Calcium dysregulation and compensation in cortical pyramidal neurons of the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) is a fatal, hereditary neurodegenerative disorder that predominantly affects striatal medium-sized spiny neurons and cortical pyramidal neurons (CPNs). It has been proposed that perturbations in Ca2+ homeostasis could play a role in CPN alterations. To test this hypothesis, we used the R6/2 mouse model of juvenile HD at different stages of disease progression; presymptomatic, early symptomatic, and late symptomatic. We combined whole-cell patch-clamp recordings of layer 2/3 CPNs with two-photon laser scanning microscopy to image somatic and dendritic Ca2+ transients associated with evoked action potentials (APs). We found that the amplitude of AP-induced Ca2+ transients recorded at the somata of CPNs was significantly reduced in presymptomatic and late symptomatic R6/2 mice compared with wild-type (WT) littermates. However, reduced amplitudes were compensated by increases in decay times, so that Ca2+ transient areas were similar between genotypes. AP-induced Ca2+ transients in CPN proximal dendrites were variable and differences did not reach statistical significance, except for reduced areas in the late symptomatic group. In late symptomatic mice, a specific store-operated Ca2+ channel antagonist, EVP4593, reduced somatic Ca2+ transient amplitude similarly in WT and R6/2 CPNs. In contrast, dantrolene, a ryanodine receptor (RyR) antagonist, and nifedipine, an L-type Ca2+ channel blocker, significantly reduced both somatic Ca2+ transient amplitude and area in R6/2 but not WT CPNs. These findings demonstrate that perturbations of Ca2+ homeostasis and compensation occur in CPNs before and after the onset of overt symptoms, and suggest RyRs and L-type Ca2+ channels as potential targets for therapeutic intervention.NEW & NOTEWORTHY We used two-photon microscopy to examine calcium influx induced by action potentials in cortical pyramidal neurons from a mouse model of Huntington's disease (HD), the R6/2. The amplitude of somatic calcium transients was reduced in R6/2 mice compared with controls. This reduction was compensated by increased decay times, which could lead to reduced calcium buffering capacity. L-type calcium channel and ryanodine receptor blockers reduced calcium transient area in HD neurons, suggesting new therapeutic avenues.

Exosomes as a potential messenger unit during heterochronic parabiosis for amelioration of Huntington's disease.

BACKGROUND: Huntington's disease (HD) starts its pathology long before clinical manifestation, however, there is no therapy to cure it completely and only a few studies have been reported for delaying the progression of HD. Recently, it has been shown that heterochronic parabiosis can modulate the neurodegenerative diseases. Despite the importance of the transportation process of positive factors during heterochronic parabiosis, there were limited understandings because the transportation process is nanoscale, which makes it difficult to identify the messenger unit. We demonstrated that heterochronic parabiosis could modulate HD in R6/2 mice model, and identified the messenger unit for transferring positive factors in the young blood serum. METHODS: R6/2 mice were surgically connected with young wild-type mice (n = 13), old wild-type mice (n = 8), or R6/2 mice (n = 6) to examine the effect of heterochronic parabiosis. Parabionts composed of 5- to 6-week-old transgenic and wild-type mice were observed for 6 weeks in a single cage. The in vitro cellular model of HD cells were treated by the blood serum of the young or old mice, and by the exosomes isolated from thereof. The in vitro cellular model of HD were developed by differentiating neural stem cells cultured from SVZ of the brain. RESULTS: After the heterochronic parabiosis, the weight loss and survival of HD mice was improved. Also, mutant Huntingtin aggregation (EM48 p < 0.005), improvement of mitochondria dysfunction (PGC-1a p < 0.05, p-CREB/CREB p < 0.005), cell death (p53 p < 0.05, Bax p < 0.05, Cleaved-caspase3 p < 0.05), and cognition (DCX p < 0.5) showed a near complete restoration. In addition, treating in vitro cellular model of HD by the exosomes from young blood serum improved mutant Huntingtin aggregation (EM48 p < 0.05), mitochondria biogenesis (p-CREB/CREB p < 0.005), cell death (p53 p < 0.05, Bax p < 0.005, Cleaved-caspase3 p < 0.05, Bcl-2 p < 0.05), and cell proliferation (WST-1 p < 0.005). CONCLUSIONS: We found that the overall pathology of HD could be improved by the shared blood circulation through heterochronic parabiosis, furthermore, we demonstrated that the exosomes could be messengers for transferring positive factors, showing the potential of exosomes from young blood for the amelioration of HD.

