mHTT Expression Research Articles

Viral vector mediated expression of mutant huntingtin in the dorsal raphe produces disease-related neuropathology but not depressive-like behaviors in wildtype mice

Huntington׳s disease (HD) is a neurodegenerative disorder caused by a mutation in the HTT gene (mHTT) encoding the protein huntingtin. An expansion in the gene׳s CAG repeat length renders a misfolded, dysfunctional protein with an abnormally long glutamine (Q) stretch at the N terminus that often incorporates into inclusion bodies and leads to neurodegeneration in many regions of the brain. HD is characterized by motor and cognitive decline as well as mood disorders, with depression being particularly common. Approximately 40% of the HD population suffers from depressive symptoms. Because these symptoms often manifest a decade or more prior to the knowledge that the person is at risk for the disease, a portion of the early depression in HD appears to be a consequence of the pathology arising from expression of the mutant gene. While the depression in HD patients is often treated with serotonin agonists, there is scant experimental evidence that the depression in HD responds well to these serotonin treatments or in a similar manner to how non-HD depression tends to respond. Additionally, at very early sub-threshold depression levels, abnormal changes in several neuronal populations are already detectable in HD patients, suggesting that a variety of brain structures may be involved. Taken together, the serotonin system is a viable candidate. However, at present there is limited evidence of the precise nuclei or circuits that play a role in HD depression. With this in mind, the current study was designed to control for the widespread brain neuropathology that occurs in HD and in transgenic mouse models of HD and focuses specifically on the influence of the midbrain dorsal raphe nucleus (DRN). The DRN provides the majority of the serotonin to the forebrain and exhibits cell loss in non-HD depression. Therefore, we employed a viral vector delivery system to investigate whether the over-expression of mHTT in the DRN׳s ventral sub-nuclei alone is sufficient to produce depressive-like behaviors. Wildtype mice were injected with an adeno-associated virus (AAV2/1) encoding HTT containing either a pathogenic (N171-82Q) or control (N171-16Q) CAG repeat length into the ventral DRN and depressive-like behaviors and motor behaviors were assessed for 12 weeks post-surgery. Quantitative PCR and immunohistochemistry (IHC) verified positive transduction in the ventral aspects of the DRN, including the ventral sub-nucleus (DRv) and interfascicular sub-nucleus (DRif). IHC demonstrated microgliosis in and around the injection site and mHTT-positive inclusions in serotonin-producing neurons and a small percentage of astrocytes in animals injected with N171-82Q compared to controls. Moreover, N171-82Q injected mice showed a 75% reduction in cells that stained positive for the serotonin synthesis enzyme, tryptophan hydroxylase-2 (TPH2) compared to controls (p<0.05). Despite mHTT-mediated pathology in the DRv and DRif, no significant changes in depressive-like behavior were detected. Consequently, we conclude that 12 weeks of N171-82Q expression in the ventral sub-nuclei of the DRN of wildtype mice causes characteristic disease-related cellular neuropathology but is not sufficient to elicit depressive-like behaviors. Ongoing studies are investigating whether a larger injection volume that transfects a larger percentage of the DRN and/or a longer time course of mHTT expression might elicit depressive-like behaviors. Moreover, mHTT expression in other regions of the brain, such as the hippocampal dentate gyrus and/or the frontal cortex might be necessary to elicit HD depression. Together, these results may prove helpful in addressing which therapeutic and/or pharmacological strategies might be most efficacious when treating depressive symptomology in patients suffering from HD.

Targets for future clinical trials in Huntington's disease: what's in the pipeline?

