Expression Of Mutant Huntingtin Research Articles

Enhanced immune response to MMP3 stimulation in microglia expressing mutant huntingtin

Huntington’s Disease (HD) is an inherited neurodegenerative disease caused by a polyglutamine expansion in the huntingtin protein. The YAC128 mouse model of HD expresses the full-length human huntingtin protein with 128 CAG repeats and replicates the phenotype and neurodegeneration that occur in HD. Several studies have implicated a role for neuroinflammation in HD pathogenesis. Studies on presymptomatic HD patients have illustrated microgliosis (activated microglia) in brain regions affected in HD. Mutant huntingtin expressing isolated primary monocytes (human HD patients) and primary macrophages (YAC128) are overactive in response to lipopolysaccharide (LPS) stimulation. In this study we demonstrate that cultured primary microglia (the resident immune cells of the brain cells) from YAC128 mice differentially express a wide number of cytokines compared to wildtype microglia cultures in response to LPS. Furthermore, this study outlines a direct interaction between mutant huntingtin and cytokine secretion in HD microglia. Increased cytokine release in YAC128 microglia can be blocked by cannabinoid activation or by mutant huntingtin knockdown with anti-sense oligonucleotide treatment. Matrix metalloprotease 3 (MMP3), an endogenous neuronal activator of microglia, also induces increased cytokine release from YAC128 microglia compared to wildtype microglia. We found elevated MMP levels in HD CSF, and MMP levels correlate with disease severity in HD. These data support a novel role for MMPs and microglial activation in HD pathogenesis. With an improved understanding of the specific cellular processes involved in HD neuroinflammation, novel therapeutic agents targeting these processes can be developed and hold great promise in the treatment of HD.

Focal expression of mutant huntingtin in the songbird basal ganglia disrupts cortico-basal ganglia networks and vocal sequences

The basal ganglia (BG) promote complex sequential movements by helping to select elementary motor gestures appropriate to a given behavioral context. Indeed, Huntington's disease (HD), which causes striatal atrophy in the BG, is characterized by hyperkinesia and chorea. How striatal cell loss alters activity in the BG and downstream motor cortical regions to cause these disorganized movements remains unknown. Here, we show that expressing the genetic mutation that causes HD in a song-related region of the songbird BG destabilizes syllable sequences and increases overall vocal activity, but leave the structure of individual syllables intact. These behavioral changes are paralleled by the selective loss of striatal neurons and reduction of inhibitory synapses on pallidal neurons that serve as the BG output. Chronic recordings in singing birds revealed disrupted temporal patterns of activity in pallidal neurons and downstream cortical neurons. Moreover, reversible inactivation of the cortical neurons rescued the disorganized vocal sequences in transfected birds. These findings shed light on a key role of temporal patterns of cortico-BG activity in the regulation of complex motor sequences and show how a genetic mutation alters cortico-BG networks to cause disorganized movements.

Mitochondria-targeted molecules MitoQ and SS31 reduce mutant huntingtin-induced mitochondrial toxicity and synaptic damage in Huntington's disease.

The objective of this study was to determine the protective effects of the mitochondria-targeted molecules MitoQ and SS31 in striatal neurons that stably express mutant huntingtin (Htt) (STHDhQ111/Q111) in Huntington's disease (HD). We studied mitochondrial and synaptic activities by measuring mRNA and the protein levels of mitochondrial and synaptic genes, mitochondrial function, and ultra-structural changes in MitoQ- and SS31-treated mutant Htt neurons relative to untreated mutant Htt neurons. We used gene expression analysis, biochemical methods, transmission electron microscopy (TEM) and confocal microscopy methods. In the MitoQ- and SS31-treated mutant Htt neurons, fission genes Drp1 and Fis1 were down-regulated, and fusion genes Mfn1, Mfn2 and Opa1 were up-regulated relative to untreated neurons, suggesting that mitochondria-targeted molecules reduce fission activity. Interestingly, the mitochondrial biogenesis genes PGC1α, PGC1β, Nrf1, Nrf2 and TFAM were up-regulated in MitoQ- and SS31-treated mutant Htt neurons. The synaptic genes synaptophysin and PSD95 were up-regulated, and mitochondrial function was normal in the MitoQ- and SS31-treated mutant Htt neurons. Immunoblotting findings of mitochondrial and synaptic proteins agreed with the mRNA findings. TEM studies revealed decreased numbers of structurally intact mitochondria in MitoQ- and SS31-treated mutant Htt neurons. These findings suggest that mitochondria-targeted molecules MitoQ and SS31 are protective against mutant Htt-induced mitochondrial and synaptic damage in HD neurons, and these mitochondria-targeted molecules are potential therapeutic molecules for the treatment of HD neurons.