Inhibition of GSK-3 ameliorates the pathogenesis of Huntington's disease.

In Huntington's disease (HD), the mutant huntingtin (mHtt) accumulates as toxic aggregates in the striatum tissue, with deleterious effects on motor-coordination and cognitive functions. Reducing the levels of mHtt is therefore a promising therapeutic strategy. We have previously reported that GSK-3 is a negative regulator of the autophagy/lysosome pathway, which is responsible for intracellular degradation, and is critically important for maintaining neuronal vitality. Thus, we hypothesized that inhibition of GSK-3 may trigger mHtt clearance thereby reducing mHtt cytotoxicity and improving HD symptoms. Here, we demonstrate that depletion or suppression of autophagy results in a massive accumulation of mHtt aggregates. Accordingly, mHtt aggregates were localized in lysosomes, but, mostly mislocalized from lysosomes in the absence of functional autophagy. Overexpression of GSK-3, particularly the α isozyme, increased the number of mHtt aggregates, while silencing GSK-3α/ß, or treatment with a selective GSK-3 inhibitor, L807mts, previously described by us, reduced the amounts of mHtt aggregates. This effect was mediated by increased autophagic and lysosomal activity. Treating R6/2 mouse model of HD with L807mts, reduced striatal mHtt aggregates and elevated autophagic and lysosomal markers. The L807mts treatment also reduced hyperglycemia and improved motor-coordination functions in these mice. In addition, L807mts restored the expression levels of Sirt1, a critical neuroprotective factor in the HD striatum, along with its targets BDNF, DRPP-32, and active Akt, all provide neurotrophic/pro-survival support and typically decline in the HD brain. Our results provide strong evidence for a role for GSK-3 in the regulation of mHtt dynamics, and demonstrate the benefits of GSK-3 inhibition in reducing mHtt toxicity, providing neuroprotective support, and improving HD symptoms.

Cortical Network Dynamics Is Altered in Mouse Models of Huntington's Disease.

Huntington's disease (HD) is a neurodegenerative disorder characterized by involuntary movements, cognitive deficits, and psychiatric disturbances. Although evidence indicates that projections from motor cortical areas play a key role in the development of dysfunctional striatal activity and motor phenotype, little is known about the changes in cortical microcircuits and their role in the development of the HD phenotype. Here we used two-photon laser-scanning microscopy to evaluate network dynamics of motor cortical neurons in layers II/III in behaving transgenic R6/2 and knock-in Q175+/- mice. Symptomatic R6/2 mice displayed increased motion manifested by a significantly greater number of motion epochs, whereas symptomatic Q175 mice displayed decreased motion. In both models, calcium transients in symptomatic mice displayed reduced amplitude, suggesting decreased bursting activity. Changes in frequency were genotype- and time-dependent; for R6/2 mice, the frequency was reduced during both motion and nonmotion, whereas in symptomatic Q175 mice, the reduction only occurred during nonmotion. In presymptomatic Q175 mice, frequency was increased during both behavioral states. Interneuronal correlation coefficients were generally decreased in both models, suggesting disrupted interneuronal communication in HD cerebral cortex. These results indicate similar and contrasting effects of the HD mutation on cortical ensemble activity depending on mouse model and disease stage.

Treatment with K6PC-5, a selective stimulator of SPHK1, ameliorates intestinal homeostasis in an animal model of Huntington's disease.

