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Effects of CypD on the motor performance of R6/2 mice. ( A ) Times before fall from rotarod at accelerating speeds (4 –40 rpm) of R6/2:CypD + / + , ( n 1⁄4 16), R6/2:CypD 2 / 2 ( n 1⁄4 22) and R6/2:CypD + / 2 ( n 1⁄4 19) mice were monitored at 60, 90 and 120 days of age. Data are presented as mean + SEM. ( B ) Motor performance of R6/2:CypD + / + ( n 1⁄4 16), R6/2:CypD 2 / 2 ( n 1⁄4 20) and R6/2:CypD + / 2 ( n 1⁄4 19) mice using a constant speed rotarod protocol (10 rpm). At different time points examined, the CypD genotype Has no significant effect on both accelerated and fixed-speed rotarod performances of R6/2 mice. Data are presented as mean + SEM.
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Huntington's disease (HD) is an incurable autosomal-dominant neurodegenerative disorder initiated by an abnormally expanded polyglutamine domain in the huntingtin protein. It is proposed that abnormal mitochondrial Ca2+ capacity results in an increased susceptibility to mitochondrial permeability transition (MPT) induction that may contribute signi...
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... in the mitochondria suspension from R6/2:CypD2/2 mice. R6/2:CypD+/+, R6/2:CypD2/2 and R6/2:CypD+/2 mice. All genotype groups were able to remain on the rod for signifi- cantly longer times with successive trials [R6/2:CypD+/+: F 9,150 ¼ 3.65, P ¼ 0.0004; R6/2:CypD2/2: F 9,190 ¼ 5.74, P , 0.0001; R6/2:CypD+/2: F 9,180 ¼ 3.73, P ¼ 0.0003] ( Fig. 5A). At 90 and 120 days of age, however, no significant effect of trial numbers was detected as mice with different genotype groups did not improve their rotarod performances with successive trials ( Fig. 5B and C (Fig. 5A and C). At all time points examined, there was no significant effect of CypD genotype on declining accel- erating ...
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... trials [R6/2:CypD+/+: F 9,150 ¼ 3.65, P ¼ 0.0004; R6/2:CypD2/2: F 9,190 ¼ 5.74, P , 0.0001; R6/2:CypD+/2: F 9,180 ¼ 3.73, P ¼ 0.0003] ( Fig. 5A). At 90 and 120 days of age, however, no significant effect of trial numbers was detected as mice with different genotype groups did not improve their rotarod performances with successive trials ( Fig. 5B and C (Fig. 5A and C). At all time points examined, there was no significant effect of CypD genotype on declining accel- erating rotarod performance of R6/2 mice ( Fig. 5A and C). We further included mouse gender as a potential variable in our analysis and analyzed male and female separately. The results of this analysis are presented in ...
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... [R6/2:CypD+/+: F 9,150 ¼ 3.65, P ¼ 0.0004; R6/2:CypD2/2: F 9,190 ¼ 5.74, P , 0.0001; R6/2:CypD+/2: F 9,180 ¼ 3.73, P ¼ 0.0003] ( Fig. 5A). At 90 and 120 days of age, however, no significant effect of trial numbers was detected as mice with different genotype groups did not improve their rotarod performances with successive trials ( Fig. 5B and C (Fig. 5A and C). At all time points examined, there was no significant effect of CypD genotype on declining accel- erating rotarod performance of R6/2 mice ( Fig. 5A and C). We further included mouse gender as a potential variable in our analysis and analyzed male and female separately. The results of this analysis are presented in Supplementary ...
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... of age, however, no significant effect of trial numbers was detected as mice with different genotype groups did not improve their rotarod performances with successive trials ( Fig. 5B and C (Fig. 5A and C). At all time points examined, there was no significant effect of CypD genotype on declining accel- erating rotarod performance of R6/2 mice ( Fig. 5A and C). We further included mouse gender as a potential variable in our analysis and analyzed male and female separately. The results of this analysis are presented in Supplementary Material (Fig. S3) and revealed that, except for one trial at 120 days in the R6/2:CypD+/+ group, there was no significant effect of gender on the accelerated ...
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... 24 h period (dark and light), or over 12 h dark and 12 h light periods. Cage activities were examined at 60 and 90 days of age. The rationale for selecting these time points was based on the outcomes of the rotarod performances. At 60 days of age, R6/2 mice of all the different CypD genotypes performed relatively well on both accelerating rotarod (Fig. 5A) and fixed speed rotarod (Fig. 5D), but presented balance and motor coordination impairments at 90 days of age ( Fig. 5B and D). We were not able to measure activity at later time points because chow mixed with water had to be placed in the bottom of cages, which was incompatible with this apparatus. At 60 days of age, there was no ...