The known genetic cause of Huntington's disease (HD) has fueled considerable progress in understanding its pathobiology and the development of therapeutic approaches aimed at correcting specific changes linked to the causative mutation. Among the most promising is reducing expression of mutant huntingtin protein (mHTT) with RNA interference or antisense oligonucleotides; human trials are now being planned. Zinc-finger transcriptional repression is another innovative method to reduce mHTT expression. Modulation of mHTT phosphorylation, chaperone upregulation, and autophagy enhancement represent attempts to alter cellular homeostasis to favor removal of mHTT. Inhibition of histone deacetylases (HDACs) remains of interest; recent work affirms HDAC4 as a target but questions the assumed centrality of its catalytic activity in HD. Phosphodiesterase inhibition, aimed at restoring synaptic function, has progressed rapidly to human trials. Deranged cellular signaling provides several tractable targets, but specificity and complexity are challenges. Restoring neurotrophic support in HD remains a key potential therapeutic approach. with several approaches being pursued, including brain-derived neurotrophic factor (BDNF) mimesis through tyrosine receptor kinase B (TrkB) agonism and monoclonal antibodies. An increasing understanding of the role of glial cells in HD has led to several new therapeutic avenues, including kynurenine monooxygenase inhibition, immunomodulation by laquinimod, CB2 agonism, and others. The complex metabolic derangements in HD remain under study, but no clear therapeutic strategy has yet emerged. We conclude that many exciting therapeutics are progressing through the development pipeline, and combining a better understanding of HD biology in human patients, with concerted medicinal chemistry efforts, will be crucial for bringing about an era of effective therapies.

Neuronal targets for reducing mutant huntingtin expression to ameliorate disease in a mouse model of Huntington's disease.

Huntington’s disease (HD) is a fatal dominantly inherited neurodegenerative disorder caused by a CAG repeat expansion leading to an elongated polyglutamine stretch in Huntingtin1. Mutant Huntingtin (mHTT) is ubiquitously expressed but elicits selective cortical and striatal neurodegeneration in HD2. The mechanistic basis for such selective neuronal vulnerability remains unclear. A necessary step towards resolving this enigma is to define the cell types in which mHTT expression is causally linked to the disease pathogenesis. Using a conditional human genomic transgenic mouse model of HD expressing full-length mHTT (BACHD)3, we genetically reduced mHTT expression in striatal, cortical, or both neuronal populations. We show that cortical mHTT reduction in BACHD partially improves motor and psychiatric-like behavioral deficits, but does not improve neurodegeneration, while mHTT reduction in both neuronal populations consistently ameliorates all behavioral deficits and selective brain atrophy in this HD model. Furthermore, mHTT reduction in cortical or striatal neurons partially ameliorates cortico-striatal synaptic deficits, while further restoration of striatal synaptic function is achieved by mHTT reduction in both neuronal cell types. Our study demonstrates distinct, but interacting roles of cortical and striatal mHTT in disease pathogenesis and suggests that optimal HD therapeutics may require targeting mHTT in both cortical and striatal neurons.

Intrajugular Vein Delivery of AAV9-RNAi Prevents Neuropathological Changes and Weight Loss in Huntington's Disease Mice

Huntington's disease (HD) is a fatal neurological disorder caused by a CAG repeat expansion in the HTT gene, which encodes a mutant huntingtin protein (mHTT). The mutation confers a toxic gain of function on huntingtin, leading to widespread neurodegeneration and inclusion formation in many brain regions. Although the hallmark symptom of HD is hyperkinesia stemming from striatal degeneration, several other brain regions are affected which cause psychiatric, cognitive, and metabolic symptoms. Additionally, mHTT expression in peripheral tissue is associated with skeletal muscle atrophy, cardiac failure, weight loss, and diabetes. We, and others, have demonstrated a prevention of motor symptoms in HD mice following direct striatal injection of adeno-associated viral vector (AAV) serotype 1 encoding an RNA interference (RNAi) construct targeting mutant HTT mRNA (mHTT). Here, we expand these efforts and demonstrate that an intrajugular vein injection of AAV serotype 9 (AAV9) expressing a mutant HTT-specific RNAi construct significantly reduced mHTT expression in multiple brain regions and peripheral tissues affected in HD. Correspondingly, this approach prevented atrophy and inclusion formation in key brain regions as well as the severe weight loss germane to HD transgenic mice. These results demonstrate that systemic delivery of AAV9-RNAi may provide more widespread clinical benefit for patients suffering from HD.