Emerging evidence indicates that Huntington's disease (HD) may be described as multi-organ pathology. In this context, we and others have contributed to demonstrate that the disease is characterized by an impairment of the homeostasis of gastro-intestinal (GI) tract. Sphingolipids represent a class of molecules involved in the regulation and maintenance of different tissues and organs including GI system. In this study, we investigated whether the alteration of Sphingosine-1-phosphate (S1P) metabolism, previously described in human HD brains and animal models, is also detectable peripherally in R6/2 HD mice. Our findings indicate, for the first time, that sphingolipid metabolism is perturbed early in the disease in the intestinal tract of HD mice and, its modulation by K6PC-5, a selective activator of S1P synthesis, preserved intestinal integrity and homeostasis. These results further support the evidence that modulation of sphingolipid pathways may represent a potential therapeutic option in HD and suggest that it has also the potential to counteract the peripheral disturbances which may usually complicate the management of the disease and affect patient's quality of life.

Effects of a Sativex-Like Combination of Phytocannabinoids on Disease Progression in R6/2 Mice, an Experimental Model of Huntington's Disease.

Several cannabinoids afforded neuroprotection in experimental models of Huntington's disease (HD). We investigated whether a 1:1 combination of botanical extracts enriched in either ∆8-tetrahydrocannabinol (∆8-THC) or cannabidiol (CBD), which are the main constituents of the cannabis-based medicine Sativex®, is beneficial in R6/2 mice (a transgenic model of HD), as it was previously shown to have positive effects in neurotoxin-based models of HD. We recorded the progression of neurological deficits and the extent of striatal deterioration, using behavioral, in vivo imaging, and biochemical methods in R6/2 mice and their corresponding wild-type mice. The mice were daily treated, starting at 4 weeks after birth, with a Sativex-like combination of phytocannabinoids (equivalent to 3 mg/kg weight of pure CBD + ∆8-THC) or vehicle. R6/2 mice exhibited the characteristic deterioration in rotarod performance that initiated at 6 weeks and progressed up to 10 weeks, and elevated clasping behavior reflecting dystonia. Treatment with the Sativex-like combination of phytocannabinoids did not recover rotarod performance, but markedly attenuated clasping behavior. The in vivo positron emission tomography (PET) analysis of R6/2 animals at 10 weeks revealed a reduced metabolic activity in the basal ganglia, which was partially attenuated by treatment with the Sativex-like combination of phytocannabinoids. Proton nuclear magnetic resonance spectroscopy (H⁺-MRS) analysis of the ex vivo striatum of R6/2 mice at 12 weeks revealed changes in various prognostic markers reflecting events typically found in HD patients and animal models, such as energy failure, mitochondrial dysfunction, and excitotoxicity. Some of these changes (taurine/creatine, taurine/N-acetylaspartate, and N-acetylaspartate/choline ratios) were completely reversed by treatment with the Sativex-like combination of phytocannabinoids. A Sativex-like combination of phytocannabinoids administered to R6/2 mice at the onset of motor symptoms produced certain benefits on the progression of striatal deterioration in these mice, which supports the interest of this cannabinoid-based medicine for the treatment of disease progression in HD patients.

Altered Aconitase 2 Activity in Huntington's Disease Peripheral Blood Cells and Mouse Model Striatum.

Huntington's disease (HD) is caused by an unstable cytosine adenine guanine (CAG) trinucleotide repeat expansion encoding a polyglutamine tract in the huntingtin protein. Previously, we identified several up- and down-regulated protein molecules in the striatum of the Hdh(CAG)150 knock-in mice at 16 months of age, a mouse model which is modeling the early human HD stage. Among those molecules, aconitase 2 (Aco2) located in the mitochondrial matrix is involved in the energy generation and susceptible to increased oxidative stress that would lead to inactivation of Aco2 activity. In this study, we demonstrate decreased Aco2 protein level and activity in the brain of both Hdh(CAG)150 and R6/2 mice. Aco2 activity was decreased in striatum of Hdh(CAG)150 mice at 16 months of age as well as R6/2 mice at 7 to 13 weeks of age. Aco2 activity in the striatum of R6/2 mice could be restored by the anti-oxidant, N-acetyl-l-cysteine, supporting that decreased Aco2 activity in HD is probably caused by increased oxidative damage. Decreased Aco2 activity was further found in the peripheral blood mononuclear cells (PBMC) of both HD patients and pre-symptomatic HD mutation (PreHD) carriers, while the decreased Aco2 protein level of PBMC was only present in HD patients. Aco2 activity correlated significantly with motor score, independence scale, and functional capacity of the Unified Huntington's Disease Rating Scale as well as disease duration. Our study provides a potential biomarker to assess the disease status of HD patients and PreHD carriers.