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... 12 h dark and 12 h light periods. Cage activities were examined at 60 and 90 days of age. The rationale for selecting these time points was based on the outcomes of the rotarod performances. At 60 days of age, R6/2 mice of all the different CypD genotypes performed relatively well on both accelerating rotarod (Fig. 5A) and fixed speed rotarod (Fig. 5D), but presented balance and motor coordination impairments at 90 days of age ( Fig. 5B and D). We were not able to measure activity at later time points because chow mixed with water had to be placed in the bottom of cages, which was incompatible with this apparatus. At 60 days of age, there was no effect of CypD genotype on the ...
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... age. The rationale for selecting these time points was based on the outcomes of the rotarod performances. At 60 days of age, R6/2 mice of all the different CypD genotypes performed relatively well on both accelerating rotarod (Fig. 5A) and fixed speed rotarod (Fig. 5D), but presented balance and motor coordination impairments at 90 days of age ( Fig. 5B and D). We were not able to measure activity at later time points because chow mixed with water had to be placed in the bottom of cages, which was incompatible with this apparatus. At 60 days of age, there was no effect of CypD genotype on the locomotor and rhythm activities of R6/2 mice (data not shown). At 90 days of age, a total of ...
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Huntington's disease (HD) is caused by a polyglutamine expansion in the Huntingtin (Htt) protein. Proteolytic cleavage of Htt into toxic N-terminal fragments is believed to be a key aspect of pathogenesis. The best characterized putative cleavage event is at amino acid 586, hypothesized to be mediated by caspase 6. A corollary of the caspase 6 clea...
Background:
Huntington's disease (HD) is caused by a tandem repeat expansion and involves progressive cognitive decline, psychiatric abnormalities and motor deficits. Disease onset and progression in HD mice can be substantially delayed by a housing environment with enhanced sensorimotor and cognitive stimulation. However, the proposed benefits of...
Citations
... We used a Ca 2+ -sensitive electrode to evaluate Ca 2+ uptake by isolated Percoll-purified brain mitochondria. Ca 2+ retention capacity (also known as Ca 2+ uptake capacity (35)(36)(37)(38)) was assessed by applying multiple pulses of 10µM Ca 2+ delivered as CaCl 2 (Fig. 2). Mitochondrial Ca 2+ uptake was monitored by following a decrease in Ca 2+ concentration in the incubation medium outside of mitochondria. ...
The mitochondrial calcium uniporter (MCU) and cyclophilin D (CyD) are key players in induction of the permeability transition pore (PTP), which leads to mitochondrial depolarization and swelling, the major sings of Ca²⁺-induced mitochondrial damage. Mitochondrial depolarization inhibits ATP production, whereas swelling results in the release of mitochondrial pro-apoptotic proteins. The extent to which simultaneous deletion of MCU and CyD inhibits PTP induction and prevents damage of brain mitochondria is not clear. Here, we investigated the effects of MCU and CyD deletion on the propensity for PTP induction using mitochondria isolated from the brains of MCU-KO, CyD-KO, and newly created MCU/CyD-double knockout (DKO) mice. Neither deletion of MCU nor of CyD affected respiration or membrane potential in mitochondria isolated from the brains of these mice. Mitochondria from MCU-KO and MCU/CyD-DKO mice displayed reduced Ca²⁺ uptake and diminished extent of PTP induction. The Ca²⁺ uptake by mitochondria from CyD-KO mice was increased compared to mitochondria from wild-type mice. Deletion of CyD prevented mitochondrial swelling and resulted in transient depolarization in response to Ca²⁺, but it did not prevent Ca²⁺-induced delayed mitochondrial depolarization. Mitochondria from MCU/CyD-DKO mice did not swell in response to Ca²⁺, but they did exhibit mild sustained depolarization. Dibucaine, an inhibitor of the Ca²⁺-activated mitochondrial phospholipase A2, attenuated, and bovine serum albumin completely eliminated the sustained depolarization. This suggests the involvement of phospholipase A2 and free fatty acids. Thus, in addition to induction of the classical PTP, alternative deleterious mechanisms may contribute to mitochondrial damage following exposure to elevated Ca²⁺.
... These studies attribute the aberrant increase in Ca i 2+ to the enhanced mitochondrial sensitivity to Ca 2+ loads. However, the recovery of the mitochondrial Ca 2+ capacity and the inhibition of mitochondrial permeability transition pore (PTP) induction failed to ameliorate either the behavioral or the neuropathological features of the disease (Perry et al., 2010). Other studies have reported an age-dependent increase in the Ca 2+ loading capacity of striatal mitochondria (Brustovetsky et al., 2005) or even no changes (Oliveira et al., 2007). ...