Genetic manipulations of mutant huntingtin in mice: new insights into Huntington's disease pathogenesis

This year (2013) marks the 20th anniversary of identification of the causal genetic mutation for Huntington's disease (HD), a landmark discovery that heralded study of the biological underpinnings of this most common dominantly inherited neurodegenerative disorder. Among the variety of model organisms used to study HD pathogenesis, the mouse model is by far the most commonly used mammalian genetic model. Much of our current knowledge regarding mutant huntingtin (mHtt)-induced disease pathogenesis in mammalian models has been obtained by studying transgenic mouse models expressing mHtt N-terminal fragments, full-length murine or human mHtt. In this review, we focus on recent progress in using novel HD mouse models with targeted mHtt expression in specific brain cell types. These models help to address the role of distinct neuronal and non-neuronal cell types in eliciting cell-autonomous or non-cell-autonomous disease processes in HD. We also describe several mHtt transgenic mouse models with targeted mutations in Htt cis-domains to address specific pathogenic hypotheses, ranging from mHtt proteolysis to post-translational modifications. These novel mouse genetic studies, through direct manipulations of the causal HD gene, utilize a reductionist approach to systematically unravel the cellular and molecular pathways that are targeted by mHtt in disease pathogenesis, and to potentially identify novel targets for therapy.

Impaired Mitochondrial Dynamics and Nrf2 Signaling Contribute to Compromised Responses to Oxidative Stress in Striatal Cells Expressing Full-Length Mutant Huntingtin

Huntington disease (HD) is an inherited neurodegenerative disease resulting from an abnormal expansion of polyglutamine in huntingtin (Htt). Compromised oxidative stress defense systems have emerged as a contributing factor to the pathogenesis of HD. Indeed activation of the Nrf2 pathway, which plays a prominent role in mediating antioxidant responses, has been considered as a therapeutic strategy for the treatment of HD. Given the fact that there is an interrelationship between impairments in mitochondrial dynamics and increased oxidative stress, in this present study we examined the effect of mutant Htt (mHtt) on these two parameters. STHdhQ111/Q111 cells, striatal cells expressing mHtt, display more fragmented mitochondria compared to STHdhQ7/Q7 cells, striatal cells expressing wild type Htt, concurrent with alterations in the expression levels of Drp1 and Opa1, key regulators of mitochondrial fission and fusion, respectively. Studies of mitochondrial dynamics using cell fusion and mitochondrial targeted photo-switchable Dendra revealed that mitochondrial fusion is significantly decreased in STHdhQ111/Q111 cells. Oxidative stress leads to dramatic increases in the number of STHdhQ111/Q111 cells containing swollen mitochondria, while STHdhQ7/Q7 cells just show increases in the number of fragmented mitochondria. mHtt expression results in reduced activity of Nrf2, and activation of the Nrf2 pathway by the oxidant tBHQ is significantly impaired in STHdhQ111/Q111 cells. Nrf2 expression does not differ between the two cell types, but STHdhQ111/Q111 cells show reduced expression of Keap1 and p62, key modulators of Nrf2 signaling. In addition, STHdhQ111/Q111 cells exhibit increases in autophagy, whereas the basal level of autophagy activation is low in STHdhQ7/Q7 cells. These results suggest that mHtt disrupts Nrf2 signaling which contributes to impaired mitochondrial dynamics and may enhance susceptibility to oxidative stress in STHdhQ111/Q111 cells.

B23 Immune dysfunction in HD human myeloid cells is caused by NFκB pathway dysregulation and is reversed by lowering HTT levels