Forkhead transcription factor FOXO3a levels are increased in Huntington disease because of overactivated positive autofeedback loop.

Huntington disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by an increased number of CAG repeats in the HTT gene coding for huntingtin. Decreased neurotrophic support and increased mitochondrial and excitotoxic stress have been reported in HD striatal and cortical neurons. The members of the class O forkhead (FOXO) transcription factor family, including FOXO3a, act as sensor proteins that are activated upon decreased survival signals and/or increased cellular stress. Using immunocytochemical screening in mouse striatal Hdh(7/7) (wild type), Hdh(7/109) (heterozygous for HD mutation), and Hdh(109/109) (homozygous for HD mutation) cells, we identified FOXO3a as a differentially regulated transcription factor in HD. We report increased nuclear FOXO3a levels in mutant Hdh cells. Additionally, we show that treatment with mitochondrial toxin 3-nitropropionic acid results in enhanced nuclear localization of FOXO3a in wild type Hdh(7/7) cells and in rat primary cortical neurons. Furthermore, mRNA levels of Foxo3a are increased in mutant Hdh cells compared with wild type cells and in 3-nitropropionic acid-treated primary neurons compared with untreated neurons. A similar increase was observed in the cortex of R6/2 mice and HD patient post-mortem caudate tissue compared with controls. Using chromatin immunoprecipitation and reporter assays, we demonstrate that FOXO3a regulates its own transcription by binding to the conserved response element in Foxo3a promoter. Altogether, the findings of this study suggest that FOXO3a levels are increased in HD cells as a result of overactive positive feedback loop.

Age-, tissue- and length-dependent bidirectional somatic CAG•CTG repeat instability in an allelic series of R6/2 Huntington disease mice.

The expansion of simple sequence CAG•CTG repeats is associated with a number of inherited disorders including Huntington disease (HD), myotonic dystrophy type 1 and several of the spinocerebellar ataxias. Inherited disease-associated alleles usually exceed 40 repeats and may be in excess of 1,000 repeats in some disorders. Inherited allele length is inversely proportional to age at onset, and frequent germline expansions account for the striking anticipation observed in affected families. Expanded disease associated alleles are also somatically unstable via a pathway that is age dependent and tissue specific, and also appears to be expansion biased. Somatic expansions are thought to contribute toward both tissue specificity and disease progression. Here we have examined the somatic mutational dynamics in brain and peripheral tissues from an allelic series of R6/2 HD transgenic mice inheriting from 52 to >700 CAG repeats. We found age-dependent, tissue-specific somatic instability, with particularly large expansions observed in the striatum and cortex. We also found a positive increase in somatic instability with increasing allele length. Surprisingly, however, the degree of somatic variation did not increase in a linear fashion, but leveled off with increasing allele length. Most unexpectedly, the almost exclusive bias toward the accumulation of expansions observed in mice inheriting smaller alleles was lost, and a high frequency of large somatic contractions was observed in mice inheriting very large alleles (>500 repeats). These data highlight the bidirectional nature of CAG•CTG repeat instability and the subtle balance that exists between expansion and contraction in vivo. Defining the dynamics and tissue specificity of expansion and contraction is important for understanding the role of genetic instability in pathophysiology and in particular the development of novel therapies based on suppressing expansions and/or promoting contractions.

Inactivation of adenosine A2A receptors reverses working memory deficits at early stages of Huntington's disease models.