Interactome network analysis identifies multiple caspase-6 interactors involved in the pathogenesis of HD.

Caspase-6 (CASP6) has emerged as an important player in Huntington disease (HD), Alzheimer disease (AD) and cerebral ischemia, where it is activated early in the disease process. CASP6 also plays a key role in axonal degeneration, further underscoring the importance of this protease in neurodegenerative pathways. As a protein's function is modulated by its protein-protein interactions, we performed a high-throughput yeast-2-hybrid (Y2H) screen against ∼17,000 human proteins to gain further insight into the function of CASP6. We identified a high-confidence list of 87 potential CASP6 interactors. From this list, 61% are predicted to contain a CASP6 recognition site. Of nine candidate substrates assessed, six are cleaved by CASP6. Proteins that did not contain a predicted CASP6 recognition site were assessed using a LUMIER assay approach, and 51% were further validated as interactors by this method. Of note, 54% of the high-confidence interactors identified show alterations in human HD brain at the mRNA level, and there is a significant enrichment for previously validated huntingtin (HTT) interactors. One protein of interest, STK3, a pro-apoptotic kinase, was validated biochemically to be a CASP6 substrate. Furthermore, our results demonstrate that in striatal cells expressing mutant huntingtin (mHTT), an increase in full length and fragment levels of STK3 are observed. We further show that caspase-3 is not essential for the endogenous cleavage of STK3. Characterization of the interaction network provides important new information regarding key pathways of interactors of CASP6 and highlights potential novel therapeutic targets for HD, AD and cerebral ischemia.

Insulin and IGF-1 regularize energy metabolites in neural cells expressing full-length mutant huntingtin

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder linked to the expression of mutant huntingtin. Bioenergetic dysfunction has been described to contribute to HD pathogenesis. Thus, treatment paradigms aimed to ameliorate energy deficits appear to be suitable candidates in HD. In previous studies, we observed protective effects of insulin growth factor-1 (IGF-1) in YAC128 and R6/2 mice, two HD mouse models, whereas IGF-1 and/or insulin halted mitochondrial-driven oxidative stress in mutant striatal cells and mitochondrial dysfunction in HD human lymphoblasts. Here, we analyzed the effect of IGF-1 versus insulin on energy metabolic parameters using striatal cells derived from HD knock-in mice and primary cortical cultures from YAC128 mice. STHdhQ111/Q111 cells exhibited decreased ATP/ADP ratio and increased phosphocreatine levels. Moreover, pyruvate levels were increased in mutant cells, most probably in consequence of a decrease in pyruvate dehydrogenase (PDH) protein expression and increased PDH phosphorylation, reflecting its inactivation. Insulin and IGF-1 treatment significantly decreased phosphocreatine levels, whereas IGF-1 only decreased pyruvate levels in mutant cells. In a different scenario, primary cortical cultures derived from YAC128 mice also displayed energetic abnormalities. We observed a decrease in both ATP/ADP and phosphocreatine levels, which were prevented following exposure to insulin or IGF-1. Furthermore, decreased lactate levels in YAC128 cultures occurred concomitantly with a decline in lactate dehydrogenase activity, which was ameliorated with both insulin and IGF-1. These data demonstrate differential HD-associated metabolic dysfunction in striatal cell lines and primary cortical cultures, both of which being alleviated by insulin and IGF-1.

