expanded polyglutamine (polyQ) domain near the N-terminus of htt, leading to a diverse population of aggregate species, including oligomers and fibrils. Furthermore, the length of the polyQ domain is directly related to onset and severity of disease. The first 17 amino acids on the N-terminus (N17) and the polyproline (polyP) domain on the C-terminal side of the polyQ domain have been shown to further modulate the aggregation process. Additionally, N17 appears to have lipid binding properties as htt interacts with a variety of membrane-containing structures present in cells, such as organelles, and interactions with these membrane surfaces may further modulate htt aggregation. To investigate the interaction between htt exon1 and lipid bilayers, in situ atomic force microscopy (AFM) was used to directly monitor the aggregation of htt exon1 constructs with varying polyQ-length or synthetic peptides with different combinations of polyQ domain flanking sequences associated with htt exon1 on supported lipid membranes comprised of total brain lipid extract. The exon1 fragments accumulated on the lipid membranes, causing disruption of the membrane, in a polyQ-length dependent manner. By adding N-terminal tags to the htt exon1 fragments, the interaction with the lipid bilayer was impeded. Synthetic peptides lacking the N17 flanking sequence had no appreciable interaction with lipid bilayers. Interestingly, polyQ peptides with the N17 flanking sequence interacted with the bilayer. This interaction was further modulated by the addition of the polyP domain.