<h3>Background</h3> Altered innate immune responses are observed in HD, with hyper-reactive central and peripheral immune cells producing increased levels of pro-inflammatory cytokine in HD patients. Bone marrow transplantation studies and KMO inhibitor administration have both shown that the immune system is a modifier of HD progression. However, a mechanistic understanding of how this relates to mHTT expression has been lacking. <h3>Aim</h3> Characterise the abnormal function of peripheral myeloid cells and examine alterations in signalling pathways responsible for those changes. <h3>Results</h3> The production of pro-inflammatory cytokines in response to LPS was elevated in both monocytes and macrophages from HD patients. Induction of mHTT exon 1 expression in a myeloid cell line showed that the hyper-reactive cytokine production is caused by a cell intrinsic effect of exon 1 mHTT expression. Significant changes in the expression levels of several key molecules in the NFκB pathway, such as IRAK1 and AKT1, were identified using PCR signalling arrays. IκB, the endogenous inhibitor of NFκB activity, was more rapidly degraded following LPS stimulation in HD patients compared to controls, potentially leading to increased NFκB activity and altered gene expression. A direct interaction between HTT and IKKγ was demonstrated by co-immunoprecipitation, suggesting the possibility of a scaffolding function of HTT in the NFκB pathway that may be altered by an expanded polyQ-repeat. Knocking-down total HTT protein levels using GeRP-delivered siRNA in human HD macrophages led to a reduction of cytokine production by LPS-stimulated HD cells. <h3>Conclusions</h3> Taken together, these data suggests that the dysfunction of primary myeloid cells from HD patients is directly caused by the expression of mHTT and its effects on the NFκB pathway. Lowering HTT levels in human macrophages can at least partially restore normal cell function, underlining the importance of HTT-lowering as a therapeutic approach.

Changes in BiP availability reveal hypersensitivity to acute endoplasmic reticulum stress in cells expressing mutant huntingtin

Huntington's disease (HD) is caused by expanded glutamine repeats within the huntingtin (Htt) protein. Mutant Htt (mHtt) in the cytoplasm has been linked to induction of the luminal endoplasmic reticulum (ER) stress pathway, the unfolded protein response (UPR). How mHtt impacts the susceptibility of the ER lumen to stress remains poorly understood. To investigate molecular differences in the ER in cells expressing mHtt, we used live-cell imaging of a sensitive reporter of the misfolded secretory protein burden, GFP fused to the ER chaperone BiP (also known as GRP78), which decreases in mobility as it binds increasing amounts of misfolded proteins. Striatal neurons expressing full-length mHtt showed no differences in BiP-GFP mobility and no evidence of UPR activation compared with wild-type cells at steady state. However, mHtt-expressing cells were acutely sensitive to misfolded secretory proteins. Treatment with ER stressors, tunicamycin or DTT, rapidly decreased BiP-GFP mobility in mHtt striatal cells and accelerated UPR activation compared with wild-type cells. mHtt-expressing cells exhibited decreased misfolded protein flux as a result of ER associated degradation (ERAD) dysfunction. Furthermore, UPR-adapted mHtt cells succumbed to misfolded protein stresses that could be tolerated by adapted wild-type cells. Thus, mHtt expression impairs misfolded secretory protein turnover, decreases the ER stress threshold, and increases cell vulnerability to insults.

Activated microglia proliferate at neurites of mutant huntingtin-expressing neurons

In Huntington's disease (HD), mutated huntingtin (mhtt) causes striatal neurodegeneration which is paralleled by elevated microglia cell numbers. In vitro corticostriatal slice and primary neuronal culture models, in which neuronal expression of mhtt fragments drives HD-like neurotoxicity, were employed to examine wild type microglia during both the initiation and progression of neuronal pathology. As neuronal pathology progressed, microglia initially localized in the vicinity of neurons expressing mhtt fragments increased in number, demonstrated morphological evidence of activation, and expressed the proliferation marker, Ki67. These microglia were positioned along irregular neurites, but did not localize with mhtt inclusions nor exacerbate mhtt fragment-induced neurotoxicity. Prior to neuronal pathology, microglia upregulated ionized calcium binding adaptor molecule 1 (Iba1), signaling a functional shift. With neurodegeneration, interleukin-6 and complement component 1q were increased. The results suggest a stimulatory, proliferative signal for microglia present at the onset of mhtt fragment-induced neurodegeneration. Thus, microglia effect a localized inflammatory response to neuronal mhtt expression that may serve to direct microglial removal of dysfunctional neurites or aberrant synapses, as is required for reparative actions in vivo.