<I>In Vitro</I> Study of Caecal and Colon Microbial Fermentation Patterns in Wild Boar (<I>Sus scrofa scrofa</I>)

The aim of this study was to evaluate wild boar (Sus scrofa scrofa) caecal and colon products of microbial activity including short chain fatty acids (SCFA), ammonia and methane concentrations. The in vitro method was applied to caecal and colon contents after 12 and 24-hour incubation with the substrate (wheat bran), or without any additive (control samples). The pH was also measured in each sample. In samples incubated with the substrate, a lower pH was noted as compared to the control (P < 0.001). In terms of the total SCFA concentration, the hindgut microbial fermentation pattern of wild boar was characterized by a high acetate level, followed by propionate and then butyrate at a ratio of 7:1.5:1. Substrate addition decreased acetate molar proportions (P < 0.001) and increased those of butyrate (P < 0.001) as well as propionate (P < 0.05). The total SCFA level in fresh, unincubated caecal samples (128 mmol/kg) was similar to that in the colon (111 mmol/kg). The ammonia concentrations were at the level of 0.8-1.5 mmol/kg of hindgut content and did not differ between the two investigated hindgut parts. Methanogenesis was also similar in the caecum and colon and after 24h was 2.69 mmol/kg and 2.27 for caecal colon control samples, respectively. The substrate increased total gas production and methane concentration (P < 0.001).

Curcumin modulates cell death and is protective in Huntington's disease model.

Huntington’s disease (HD) is a progressive, dominantly inherited neurological disorder caused by an abnormal expansion of polyglutamine (polyQ) repeat within the Huntingtin (Htt) protein with no disease modifying treatments. In a Drosophila model of HD, expression of mutant Huntingtin (Htt) protein with expanded polyQ leads to formation of inclusion bodies (IBs), increase in cellular toxicity, progression of motor disabilities and reduced viability. Multiple cellular events such as oxidative stress, mitochondrial dysfunction, inflammation and transcriptional dysregulation are reported to contribute to pathology, however, till date there are no disease-modifying treatments with least side effects. Therefore, we investigated effect of the phytochemical curcumin on HD pathogenesis. Curcumin, a phytochemical and commonly used ingredient in Asian food has a wide spectrum of anti-oxidant, anti-inflammatory and anti-fibrilogenic properties. In this study, we provide evidence that curcumin significantly ameliorates disease symptoms in a Drosophila model of HD by suppressing cell death and can be a key to halting the progression of Huntington’s disease with least side effects.

Vulnerability of newly synthesized proteins to proteostasis stress

The capacity of the cell to produce, fold and degrade proteins relies on components of the proteostasis network. Multiple types of insults can impose a burden on this network, causing protein misfolding. Using thermal stress, a classic example of acute proteostatic stress, we demonstrate that ∼5-10% of the soluble cytosolic and nuclear proteome in human HEK293 cells is vulnerable to misfolding when proteostatic function is overwhelmed. Inhibiting new protein synthesis for 30 min prior to heat-shock dramatically reduced the amount of heat-stress induced polyubiquitylation, and reduced the misfolding of proteins identified as vulnerable to thermal stress. Following prior studies in C. elegans in which mutant huntingtin (Q103) expression was shown to cause the secondary misfolding of cytosolic proteins, we also demonstrate that mutant huntingtin causes similar 'secondary' misfolding in human cells. Similar to thermal stress, inhibiting new protein synthesis reduced the impact of mutant huntingtin on proteostatic function. These findings suggest that newly made proteins are vulnerable to misfolding when proteostasis is disrupted by insults such as thermal stress and mutant protein aggregation.

