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

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

Ex vivo 100 µm isotropic diffusion MRI-based tractography of connectivity changes in the end-stage R6/2 mouse model of Huntington's disease.

Background: Huntington's disease is a progressive neurodegenerative disorder. Brain atrophy, as measured by volumetric magnetic resonance imaging (MRI), is a downstream consequence of neurodegeneration, but microstructural changes within brain tissue are expected to precede this volumetric decline. The tissue microstructure can be assayed non-invasively using diffusion MRI, which also allows a tractographic analysis of brain connectivity. Methods: We here used ex vivo diffusion MRI (11.7 T) to measure microstructural changes in different brain regions of end-stage (14 weeks of age) wild type and R6/2 mice (male and female) modeling Huntington's disease. To probe the microstructure of different brain regions, reduce partial volume effects and measure connectivity between different regions, a 100 µm isotropic voxel resolution was acquired. Results: Although fractional anisotropy did not reveal any difference between wild-type controls and R6/2 mice, mean, axial, and radial diffusivity were increased in female R6/2 mice and decreased in male R6/2 mice. Whole brain streamlines were only reduced in male R6/2 mice, but streamline density was increased. Region-to-region tractography indicated reductions in connectivity between the cortex, hippocampus, and thalamus with the striatum, as well as within the basal ganglia (striatum-globus pallidus-subthalamic nucleus-substantia nigra-thalamus). Conclusions: Biological sex and left/right hemisphere affected tractographic results, potentially reflecting different stages of disease progression. This proof-of-principle study indicates that diffusion MRI and tractography potentially provide novel biomarkers that connect volumetric changes across different brain regions. In a translation setting, these measurements constitute a novel tool to assess the therapeutic impact of interventions such as neuroprotective agents in transgenic models, as well as patients with Huntington's disease.

Implantation of undifferentiated and pre-differentiated human neural stem cells in the R6/2 transgenic mouse model of Huntington's disease.

BACKGROUND: Cell therapy is a potential therapeutic approach for several neurodegenetative disease, including Huntington Disease (HD). To evaluate the putative efficacy of cell therapy in HD, most studies have used excitotoxic animal models with only a few studies having been conducted in genetic animal models. Genetically modified animals should provide a more accurate representation of human HD, as they emulate the genetic basis of its etiology. RESULTS: In this study, we aimed to assess the therapeutic potential of a human striatal neural stem cell line (STROC05) implanted in the R6/2 transgenic mouse model of HD. As DARPP-32 GABAergic output neurons are predominately lost in HD, STROC05 cells were also pre-differentiated using purmorphamine, a hedgehog agonist, to yield a greater number of DARPP-32 cells. A bilateral injection of 4.5x105 cells of either undifferentiated or pre-differentiated DARPP-32 cells, however, did not affect outcome compared to a vehicle control injection. Both survival and neuronal differentiation remained poor with a mean of only 161 and 81 cells surviving in the undifferentiated and differentiated conditions respectively. Only a few cells expressed the neuronal marker Fox3. CONCLUSIONS: Although the rapid brain atrophy and short life-span of the R6/2 model constitute adverse conditions to detect potentially delayed treatment effects, significant technical hurdles, such as poor cell survival and differentiation, were also sub-optimal. Further consideration of these aspects is therefore needed in more enduring transgenic HD models to provide a definite assessment of this cell line's therapeutic relevance. However, a combination of treatments is likely needed to affect outcome in transgenic models of HD.

Intraperitoneal delivery of acetate-encapsulated liposomal nanoparticles for neuroprotection of the penumbra in a rat model of ischemic stroke.