Modifications of p53 and the DNA Damage Response in Cells Expressing Mutant Form of the Protein Huntingtin

Huntington's disease (HD) occurs through an expansion of the trinucleotide repeat in the HD gene resulting in the lengthening of the polyglutamine stretch within the N terminus of the protein, huntingtin (Htt). While the function of the protein is still being fully elucidated, we have shown that genomic DNA damage is associated with the expression of mutant Htt (mHtt) in a time-dependent fashion. With the accumulation of mHtt and its development into a micro-aggregated complex, the initiation of genomic damage engages a cellular stress signal that activates the DNA damage and stress response pathway. Here we explore the modifications and activation of p53 and keystone regulators of the cell stress response pathway using expression of a fragment of mHtt in HEK293T cells. We find an increase in phosphorylated p53 at serine 15 (S15), diminished acetylation at lysine 382 (K382), altered ubiquitination pattern, and oligomerization activity as a function of mHtt expression. As one might predict, upstream regulators of p53, such as CREB-binding protein/p300 and MDM2, are also seen to be affected by the expression of mHtt, albeit in different ways. These data suggest a possible relationship between p53 and the slow accumulation of DNA damage resulting from the expression of mHtt. The lack of a proper p53-mediated signaling cascade or its alteration in the presence of DNA damage may contribute to the slow progression of cellular dysfunction which is a hallmark of HD pathology.

Transcription deregulation and mitochondria crosstalk in Huntington's disease

Event Abstract Back to Event Transcription deregulation and mitochondria crosstalk in Huntington's disease Tatiana Rosado-Rosenstock1, Mario Noro-Laco1, 2*, Oliveira C. R1, 2 and Ana Cristina-Rego1, 2 1 University of Coimbra, Center for Neuroscience and Cell Biology, Portugal 2 University of Coimbra, Faculty of Medicine, Portugal Huntington’s Disease (HD) is an autosomal-dominant neurodegenerative disorder characterized by neuronal loss in the striatum. HD is caused by an expansion of the trinucleotide CAG in the HD gene, responsible for the translation of mutant huntingtin (mhtt) with a long polyglutamine stretch in its N-terminal. As a consequence, mhtt interacts with several other proteins, such as caspase 3 and glyceraldehyde 3-phosphate dehydrogenase. Since mhtt can also interact with many transcriptional factors and because some of the mechanisms related to the neurodegeneration observed in HD are associated with transcriptional deregulation and mitochondrial dysfunction, the objective of this work is to investigate changes in expression of transcription factors and nuclear-encoded candidate proteins that may influence mitochondrial function in a cellular model of HD. For this purpose we used striatal cells derived from HD knock-in mice expressing full-length mutant huntingtin with 111 glutamines (Q111) versus striatal cells expressing normal huntingtin (Q7). No significant changes were observed in total levels of CBP, p65 and p53 between both cell lines. However, we determined a significant decrease in the expression of CBP and p53 in nuclear fractions isolated from Q111 compared to Q7 cells. Moreover, we observed a significant decrease in total CREB levels in Q111 cells, but no changes were observed in the ratio P-CREB/CREB. Additionally, there was a significant decrease in the mitochondrial levels of prohibitin-1 in Q111, in comparison to Q7 cells. Since prohibitin is a nuclear-encoded protein localized in the inner mitochondrial membrane that seems to act as chaperone for the stabilization of mitochondrial proteins preventing their misfolding, our data suggest that full-length mhtt affects not only the transcription of nuclear encoded proteins, including mitochondrial proteins, but also mitochondrial protein stabilization, which may underlie changes in mitochondrial function. These alterations in transcription and in the overall quality of the protein content inside the cell could trigger cell death. However, levels of pro-apoptotic protein, Bax, were lower in mitochondrial extracts of Q111 cell line. These data corroborate our results from brain mitochondria extracted from YAC mice expressing full-length human mHtt with 128 glutamines (Q128). The YAC 128 mitochondrial extracts have shown to be more enriched in anti-apoptotic proteins Bcl-2 and displayed higher levels of cytochrome C and AIF. Altogether, our results indicate the existence of transcriptional deregulation and protein destabilization under mHtt expression, although the prolonged exposure to this pathological protein might trigger compensatory mechanisms, which would account for the increased resilience to cell death activation. Conference: 11th Meeting of the Portuguese Society for Neuroscience, Braga, Portugal, 4 Jun - 6 Jun, 2009. Presentation Type: Oral Presentation Topic: Symposium 3 – Neurodegenerative Disorders Citation: Rosado-Rosenstock T, Noro-Laco M, R OC and Cristina-Rego A (2009). Transcription deregulation and mitochondria crosstalk in Huntington's disease. Front. Neurosci. Conference Abstract: 11th Meeting of the Portuguese Society for Neuroscience. doi: 10.3389/conf.neuro.01.2009.11.010 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 05 Aug 2009; Published Online: 05 Aug 2009. * Correspondence: Mario Noro-Laco, University of Coimbra, Center for Neuroscience and Cell Biology, Alicante, Portugal, Portugal.mariolnl@cnc.cj.uc.pt Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Tatiana Rosado-Rosenstock Mario Noro-Laco Oliveira C R Ana Cristina-Rego Google Tatiana Rosado-Rosenstock Mario Noro-Laco Oliveira C R Ana Cristina-Rego Google Scholar Tatiana Rosado-Rosenstock Mario Noro-Laco Oliveira C R Ana Cristina-Rego PubMed Tatiana Rosado-Rosenstock Mario Noro-Laco Oliveira C R Ana Cristina-Rego Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Mutant huntingtin inhibits clathrin-independent endocytosis and causes accumulation of cholesterol in vitro and in vivo