P75NTR in Huntington's disease: beyond the basal ganglia

Huntington's disease (HD) is a fatal neurodegenerative disorder with a characteristic phenotype including chorea and dystonia, uncoordinated fine movements, cognitive decline and psychiatric disturbances. Even though the clinical diagnosis of HD relies on the manifestation of motor abnormalities, the associated memory impairments have been growing in prominence. Indeed, cognitive deficits are evident along all the disease process even in the prodrome before any motor diagnosis is given. These clinical signs have been mainly attributed to corticostriatal dysfunction being deficits of neurotrophic support, caused by either reduced levels of brain-derived neurotrophic factor (BDNF) [1] or decreased TrkB and aberrant p75NTR signalling [2, 3], one of the major pathogenic mechanism involved. However, in recent years has emerged the idea that cognitive decline in HD is likely a reflection of a widespread brain circuitry defect rather than a basal ganglia dysfunction per se. In this regard, in our recent studies we shed new light on the contribution of the hippocampal circuitry to synaptic and memory decline in HD. We demonstrated hippocampal dysfunction in a precise genetic HD mouse model that expresses endogenous levels of mutant huntingtin, HdhQ7/Q111 mice, manifested as alterations in spatial, recognition and associative memories. To get insight into the molecular mechanisms underlying such defects, we focused on p75NTR since growing evidence indicate that p75NTR plays an antagonistic role in synaptic plasticity. We demonstrated up-regulation of p75NTR in the hippocampus of distinct HD mouse models and in human brain without evident changes in the cortex, which extends our previous data showing increased p75NTR expression in the HD striatum [4]. In agreement with a critical role of aberrant p75NTR expression in hippocampal dysfunction we found preserved spatial, recognition and associative memories in new double-mutant mice expressing mutant huntingtin but “physiological” levels of p75NTR levels (HdhQ7/Q111:p75+/−mice). How aberrant p75NTR levels may mediate synaptic and memory deficits in HD is an intriguing question. On one hand, our results indicated that p75NTR directly or indirectly regulate the expression of different synaptic-related proteins previously implicated in HD synaptic and/or cognitive deficits, such as CBP, CamKII, GluA1 or BDNF since memory improvements in double mutant HdhQ7/Q111:p75+/−mice correlated with a recovery of the expression and/or phosphorylation of these molecules. On the other, the loss of dendritic spines in CA1 pyramidal neurons exhibited by HdhQ7/Q111 mutant mice was also prevented by normalization of p75NTR levels. Altogether, these data suggest that synaptic and memory deficits in HD could be related with a reduction in proteins involved in synaptic function and in the number and complexity of hippocampal dendritic spines in agreement with a role of p75NTR as a negative regulator of dendritic spine dynamics and synaptic activity. We further built on work showing that p75NTRcontributes to synaptic dysfunction and memory decline in HD by deregulation of RhoA activity, a small GTPase with complex effects on spines and thereby in synaptic plasticity [5]. Interestingly, such increase was reversed in double mutant HdhQ7/Q111:p75+/−mice suggesting a direct link between aberrant p75NTR activity, dendritic spine loss and aberrant RhoA activity. In agreement with our findings Plotkin and colleagues [3] have demonstrated that plasticity in indirect pathway spiny projection neurons (iSPNs) from BACHD mutant mice can be rescued by inhibition of the p75NTR-RhoA signalling suggesting that early corticostriatal dysfunction in HD could also be attributable to a correctable defect in BDNF signalling. Altogether, this evidence strongly suggests that, in the early stages of the disease, p75NTR antagonism should be considered an effective therapeutic strategy for restoring BDNF neuroprotective and synaptic functions. However, remains to be investigated whether therapeutic approaches targeting p75NTR inhibition should also integrate TrkB activation to produce the most favourable benefits on motor and cognitive impairments. Moreover, we have recently demonstrated that fingolimod, a compound used as an immunomodulator in Multiple Sclerosis patients, restores hippocampal synaptic plasticity and improves memory function in a mouse model of Huntington disease acting through down-regulation of TNFα and p75NTR [6]. These last evidence open the question whether p75NTR dysregulation is triggered by neuroinflammation at very early stages and whether modulation of the inflammatory response of microglia and astroglia in HD may contribute to antagonize the deleterious effects of aberrant p75NTR induced RhoA activity in synaptic plasticity. So far, our studies strongly implicates p75NTR-RhoA hippocampal dysfunction on cognitive decline in HD stressing the need to explore therapeutic strategies that target the identified pathways, not only in the striatum but also in other brain areas besides the basal ganglia which has been underestimated and are strongly involved in HD cognitive pathology