Mitochondria-associated membranes (MAMs) are dynamic structures that communicate endoplasmic reticulum (ER) and mitochondria allowing calcium transfer between these two organelles. Since calcium dysregulation is an important hallmark of several neurodegenerative diseases, disruption of MAMs has been speculated to contribute to pathological features associated with these neurodegenerative processes. In Huntington's disease (HD), mutant huntingtin induces the selective loss of medium spiny neurons within the striatum. The cause of this specific susceptibility remain unclear. However, defects on mitochondrial dynamics and bioenergetics have been proposed as critical contributors, causing accumulation of fragmented mitochondria and subsequent Ca²⁺ homeostasis alterations. In the present work, we show that aberrant Drp1-mediated mitochondrial fragmentation within the striatum of HD mutant mice, forces mitochondria to place far away from the ER disrupting the ER-mitochondria association and therefore causing drawbacks in Ca²⁺ efflux and an excessive production of mitochondria superoxide species. Accordingly, inhibition of Drp1 activity by Mdivi-1 treatment restored ER-mitochondria contacts, mitochondria dysfunction and Ca²⁺ homeostasis.
In sum, our results give new insight on how defects on mitochondria dynamics may contribute to striatal vulnerability in HD and highlights MAMs dysfunction as an important factor involved in HD striatal pathology.
... This idea was reinforced by the fact that CypD is upregulated in Huntington's patients and that this upregulation increased as Huntington's disease progressed ( Shirendeb et al., 2011). However, other reports did not find such significant contribution of mPTP to mitochondrial injury in Huntington's disease, demonstrating that genetic inactivation of CypD does not modify the onset and the progression of the disease in mice Pellman, Hamilton, Brustovetsky, & Brustovetsky, 2015;Perry et al., 2010). ...
Les phénomènes d’ischémie-reperfusion sont rencontrés dans plusieurs situations physiopathologiques. Le seul traitement de l’ischémie repose sur une restauration précoce du flux sanguin. Paradoxalement, la reperfusion génère des lésions supplémentaires, appelées « lésions de reperfusion », dont la mitochondrie est un acteur majeur via l’ouverture du pore de transition de perméabilité mitochondrial (mPTP). L’ouverture du mPTP est principalement modulée par la cyclophiline D (CypD), une protéine de la matrice mitochondriale, dont l’inhibition pharmacologique par la cyclosporine A (CsA) permet de limiter l’ouverture du pore. Cette inhibition, obtenue in vitro et in vivo, permet de réduire les lésions de reperfusion. Néanmoins, de récents essais cliniques n’ont pas permis de confirmer ce bénéfice dans le cadre de l’infarctus du myocarde, soulignant la nécessité de développer de nouveaux inhibiteurs du mPTP. Dans ce travail, nous avons étudié l’effet de nouveaux ligands de la CypD sur l’ouverture du mPTP. Ces petites molécules innovantes, de structure radicalement différente de la CsA inhibent l’ouverture du mPTP de mitochondries isolées et le dérivé le plus actif, le C31, permet une inhibition plus efficace du mPTP que la CsA. Le C31 inhibe également le mPTP au niveau cellulaire, dans des hépatocytes primaires et dans des cardiomyocytes isolés. In vivo, le C31 atteint les mitochondries hépatiques et protège le foie dans un modèle d’ischémie-reperfusion hépatique. Cependant, la stabilité métabolique du C31 ne lui permet pas d’atteindre le cœur. La poursuite du développement de ces inhibiteurs pourrait aboutir à de nouveaux candidats pour protéger les organes des lésions de reperfusion.
... They do not reproduce some major features of HD pathology, such as serious neuronal degeneration in the striatum (Crook and Housman, 2011;Pouladi et al., 2013;Menalled and Brunner, 2014). R6/2 mice are well known to have limited lifespan (Perry et al., 2010). Nonetheless, these models have been widely used to study HD pathogenesis and for therapeutic trials (Crook and Housman, 2011;Pouladi et al., 2013;Menalled and Brunner, 2014). ...