We show that the mutant Huntington's disease (HD) protein (mhtt) specifically inhibits endocytosis in primary striatal neurons. Unexpectedly, mhtt does not inhibit clathrin-dependent endocytosis as was anticipated based on known interacting partners. Instead, inhibition occurs through a non-clathrin, caveolar-related pathway. Expression of mhtt inhibited internalization of BODIPY-lactosylceramide (LacCer), which is internalized by a caveolar-related mechanism. In contrast, endocytosis of Alexa Fluor 594-transferrin (Tfn) and epidermal growth factor, internalized through clathrin pathway, was unaffected by mhtt expression. Caveolin-1 (cav1), the major structural protein of caveolae binds cholesterol and is responsible for its trafficking inside cells. Mhtt interacts with cav-1 and caused a striking accumulation of intracellular cholesterol. Cholesterol accumulated in cultured neurons expressing mhtt in vitro and in brains of mhtt-expressing animals in vivo, and was observed after induction of mhtt expression in PC-12 cell lines. The accumulation occurred only when mhtt and cav1 were simultaneously expressed in cells. Knockdown of cav1 in mhtt-expressing neurons blocked cholesterol accumulation and restored LacCer endocytosis. Thus, mhtt and cav1 functionally interact to cause both cellular defects. These data provide the first direct link between mhtt and caveolar-related endocytosis and also suggest a possible mechanism for HD neurotoxicity where cholesterol homeostasis is perturbed.

Pathological Cell-Cell Interactions Elicited by a Neuropathogenic Form of Mutant Huntingtin Contribute to Cortical Pathogenesis in HD Mice

Expanded polyglutamine (polyQ) proteins in Huntington's disease (HD) as well as other polyQ disorders are known to elicit a variety of intracellular toxicities, but it remains unclear whether polyQ proteins can elicit pathological cell-cell interactions which are critical to disease pathogenesis. To test this possibility, we have created conditional HD mice expressing a neuropathogenic form of mutant huntingtin (mhtt-exon1) in discrete neuronal populations. We show that mhtt aggregation is a cell-autonomous process. However, progressive motor deficits and cortical neuropathology are only observed when mhtt expression is in multiple neuronal types, including cortical interneurons, but not when mhtt expression is restricted to cortical pyramidal neurons. We further demonstrate an early deficit in cortical inhibition, suggesting that pathological interactions between interneurons and pyramidal neurons may contribute to the cortical manifestation of HD. Our study provides genetic evidence that pathological cell-cell interactions elicited by neuropathogenic forms of mhtt can critically contribute to cortical pathogenesis in a HD mouse model.