Synaptic scaling up in medium spiny neurons of aged BACHD mice: A slow-progression model of Huntington's disease

Huntington's disease (HD) is an autosomal dominant disease that develops in midlife (~40years-old at onset) and then progresses slowly. It is still unclear how striatal medium spiny neurons (MSNs), the most vulnerable neurons in HD, maintain their function for decades despite the chronic expression of mutant huntingtin (mHTT). In this study, we used aged BACHD mice, a HD model expressing the full-length human mHTT gene, to investigate the molecular, morphological and functional properties of striatal MSNs.We report that the density of dendritic spines in MSNs is substantially lower in aged BACHD mice than in wild-type (WT) mice, in the absence of major dendritic changes and neuronal loss. This spine loss is accompanied by changes in transcription, resulting in a low expression of the striatum-specific G protein-coupled receptor 88 (Gpr88) as well as a reorganization of the composition of AMPAR subunits (high Gria1/Gria2 mRNA ratio). We also detected functional changes in BACHD MSNs. Notably, BACHD MSNs were hyperexcitable and the amplitude of AMPAR-mediated synaptic currents was higher than in WT MSNs. Altogether, these data show that both the intrinsic properties and the strength of the remaining synapses are modified in MSNs with low dendritic spine density in aged BACHD mice. These homeostatic mechanisms may compensate for the substantial loss of synaptic inputs and thus alleviate the deleterious effects of mHTT expression on the activity of MSNs and also possibly on the motor phenotype in aged BACHD.

Early-onset sleep defects in Drosophila models of Huntington's disease reflect alterations of PKA/CREB signaling.

Huntington's disease (HD) is a progressive neurological disorder whose non-motor symptoms include sleep disturbances. Whether sleep and activity abnormalities are primary molecular disruptions of mutant Huntingtin (mutHtt) expression or result from neurodegeneration is unclear. Here, we report Drosophila models of HD exhibit sleep and activity disruptions very early in adulthood, as soon as sleep patterns have developed. Pan-neuronal expression of full-length or N-terminally truncated mutHtt recapitulates sleep phenotypes of HD patients: impaired sleep initiation, fragmented and diminished sleep, and nighttime hyperactivity. Sleep deprivation of HD model flies results in exacerbated sleep deficits, indicating that homeostatic regulation of sleep is impaired. Elevated PKA/CREB activity in healthy flies produces patterns of sleep and activity similar to those in our HD models. We were curious whether aberrations in PKA/CREB signaling were responsible for our early-onset sleep/activity phenotypes. Decreasing signaling through the cAMP/PKA pathway suppresses mutHtt-induced developmental lethality. Genetically reducing PKA abolishes sleep/activity deficits in HD model flies, restores the homeostatic response and extends median lifespan. In vivo reporters, however, show dCREB2 activity is unchanged, or decreased when sleep/activity patterns are abnormal, suggesting dissociation of PKA and dCREB2 occurs early in pathogenesis. Collectively, our data suggest that sleep defects may reflect a primary pathological process in HD, and that measurements of sleep and cAMP/PKA could be prodromal indicators of disease, and serve as therapeutic targets for intervention.

Both Orai1 and TRPC1 are Involved in Excessive Store-Operated Calcium Entry in Striatal Neurons Expressing Mutant Huntingtin Exon 1.