Huntington’s disease (HD) is an autosomal-dominant inherited neurodegenerative disorder characterized by motor, psychiatric and cognitive symptoms. HD is caused by an expansion of CAG repeats in the huntingtin (HTT) gene in various areas of the brain including striatum. There are few suitable animal models to study the pathogenesis of HD and validate therapeutic strategies. Recombinant adeno-associated viral (AAV) vectors successfully transfer foreign genes to the brain of adult mammalians. In this article, we report a novel mouse model of HD generated by bilateral intrastriatal injection of AAV vector serotype DJ (AAV-DJ) containing N171-82Q mutant HTT (82Q) and N171-18Q wild type HTT (18Q; sham). The AAV-DJ-82Q model displayed motor dysfunctions in pole and rotarod tests beginning 4 weeks after viral infection in juvenile mice (8 weeks after birth). They showed behaviors reflecting neurodegeneration. They also showed increased apoptosis, robust glial activation and upregulated representative inflammatory cytokines (tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6), mediators (cyclooxygenase-2 and inducible nitric oxide synthase) and signaling pathways (nuclear factor kappa B and signal transducer and activator of transcription 3 (STAT3)) in the striatum at 10 weeks after viral infection (14 weeks after birth) via successful transfection of mutant HTT into neurons, microglia, and astrocytes in the striatum. However, little evidence of any of these events was found in mice infected with the AAV-DJ-18Q expressing construct. Intrastriatal injection of AAV-DJ-82Q might be useful as a novel in vivo model to investigate the biology of truncated N-terminal fragment (N171) in the striatum and to explore the efficacy of therapeutic strategies for HD.
... This idea was reinforced by the fact that CypD is upregulated in Huntington's patients and that this upregulation increased as Huntington's disease progressed (Shirendeb et al., 2011). However, other reports did not find such significant contribution of mPTP to mitochondrial injury in Huntington's disease, demonstrating that genetic inactivation of CypD does not modify the onset and the progression of the disease in mice Pellman, Hamilton, Brustovetsky, & Brustovetsky, 2015;Perry et al., 2010). ...
The cellular mechanisms responsible for aging are poorly understood. Aging is considered as a degenerative process induced by the accumulation of cellular lesions leading progressively to organ dysfunction and death. The free radical theory of aging has long been considered the most relevant to explain the mechanisms of aging. As the mitochondrion is an important source of reactive oxygen species (ROS), this organelle is regarded as a key intracellular player in this process and a large amount of data supports the role of mitochondrial ROS production during aging. Thus, mitochondrial ROS, oxidative damage, aging, and aging‐dependent diseases are strongly connected. However, other features of mitochondrial physiology and dysfunction have been recently implicated in the development of the aging process. Here, we examine the potential role of the mitochondrial permeability transition pore (mPTP) in normal aging and in aging‐associated diseases.
... Mitochondrial calcium handling defects are an important contributor to mitochondrial injury in HD. These deficiencies have been shown in different models of HD, such as lymphoblasts from HD patients, mitochondria extracted from the brains of transgenic mice that expressed mHtt, and skeletal muscle cells obtained from R2/6 HD mice [12,18,57,58]. These models present increased sensitivity to calcium stress, which leads to mitochondrial depolarization, increased ROS levels, and failure of mitochondrial respiration [12,18,57,58]. ...
... These deficiencies have been shown in different models of HD, such as lymphoblasts from HD patients, mitochondria extracted from the brains of transgenic mice that expressed mHtt, and skeletal muscle cells obtained from R2/6 HD mice [12,18,57,58]. These models present increased sensitivity to calcium stress, which leads to mitochondrial depolarization, increased ROS levels, and failure of mitochondrial respiration [12,18,57,58]. Furthermore, the presence of mHtt at physiological levels in clonal striatal cells has been shown to lead to significant deficits in mitochondrial calcium handling, and subsequently mitochondrial depolarization, oxidative stress, and cell death [15e17]. ...
... CypD was also observed to be upregulated in HD patients, and this upregulation increased as HD progressed [56]. Of relevance, cortical mitochondria isolated from the crossing between R6/2 with CypD-knockout mice exhibited increased Ca 2þ uptake capacity; however no behavioral improvement or alleviation of HD neuropathology was observed [57]. ...
Mitochondria play a relevant role in Ca2+ buffering, governing energy metabolism and neuronal function. Huntington's disease (HD) and Alzheimer's disease (AD) are two neurodegenerative disorders that, although clinically distinct, share pathological features linked to selective brain damage. These include mitochondrial dysfunction, intracellular Ca2+ deregulation and mitochondrial Ca2+ handling deficits. Both diseases are associated with misfolding and aggregation of specific proteins that physically interact with mitochondria and interfere with endoplasmic reticulum (ER)/mitochondria-contact sites. Cumulating evidences indicate that impairment of mitochondrial Ca2+ homeostasis underlies the susceptibility to selective neuronal death observed in HD and AD; however data obtained with different models and experimental approaches are not always consistent. In this review, we explore the recent literature on deregulation of mitochondrial Ca2+ handling underlying the interplay between mitochondria and ER in HD and AD-associated neurodegeneration.