Cognitive impairments in Huntington's disease (HD) are attributed to a dysfunction of the cortico-striatal pathway and significantly affect the quality of life of the patients, but this has not been a therapeutic focus in HD to date. We postulated that adenosine A(2A) receptors (A(2A)R), located at pre- and post-synaptic elements of the cortico-striatal pathways, modulate striatal neurotransmission and synaptic plasticity and cognitive behaviors. To critically evaluate the ability of A(2A)R inactivation to prevent cognitive deficits in early HD, we cross-bred A(2A)R knockout (KO) mice with two R6/2 transgenic lines of HD (CAG120 and CAG240) to generate two double transgenic R6/2-CAG120-A(2A)R KO and R6/2-CAG240-A(2A)R KO mice and their corresponding wild-type (WT) littermates. Genetic inactivation of A(2A)R prevented working memory deficits induced by R6/2-CAG120 at post-natal week 6 and by R6/2-CAG240 at post-natal month 2 and post-natal month 3, without modifying motor deficits. Similarly the A2(A)R antagonist KW6002 selectively reverted working memory deficits in R6/2-CAG240 mice at post-natal month 3. The search for possible mechanisms indicated that the genetic inactivation of A(2A)R did not affect ubiquitin-positive neuronal inclusions, astrogliosis or Thr-75 phosphorylation of DARPP-32 in the striatum. Importantly, A(2A)R blockade preferentially controlled long-term depression at cortico-striatal synapses in R6/2-CAG240 at post-natal week 6. The reported reversal of working memory deficits in R6/2 mice by the genetic and pharmacological inactivation of A(2A)R provides a proof-of-principle for A(2A)R as novel targets to reverse cognitive deficits in HD, likely by controlling LTD deregulation.

Genetic rescue of CB1 receptors on medium spiny neurons prevents loss of excitatory striatal synapses but not motor impairment in HD mice.

Huntington's disease (HD) is caused by an expanded polyglutamine repeat in huntingtin protein that disrupts synaptic function in specific neuronal populations and results in characteristic motor, cognitive and affective deficits. Histopathological hallmarks observed in both HD patients and genetic mouse models include the reduced expression of synaptic proteins, reduced medium spiny neuron (MSN) dendritic spine density and decreased frequency of spontaneous excitatory post-synaptic currents (sEPSCs). Early down-regulation of cannabinoid CB1 receptor expression on MSN (CB1(MSN)) is thought to participate in HD pathogenesis. Here we present a cell-specific genetic rescue of CB1(MSN) in R6/2 mice and report that treatment prevents the reduction of excitatory synaptic markers in the striatum (synaptophysin, vGLUT1 and vGLUT2), of dendritic spine density on MSNs and of MSN sEPSCs, but does not prevent motor impairment. We conclude that loss of excitatory striatal synapses in HD mice is controlled by CB1(MSN) and can be uncoupled from the motor phenotype.

Expression, pharmacology and functional activity of adenosine A1 receptors in genetic models of Huntington's disease.

Adenosine A1 receptor (A1R) stimulation exerts beneficial effects in response to various insults to the brain and, although it was found neuroprotective in a lesional model of Huntington's disease (HD), the features of this receptor in genetic models of HD have never been explored. In the present study we characterized the expression, affinity and functional effects of A1Rs in R6/2 mice (the most widely used transgenic model of HD) and in a cellular model of HD. Binding studies revealed that the density of A1Rs was significantly reduced in the cortex and the striatum of R6/2 mice compared to age-matched wild-type (WT), while receptor affinity was unchanged. The selective A1R agonist cyclopentyladenosine (CPA, 300nM) was significantly more effective in reducing synaptic transmission in corticostriatal slices from symptomatic R6/2 than in age-matched WT mice. Such an effect was due to a stronger inhibition of glutamate release from the pre-synaptic terminal. The different functional activities of A1Rs in HD mice were associated also to a different intracellular signaling pathway involved in the synaptic effect of CPA. In fact, while the PKA pathway was involved in both genotypes, p38 MAPK inhibitor SB203580 partially prevented synaptic effects of CPA in R6/2, but not in WT, mice; moreover, CPA differently modulated the phosphorylation status of p38 in the two genotypes. In vitro studies confirmed a different behavior of A1Rs in HD: CPA (100 nM for 5h) modulated cell viability in STHdh(Q111/Q111) (mhttHD cells), without affecting the viability of STHdh(Q7/Q7) (wthtt cells). This effect was prevented by the application of SB203580. Our results demonstrate that in the presence of the HD mutation A1Rs undergo profound changes in terms of expression, pharmacology and functional activity. These changes have to be taken in due account when considering A1Rs as a potential therapeutic target for this disease.