It has been previously reported that N-terminus of mutant huntingtin (product of the 1st exon) is sufficient to cause a Huntington's disease (HD) pathological phenotype. In view of recent data suggesting that improper regulation of store-operated calcium (SOC) channels is involved in neurodegenerative processes, we investigated influence of expression of the mutant huntingtin N-terminal fragment (Htt138Q-1exon) on SOC entry (SOCE) in mouse neuroblastoma cells (Neuro-2a) and in primary culture of medium spiny neurons (MSNs) isolated from mice. The results show that SOCE in these cells is enhanced upon lentiviral expression of the Htt138Q-1exon. Moreover, we demonstrated that RNAi-mediated knockdown of TRPC1, Orai1, or STIM1 proteins leads to dramatic reduction of abnormal SOCE in both Neuro-2a and MSNs, expressing Htt138Q-1exon. Thus, we concluded that abnormal SOCE in these cells is maintained by both TRPC1- and Orai1-containing channels and required STIM1 for its activation. Furthermore, EVP4593 compound previously tested as a potential anti-HD drug in a Drosophila screening system has proved to be capable of reducing SOCE to the normal level in MSNs expressing the Htt138Q-1exon.

Out of one mutation, many Huntington's disease effects

In a thoughtful and wide-ranging Personal View, 1 Carroll JB Bates GP Steffan J Saft C Tabrizi SJ Treating the whole body in Huntington's disease. Lancet Neurol. 2015; 14: 1135-1142 Scopus (104) Google Scholar Jeffrey Carroll and colleagues discuss the growing literature on peripheral manifestations of Huntington's disease. As they point out, a substantial amount of evidence suggests extra-CNS abnormalities in Huntington's disease. In retrospect, these results are not surprising in view of the wide expression of mutant huntingtin in the brain and many other organs. 2 Strong TV Tagle DA Valdes JM et al. Widespread expression of the human and rat Huntington's disease gene in brain and non-neural tissues. Nat Genet. 1993; 5: 259-265 Crossref PubMed Scopus (324) Google Scholar Carroll and colleagues suggest plausibly that these abnormalities represent opportunities for useful clinical interventions. Modification of some peripheral manifestations, notably inflammatory responses, changes in kynurenine metabolism, or liver dysfunction, could modulate relevant CNS processes. They also suggest that targeting peripheral processes, even if such interventions have no CNS effects, might produce useful symptomatic benefits. Treating the whole body in Huntington's diseaseHuntington's disease is a genetic neurodegenerative disorder with symptoms that are linked to the progressive dysfunction and neuronal death in corticostriatal circuits. The causative gene (mutated HTT) is widely expressed outside the CNS and several peripheral signs of disease, including weight loss and increased proinflammatory signalling, are often seen; however, their importance in the pathophysiology of Huntington's disease is not clear. Studies in animals have shown that features of the disease involving the CNS, including synapse loss and behavioural alterations, are susceptible to modulation by treatments that target tissues and organs outside the CNS. Full-Text PDF

Mutant Huntingtin Downregulates Myelin Regulatory Factor-Mediated Myelin Gene Expression and Affects Mature Oligodendrocytes

Growing evidence indicates that non-neuronal mutant huntingtin toxicity plays an important role in Huntington's disease (HD); however, whether and how mutant huntingtin affects oligodendrocytes, which are vitally important for neural function and axonal integrity, remains unclear. We first verified the presence of mutant huntingtin in oligodendrocytes in HD140Q knockin mice. We then established transgenic mice (PLP-150Q) that selectively express mutant huntingtin in oligodendrocytes. PLP-150Q mice show progressive neurological symptoms and early death, as well as age-dependent demyelination and reduced expression of myelin genes that are downstream of myelin regulatory factor (MYRF or MRF), a transcriptional regulator that specifically activates and maintains the expression of myelin genes in mature oligodendrocytes. Consistently, mutant huntingtin binds abnormally to MYRF and affects its transcription activity. Our findings suggest that dysfunction of mature oligodendrocytes is involved in HD pathogenesis and may also make a good therapeutic target.