BACKGROUND: Ischemic stroke is a devastating condition, with metabolic derangement and persistent inflammation enhancing the initial insult of ischaemia. Recombinant tissue plasminogen remains the only effective treatment but limited as therapy must commence soon after the onset of symptoms. PURPOSE: We investigated whether acetate, which modulates many pathways including inflammation, may attenuate brain injury in stroke. As acetate has a short blood half-life and high amounts irritate the gastrointestinal tract, acetate was administered encapsulated in a liposomal nanoparticle (liposomal-encapsulated acetate, LITA). METHODS: Transient ischemia was induced by 90 mins middle-cerebral artery occlusion (MCAO) in Sprague-Dawley rats, and LITA or control liposomes given intraperitoneally at occlusion and daily for up to two weeks post-MCAO. Magnetic resonance imaging (MRI) was used to estimate lesion volume at 24 h, 1 and 2 weeks post-MCAO and anterior lateral ventricular volume (ALVv) at 2 weeks post-MCAO. Locomotive behaviour was tested prior to the final MRI scan. After the final scan, brains were collected, and immunohistochemistry was performed. RESULTS: Lesion volumes were decreased by ~80% from 24 h to one-week post-MCAO, in both control and LITA groups (P⩽0.05). However, the lesion was increased by ~50% over the subsequent 1 to 2 weeks after MCAO in the control group (from 24.1±10.0 to 58.7±28.6 mm3; P⩽0.05) but remained unchanged in the LITA group. ALVv were also attenuated by LITA treatment at 2 weeks post-MCAO (177.2±11.9% and 135.3±10.9% of contralateral ALVv for control and LITA groups, respectively; P⩽0.05). LITA-treated animals also appeared to have improved motor activity, moving with greater average velocity than control animals. Microglial immunoreactivity was ~40% lower in the LITA group compared to the control group (P⩽0.05), but LITA did not modulate neurogenesis, apoptosis, histone acetylation and lipid peroxidation. CONCLUSION: LITA appears to attenuate the harmful chronic neuroinflammation observed during brain remodeling after a focal ischemic insult.

What do we know about the long-term consequences of stress on ageing and the progression of age-related neurodegenerative disorders?

The aim of this paper is to review evidences that stressful events throughout life can have a long-term impact on ageing and the progression of Alzheimer's disease. As early as the prenatal or neonatal period, stress can alter the rate of cognitive decline and neurodegenerative changes in the brain in a stressor-dependent manner, with prenatal restraint and maternal separation usually causing damage to the brain, whereas neonatal handling was found protective. The occurrence of negative outcomes of early stress can, however, be reversed by subsequent events known to be beneficial to the ageing process. After the early developmental period, it is currently unknown how stress will impact on the ageing process, due to a lack of studies. On the other hand, there is evidence of a lack of plasticity of the brain monoaminergic systems in response to stress with age, and of age-dependent changes in the immediate impact of stress, which is greater in subjects vulnerable to age-related cognitive decline. In addition, vulnerability to stress enhances the risk of developing Alzheimer's disease in humans and chronic substantial stress in animal models of the disease accelerates both the onset and progression of pathological markers in the brain. In an attempt to integrate these findings, a hypothesis is presented here whereby stress, in susceptible individuals, would precipitate age-related cognitive decline and hippocampal integrity during normal and pathological ageing, but will only affect the progression of pathological markers of Alzheimer's disease in the presence of other risk factors to this neuropathological disorder.

Correlations of Behavioral Deficits with Brain Pathology Assessed through Longitudinal MRI and Histopathology in the HdhQ150/Q150 Mouse Model of Huntington's Disease.

A variety of mouse models have been developed that express mutant huntingtin (mHTT) leading to aggregates and inclusions that model the molecular pathology observed in Huntington's disease. Here we show that although homozygous HdhQ150 knock-in mice developed motor impairments (rotarod, locomotor activity, grip strength) by 36 weeks of age, cognitive dysfunction (swimming T maze, fear conditioning, odor discrimination, social interaction) was not evident by 94 weeks. Concomitant to behavioral assessments, T2-weighted MRI volume measurements indicated a slower striatal growth with a significant difference between wild type (WT) and HdhQ150 mice being present even at 15 weeks. Indeed, MRI indicated significant volumetric changes prior to the emergence of the "clinical horizon" of motor impairments at 36 weeks of age. A striatal decrease of 27% was observed over 94 weeks with cortex (12%) and hippocampus (21%) also indicating significant atrophy. A hypothesis-free analysis using tensor-based morphometry highlighted further regions undergoing atrophy by contrasting brain growth and regional neurodegeneration. Histology revealed the widespread presence of mHTT aggregates and cellular inclusions. However, there was little evidence of correlations between these outcome measures, potentially indicating that other factors are important in the causal cascade linking the molecular pathology to the emergence of behavioral impairments. In conclusion, the HdhQ150 mouse model replicates many aspects of the human condition, including an extended pre-manifest period prior to the emergence of motor impairments.