Progressive sleep and electroencephalogram changes in mice carrying the Huntington's disease mutation.

Sleep disturbances in Huntington's disease may be deleterious to the cognitive performance, affective behaviour, and general well-being of patients, but a comprehensive description of the progression of changes in sleep and electroencephalogram in Huntington's disease has never been conducted. Here we studied sleep and electroencephalogram disturbances in a transgenic mouse model of Huntington's disease (R6/2 mice). We implanted 10 R6/2 mice and five wild-type littermates with electromyography electrodes, frontofrontal and frontoparietal electroencephalogram electrodes and then recorded sleep/wake behaviour at presymptomatic, symptomatic and late stages of the disease. In addition to sleep-wake scoring, we performed a spectral analysis of the sleep electroencephalogram. We found that sleep and electroencephalogram were already significantly disrupted in R6/2 mice at 9 weeks of age (presymptomatic stage). By the time they were symptomatic, R6/2 mice were unable to maintain long periods of wakefulness and had an increased propensity for rapid eye movement sleep. In addition, the peak frequency of theta rhythm was shifted progressively from 7 Hz to 6 Hz during rapid eye movement sleep, whereas slow wave activity decreased gradually during non-rapid eye movement sleep. Finally, as the disease progressed, an abnormal electroencephalogram gamma activity (30-40 Hz) emerged in R6/2 mice irrespective of sleep states. This is reminiscent of the increased gamma power described in schizophrenic patients during sleep and events of psychosis. Gaining a better understanding of sleep and electroencephalogram changes in patients with Huntington's disease should be a priority, since it will enable clinicians to initiate appropriate investigations and to instigate treatments that could dramatically improve patients' quality of life.

Intravenous MSC-Treatment Improves Impaired Brain Functions in the R6/2 Mouse Model of Huntington's Disease via Recovered Hepatic Pathological Changes.

Huntington's disease (HD), a congenital neurodegenerative disorder, extends its pathological damages beyond the nervous system. The systematic manifestation of HD has been extensively described in numerous studies, including dysfunction in peripheral organs and peripheral inflammation. Gut dysbiosis and the gut-liver-brain axis have garnered greater emphasis in neurodegenerative research, and increased plasma levels of pro-inflammatory cytokines have been identified in HD patients and various in vivo models, correlating with disease progression. In the present study, we investigated hepatic pathological markers in the liver of R6/2 mice which convey exon 1 of the human mutant huntingtin gene. Furthermore, we evaluated the impact of intravenously administered Mesenchymal Stromal Cells (MSCs) on the liver enzymes, changes in hepatic inflammatory markers, as well as brain pathology and behavioral deficits in R6/2 mice. Our results revealed altered enzyme expression and increased levels of inflammatory mediators in the liver of R6/2 mice, which were significantly attenuated in the MSC-treated R6/2 mice. Remarkably, neuronal pathology and altered motor activities in the MSC-treated R6/2 mice were significantly ameliorated, despite the absence of MSCs in the postmortem brain. Our data highlight the importance of hepatic pathological changes in HD, providing a potential therapeutic approach. Moreover, the data open new perspectives for the search in blood biomarkers correlating with liver pathology in HD.

BDNF prevents NMDA-induced toxicity in models of Huntington's disease: the effects are genotype specific and adenosine A2A receptor is involved.