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.

C-Fos Protects Neurons Through a Noncanonical Mechanism Involving HDAC3 Interaction: Identification of a 21-Amino Acid Fragment with Neuroprotective Activity.

Proteins belonging to the AP-1 family of transcription factors are known to be involved in the regulation of neuronal viability. While strides have been made to elucidate the mechanisms of how individual members regulate cell death, much remains unknown. We find that the expression of one AP-1 member, c-Fos, is reduced in cerebellar granule neurons (CGNs) induced to die by low potassium (LK) treatment. Restoration and increase of this expression protect CGNs against LK-induced death, whereas knockdown induces death of otherwise healthy neurons. Furthermore, forced expression can protect cortical neurons against homocysteic acid (HCA)-induced toxicity. Taken together, this suggests that c-Fos is necessary for neuronal survival and that elevating c-Fos expression has a neuroprotective effect. Consistent with this idea is the finding that c-Fos expression is reduced selectively in the striatum in two separate mouse models of Huntington's disease and forced expression protects against neuronal death resulting from mutant huntingtin (mut-Htt) expression. Interestingly, neuroprotection by c-Fos does not require its DNA-binding, transcriptional, or heteromerization domains. However, this protective activity can be inhibited by pharmacological inhibition of c-Abl, CK-I, and MEK-ERK signaling. Additionally, expression of point mutant forms of this protein has identified that mutation of a tyrosine residue, Tyr345, can convert c-Fos from neuroprotective to neurotoxic. We show that c-Fos interacts with histone deacetylase-3 (HDAC3), a protein that contributes to mut-Htt neurotoxicity and whose overexpression is sufficient to promote neuronal death. When co-expressed, c-Fos can protect against HDAC3 neurotoxicity. Finally, our study identifies a 21-amino acid region at the C-terminus of c-Fos that is sufficient to protect neurons against death induced by LK, HCA treatment, or mut-Htt expression when expressed via a plasmid transfection or as a cell-permeable peptide. This cell-permeable peptide, designated as Fos-CTF, could have potential as a therapeutic agent for neurodegenerative diseases.

Artificial miRNAs Targeting Mutant Huntingtin Show Preferential Silencing In Vitro and In Vivo.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disease caused by CAG repeat expansion in exon 1 of huntingtin (HTT). Studies in mouse models of HD with a regulated mutant transgene show that continuous mutant allele expression is required for behavioral and pathological signs; when mutant HTT expression declined, neuronal degeneration improved. To date, it is unknown whether neural cells in the adult human brain can tolerate reduction in both normal and mutant alleles. Thus, it may be important to develop allele-specific silencing approaches. Several siRNA sequences targeting the CAG expanded motif or prevalent single-nucleotide polymorphisms (SNPs) in linkage disequilibrium with the mutant allele have been designed and their selectivity demonstrated in vitro. However, it is unknown whether these allele-specific siRNAs will retain their specificity when expressed from artificial RNAi platforms. Here, we designed CAG- and SNP- targeting artificial miRNAs and demonstrate that some, but not all, retained their selectivity in vitro using an allele-specific reporter system and in vivo in a transgenic mouse model developed to express normal and mutant human HTT alleles.

JAZ (Znf346), a SIRT1-interacting Protein, Protects Neurons by Stimulating p21 (WAF/CIP1) Protein Expression