Stall in Canonical Autophagy-Lysosome Pathways Prompts Nucleophagy-Based Nuclear Breakdown in Neurodegeneration.

The terminal stages of neuronal degeneration and death in neurodegenerative diseases remain elusive. Autophagy is an essential catabolic process frequently failing in neurodegeneration. Selective autophagy routes have recently emerged, including nucleophagy, defined as degradation of nuclear components by autophagy. Here, we show that, in a mouse model for the polyglutamine disease dentatorubral-pallidoluysian atrophy (DRPLA), progressive acquirement of an ataxic phenotype is linked to severe cerebellar cellular pathology, characterized by nuclear degeneration through nucleophagy-based LaminB1 degradation and excretion. We find that canonical autophagy is stalled in DRPLA mice and in human fibroblasts from patients of DRPLA. This is evidenced by accumulation of p62 and downregulation of LC3-I/II conversion as well as reduced Tfeb expression. Chronic autophagy blockage in several conditions, including DRPLA and Vici syndrome, an early-onset autolysosomal pathology, leads to the activation of alternative clearance pathways including Golgi membrane-associated and nucleophagy-based LaminB1 degradation and excretion. The combination of these alternative pathways and canonical autophagy blockade, results in dramatic nuclear pathology with disruption of the nuclear organization, bringing about terminal cell atrophy and degeneration. Thus, our findings identify a novel progressive mechanism for the terminal phases of neuronal cell degeneration and death in human neurodegenerative diseases and provide a link between autophagy block, activation of alternative pathways for degradation, and excretion of cellular components.

Myostatin inhibition prevents skeletal muscle pathophysiology in Huntington's disease mice.

Huntington's disease (HD) is an inherited neurodegenerative disorder of which skeletal muscle atrophy is a common feature, and multiple lines of evidence support a muscle-based pathophysiology in HD mouse models. Inhibition of myostatin signaling increases muscle mass, and therapeutic approaches based on this are in clinical development. We have used a soluble ActRIIB decoy receptor (ACVR2B/Fc) to test the effects of myostatin/activin A inhibition in the R6/2 mouse model of HD. Weekly administration from 5 to 11 weeks of age prevented body weight loss, skeletal muscle atrophy, muscle weakness, contractile abnormalities, the loss of functional motor units in EDL muscles and delayed end-stage disease. Inhibition of myostatin/activin A signaling activated transcriptional profiles to increase muscle mass in wild type and R6/2 mice but did little to modulate the extensive Huntington's disease-associated transcriptional dysregulation, consistent with treatment having little impact on HTT aggregation levels. Modalities that inhibit myostatin signaling are currently in clinical trials for a variety of indications, the outcomes of which will present the opportunity to assess the potential benefits of targeting this pathway in HD patients.

Correlations of behavioral deficits with brain pathology assessed through longitudinal MRI and histopathology in the R6/2 mouse model of HD.