NMDA receptor-mediated excitotoxicity is thought to play a pivotal role in the pathogenesis of Huntington's disease (HD). The neurotrophin brain-derived neurotrophic factor (BDNF), which is also highly involved in HD and whose effects are modulated by adenosine A2 ARs, influences the activity and expression of striatal NMDA receptors. In electrophysiology experiments, we investigated the role of BDNF toward NMDA-induced effects in HD models, and the possible involvement of A2ARs. In corticostriatal slices from wild-type mice and age-matched symptomatic R6/2 mice (a model of HD), NMDA application (75 µM) induced a transient or a permanent (i.e., toxic) reduction of field potential amplitude, respectively. BDNF (10 ng/mL) potentiated NMDA effects in wild-type, while it protected from NMDA toxicity in R6/2 mice. Both effects of BDNF were prevented by A2 AR blockade. The protective effect of BDNF against NMDA-induced toxicity was reproduced in a cellular model of HD. These findings may have very important implications for the neuroprotective potential of BDNF and A2 AR ligands in HD.

The Urine Metabolome of R6/2 and zQ175DN Huntington's Disease Mouse Models.

Huntington's disease (HD) is caused by the expansion of a polyglutamine (polyQ)-encoding tract in exon 1 of the huntingtin gene to greater than 35 CAG repeats. It typically has a disease course lasting 15-20 years, and there are currently no disease-modifying therapies available. Thus, there is a need for faithful mouse models of HD to use in preclinical studies of disease mechanisms, target validation, and therapeutic compound testing. A large variety of mouse models of HD were generated, none of which fully recapitulate human disease, complicating the selection of appropriate models for preclinical studies. Here, we present the urinary liquid chromatography-high-resolution mass spectrometry analysis employed to identify metabolic alterations in transgenic R6/2 and zQ175DN knock-in mice. In R6/2 mice, the perturbation of the corticosterone metabolism and the accumulation of pyrraline, indicative of the development of insulin resistance and the impairment of pheromone excretion, were observed. Differently from R6/2, zQ175DN mice showed the accumulation of oxidative stress metabolites. Both genotypes showed alterations in the tryptophan metabolism. This approach aims to improve our understanding of the molecular mechanisms involved in HD neuropathology, facilitating the selection of appropriate mouse models for preclinical studies. It also aims to identify potential biomarkers specific to HD.

Evidences for Mutant Huntingtin Inducing Musculoskeletal and Brain Growth Impairments via Disturbing Testosterone Biosynthesis in Male Huntington Disease Animals.

Body weight (BW) loss and reduced body mass index (BMI) are the most common peripheral alterations in Huntington disease (HD) and have been found in HD mutation carriers and HD animal models before the manifestation of neurological symptoms. This suggests that, at least in the early disease stage, these changes could be due to abnormal tissue growth rather than tissue atrophy. Moreover, BW and BMI are reported to be more affected in males than females in HD animal models and patients. Here, we confirmed sex-dependent growth alterations in the BACHD rat model for HD and investigated the associated contributing factors. Our results showed growth abnormalities along with decreased plasma testosterone and insulin-like growth factor 1 (IGF-1) levels only in males. Moreover, we demonstrated correlations between growth parameters, IGF-1, and testosterone. Our analyses further revealed an aberrant transcription of testosterone biosynthesis-related genes in the testes of BACHD rats with undisturbed luteinizing hormone (LH)/cAMP/PKA signaling, which plays a key role in regulating the transcription process of some of these genes. In line with the findings in BACHD rats, analyses in the R6/2 mouse model of HD showed similar results. Our findings support the view that mutant huntingtin may induce abnormal growth in males via the dysregulation of gene transcription in the testis, which in turn can affect testosterone biosynthesis.

Exposure of R6/2 mice in an enriched environment augments P42 therapy efficacy on Huntington's disease progression.