SIRT1, a class III histone deacetylase, protects neurons in various models of neurodegenerative diseases. We previously described that neuroprotection by SIRT1 is independent of its catalytic activity. To elucidate how SIRT1 protects neurons, we performed a mass spectrometric screen to find SIRT1-interacting proteins. One of the proteins identified was JAZ (Znf346), a member of a new class of Cys-2-His-2 zinc finger proteins. To investigate the significance of JAZ in the regulation of neuronal survival, we overexpressed it in neurons. We found that JAZ protects cerebellar granule neurons against potassium deprivation-induced death and cortical neurons from death resulting from oxidative stress. JAZ also protects neurons against toxicity induced by mutant huntingtin and mutant ataxin-1 expression. Although expression of endogenous JAZ does not change in neurons primed to die, knockdown of its expression promotes death of otherwise healthy neurons. In contrast to its protective effect in neurons, overexpression of JAZ in different cell lines promotes death. We find that JAZ suppresses cell cycle progression, thereby explaining its contrasting effect in postmitotic neurons versus proliferating cell lines. Although not affecting the expression of several cyclins, overexpression of JAZ stimulates expression of p21 (WAF1/CIP1), a cell cycle inhibitor known to have neuroprotective effects. Results of chromatin immunoprecipitation and transcriptional assays indicate that the stimulatory effect of JAZ on p21 expression is mediated at the transcriptional level. Furthermore, knockdown of p21 expression inhibits the neuroprotective effect of JAZ. Together, our results suggest that JAZ protects neurons by inhibiting cell cycle re-entry through the transcriptional stimulation of p21 expression.

Inhibition of Excessive Monoamine Oxidase A/B Activity Protects Against Stress-induced Neuronal Death in Huntington Disease.

Monoamine oxidases (MAO) are important components of the homeostatic machinery that maintains the levels of monoamine neurotransmitters, including dopamine, in balance. Given the imbalance in dopamine levels observed in Huntington disease (HD), the aim of this study was to examine MAO activity in a mouse striatal cell model of HD and in human neural cells differentiated from control and HD patient-derived induced pluripotent stem cell (hiPSC) lines. We show that mouse striatal neural cells expressing mutant huntingtin (HTT) exhibit increased MAO expression and activity. We demonstrate using luciferase promoter assays that the increased MAO expression reflects enhanced epigenetic activation in striatal neural cells expressing mutant HTT. Using cellular stress paradigms, we further demonstrate that the increase in MAO activity in mutant striatal neural cells is accompanied by enhanced susceptibility to oxidative stress and impaired viability. Treatment of mutant striatal neural cells with MAO inhibitors ameliorated oxidative stress and improved cellular viability. Finally, we demonstrate that human HD neural cells exhibit increased MAO-A and MAO-B expression and activity. Altogether, this study demonstrates abnormal MAO expression and activity and suggests a potential use for MAO inhibitors in HD.Electronic supplementary materialThe online version of this article (doi:10.1007/s12035-014-8974-4) contains supplementary material, which is available to authorized users.

Hypothalamic alterations in Huntington's disease patients: comparison with genetic rodent models.

Unintended weight loss, sleep and circadian disturbances and autonomic dysfunction are prevalent features of Huntington's disease (HD), an autosomal dominantly inherited neurodegenerative disorder caused by an expanded CAG repeat sequence in the HTT gene. These features form a substantial contribution to disease burden in HD patients and appear to be accompanied by a number of neuroendocrine and metabolic changes, pointing towards hypothalamic pathology as a likely underlying mechanism. Neuronal inclusion bodies of mutant huntingtin, which are hallmarks of the disease, occur throughout the hypothalamus, and indicate local mutant huntingtin expression that could interfere with hypothalamic neuropeptide production. Also, several genetic rodent models of HD show features that could be related to hypothalamic pathology, such as weight loss and circadian rhythm disturbances. In these rodents, several hypothalamic neuropeptide populations are affected. In the present review, we summarise the changes in genetic rodent models of HD for individual hypothalamic nuclei, compare these observations to the hypothalamic changes that occur in HD patients, and make an inventory of the work that still needs to be done. Surprisingly, there is only limited overlap in the hypothalamic changes reported in HD patients and genetic rodent models. At present, the only similarity between the hypothalamic alterations in HD patients and genetic rodent models is a decrease in the number of orexin-expressing neurones in the lateral hypothalamus. Possible reasons for these discrepancies, as well as potential consequences for the development of novel therapeutic strategies, are discussed.