... Starting at 30 weeks of age mice were tested in a Bioseb Automatic foot misplacement apparatus (EB-Instruments, Tampa, FL). Mice walked on a 79 cm horizontal ladder (1 rung/cm) through a lit corridor (50 mm wide with opaque walls) to a shaded area and the number of missteps and time to traverse ladder was averaged over three trials. In cage activity was determined as we previously described (28) with 24 h of acclimation followed by 3 days and nights of data acquisition which was averaged for each mouse. Open field test was performed as previously described (28) in a 50 × 50 cm square arena with walls and floor made of plexiglass. ...
... In cage activity was determined as we previously described (28) with 24 h of acclimation followed by 3 days and nights of data acquisition which was averaged for each mouse. Open field test was performed as previously described (28) in a 50 × 50 cm square arena with walls and floor made of plexiglass. Distance traveled and position during 4 min trials was determined by computerized video recording and the Ethovision 3.1 software (Noldus Information Technology, Wageningen, the Netherlands). ...
Identifying molecular drivers of pathology provides potential therapeutic targets. Differentiating between drivers and coincidental
molecular alterations presents a major challenge. Variation unrelated to pathology further complicates transcriptomic, proteomic
and metabolomic studies which measure large numbers of individual molecules. To overcome these challenges towards the goal
of determining drivers of Huntington's Disease (HD), we generated an allelic series of HD knock-in mice with graded levels
of phenotypic severity for comparison with molecular alterations. RNA sequencing analysis of this series reveals high numbers
of transcripts with level alterations that do not correlate with phenotypic severity. These discorrelated molecular changes
are unlikely to be drivers of pathology allowing an exclusion-based strategy to provide a short list of driver candidates.
Further analysis of the data shows that a majority of transcript level changes in HD knock-in mice involve alteration of the
rate of mRNA processing and/or degradation rather than solely being due to alteration of transcription rate. The overall strategy
described can be applied to assess the influence of any molecular change on pathology for diseases where different mutations
cause graded phenotypic severity.
... As described previously by Van Groen (Handattu et al., 2009;Perry et al., 2010) and others (Rogers et al., 1999(Rogers et al., , 2001, a modified comprehensive phenotyping protocol (SHIRPA) was used at 12 weeks of age. Mice were assessed for locomotor activity (Open field test), balance and coordination (Rotarod test) anxiety (Elevated Plus Maze), spatial learning and memory (Novel Object Recognition test, Morris Water Maze Task), and vision (Cliff test). ...
... handling, argued against facilitation of PTP induction by mHtt, and did not support involvement of PTP in HD pathogenesis. In line with this, Perry et al. (2010) found that R6/2 mice crossed with CyD-knockout mice and, thus, expressing reduced or no CyD, had increased neuronal mitochondrial Ca 2+ uptake capacity without a sign of improvement in either behavioral or neuropathological features of HD. The authors concluded that increased Ca 2+ capacity of neuronal mitochondria is not beneficial in R6/2 mice. ...
We investigated Ca(2+) handling in isolated brain synaptic and nonsynaptic mitochondria and in cultured striatal neurons from the YAC128 mouse model of Huntington's disease (HD). Both synaptic and nonsynaptic mitochondria from 2- and 12-month-old YAC128 mice had larger Ca(2+) uptake capacity than mitochondria from YAC18 and wild-type FVB/NJ mice. Synaptic mitochondria from 12-month-old YAC128 mice had further augmented Ca(2+) capacity compared with mitochondria from 2-month-old YAC128 mice and age-matched YAC18 and FVB/NJ mice. This increase in Ca(2+) uptake capacity correlated with an increase in the amount of mutant huntingtin protein (mHtt) associated with mitochondria from 12-month-old YAC128 mice. We speculate that this may happen due to mHtt-mediated sequestration of free fatty acids thereby increasing resistance of mitochondria to Ca(2+) -induced damage. In experiments with striatal neurons from YAC128 and FVB/NJ mice, brief exposure to 25 or 100μM glutamate produced transient elevations in cytosolic Ca(2+) followed by recovery to near resting levels. Following recovery of cytosolic Ca(2+) , mitochondrial depolarization with FCCP produced comparable elevations in cytosolic Ca(2+) , suggesting similar Ca(2+) release and, consequently, Ca(2+) loads in neuronal mitochondria from YAC128 and FVB/NJ mice. Together, our data argue against a detrimental effect of mHtt on Ca(2+) handling in brain mitochondria of YAC128 mice. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