Huntington's disease (HD) is due to a mutation in the gene encoding for Huntingtin protein generating polyQ domain extension. Mutant Htt (mHtt) leads to important dysfunction of the BDNF/TrkB signaling. We previously described the 23aa Htt fragment P42, that attenuated the pathological phenotypes induced by mHtt. We reported that, in the R6/2 mouse model of HD, P42 rescued striatal TrkB level but marginally increased cortical BDNF. In the present study, our aim was to address P42 neuroprotection in presence of an external input of BDNF. We combined P42 administration with environmental enrichment (EE), induced by training in the Hamlet test. We examined the consequences of P42 + EE combination on different phenotypes in R6/2 HD mice: motor and cognitive performances, recorded at early and late pathological stages, and analyzed aggregated mHtt and BDNF levels in forebrain structures. Hamlet exploration (i.e., entries in Run, Hide, Eat, Drink and Interact houses) was gradually impaired in R6/2 mice, but maintained by P42 treatment until week 8. Topographic memory alteration measured at week 7 was attenuated by P42. Motor performances (rotarod) were significantly ameliorated by the P42 + EE combination until late stage (week 12). The P42 + EE combination also significantly decreased aggregated Htt levels in the hippocampus, striatum and cortex, and increased BDNF levels in the cortex and striatum. We concluded that combination between P42 treatment, known to increase TrkB striatal expression, and a BDNF-enhancing therapy such as EE efficiently delayed HD pathology in R6/2 mice. Use of dual therapies might be a pertinent strategy to fight neurodegeneration in HD.

Intranasal Administration of Mesenchymal Stem Cells Ameliorates the Abnormal Dopamine Transmission System and Inflammatory Reaction in the R6/2 Mouse Model of Huntington Disease.

Intrastriatal administration of mesenchymal stem cells (MSCs) has shown beneficial effects in rodent models of Huntington disease (HD). However, the invasive nature of surgical procedure and its potential to trigger the host immune response may limit its clinical use. Hence, we sought to evaluate the non-invasive intranasal administration (INA) of MSC delivery as an effective alternative route in HD. GFP-expressing MSCs derived from bone marrow were intranasally administered to 4-week-old R6/2 HD transgenic mice. MSCs were detected in the olfactory bulb, midbrain and striatum five days post-delivery. Compared to phosphate-buffered saline (PBS)-treated littermates, MSC-treated R6/2 mice showed an increased survival rate and attenuated circadian activity disruption assessed by locomotor activity. MSCs increased the protein expression of DARPP-32 and tyrosine hydroxylase (TH) and downregulated gene expression of inflammatory modulators in the brain 7.5 weeks after INA. While vehicle treated R6/2 mice displayed decreased Iba1 expression and altered microglial morphology in comparison to the wild type littermates, MSCs restored both, Iba1 level and the thickness of microglial processes in the striatum of R6/2 mice. Our results demonstrate significantly ameliorated phenotypes of R6/2 mice after MSCs administration via INA, suggesting this method as an effective delivering route of cells to the brain for HD therapy.

Supplemental Treatment for Huntington's Disease with miR-132 that Is Deficient in Huntington's Disease Brain.

Huntington's disease (HD) is an intractable neurodegenerative disorder caused by mutant Huntingtin (HTT) proteins that adversely affect various biomolecules and genes. MicroRNAs (miRNAs), which are functional small non-coding RNAs, are also affected by mutant HTT proteins. Here, we show amelioration in motor function and lifespan of HD-model mice, R6/2 mice, by supplying miR-132 to HD brains using a recombinant adeno-associated virus (rAAV) miRNA expression system. miR-132 is an miRNA related to neuronal maturation and function, but the level of miR-132 in the brain of R6/2 mice was significantly lower than that of wild-type mice. Our miR-132 supplemental treatment, i.e., supplying miR-132 to the brain, produced symptomatic improvement or retarded disease progression in R6/2 mice; interestingly, it had little effect on disease-causing mutant HTT mRNA expression and its products. Therefore, the findings suggest that there may be a therapeutic way to treat HD without inhibiting and/or repairing disease-causing HTT genes and gene products. Although miR-132 supplement may not be a definitive treatment for HD, it may become a therapeutic method for relieving HD symptoms and delaying HD progression.