Huntington's disease (HD) is caused by the expansion of a CAG repeat in the huntingtin (HTT) gene. The R6/2 mouse model of HD expresses a mutant version of exon 1 HTT and develops motor and cognitive impairments, a widespread huntingtin (HTT) aggregate pathology and brain atrophy. Despite the vast number of studies that have been performed on this model, the association between the molecular and cellular neuropathology with brain atrophy, and with the development of behavioral phenotypes remains poorly understood. In an attempt to link these factors, we have performed longitudinal assessments of behavior (rotarod, open field, passive avoidance) and of regional brain abnormalities determined through magnetic resonance imaging (MRI) (whole brain, striatum, cortex, hippocampus, corpus callosum), as well as an end-stage histological assessment. Detailed correlative analyses of these three measures were then performed. We found a gender-dependent emergence of motor impairments that was associated with an age-related loss of regional brain volumes. MRI measurements further indicated that there was no striatal atrophy, but rather a lack of striatal growth beyond 8 weeks of age. T2 relaxivity further indicated tissue-level changes within brain regions. Despite these dramatic motor and neuroanatomical abnormalities, R6/2 mice did not exhibit neuronal loss in the striatum or motor cortex, although there was a significant increase in neuronal density due to tissue atrophy. The deposition of the mutant HTT (mHTT) protein, the hallmark of HD molecular pathology, was widely distributed throughout the brain. End-stage histopathological assessments were not found to be as robustly correlated with the longitudinal measures of brain atrophy or motor impairments. In conclusion, modeling pre-manifest and early progression of the disease in more slowly progressing animal models will be key to establishing which changes are causally related.

Correlations of behavioral deficits with brain pathology assessed through longitudinal MRI and histopathology in the R6/1 mouse model of Huntington's disease.

Huntington's disease (HD) is caused by the expansion of a CAG repeat in the huntingtin (HTT) gene. The R6 mouse models of HD express a mutant version of exon 1 HTT and typically develop motor and cognitive impairments, a widespread huntingtin (HTT) aggregate pathology and brain atrophy. Unlike the more commonly used R6/2 mouse line, R6/1 mice have fewer CAG repeats and, subsequently, a less rapid pathological decline. Compared to the R6/2 line, fewer descriptions of the progressive pathologies exhibited by R6/1 mice exist. The association between the molecular and cellular neuropathology with brain atrophy, and with the development of behavioral phenotypes remains poorly understood in many models of HD. In attempt to link these factors in the R6/1 mouse line, we have performed detailed assessments of behavior and of regional brain abnormalities determined through longitudinal, in vivo magnetic resonance imaging (MRI), as well as an end-stage, ex vivo MRI study and histological assessment. We found progressive decline in both motor and non-motor related behavioral tasks in R6/1 mice, first evident at 11 weeks of age. Regional brain volumes were generally unaffected at 9 weeks, but by 17 weeks there was significant grey matter atrophy. This age-related brain volume loss was validated using a more precise, semi-automated Tensor Based morphometry assessment. As well as these clear progressive phenotypes, mutant HTT (mHTT) protein, the hallmark of HD molecular pathology, was widely distributed throughout the R6/1 brain and was accompanied by neuronal loss. Despite these seemingly concomitant, robust pathological phenotypes, there appeared to be little correlation between the three main outcome measures: behavioral performance, MRI-detected brain atrophy and histopathology. In conclusion, R6/1 mice exhibit many features of HD, but the underlying mechanisms driving these clear behavioral disturbances and the brain volume loss, still remain unclear.

Novel cage stress alters remote contextual fear extinction and regional T2 magnetic resonance relaxation times in TASTPM mice overexpressing amyloid.

We have previously shown that repeated exposure to mild novel cage stress prevents the onset of recent contextual fear memory deficits and attenuated amyloid deposition in the TASTPM mouse model of Alzheimer's disease. Here, we extended this investigation to remote contextual fear memory and extinction. TASTPM and wild-type mice acquired contextual fear at 4 months of age. Retention and extinction of contextual fear were assessed at 5.5 months prior to in vivo MRI assessment of regional T2 relaxation times and brain volumes followed by immunostaining to determine amyloid plaque load. Remote contextual fear memory was preserved in TASTPM mice regardless of the stress condition. Stress impaired extinction in wild-type mice but facilitated this process in TASTPM mice. Genotype-dependent effects of stress were observed on regional T2 times which were prolonged in the subiculum and thalamus of stressed TASTPM, possibly reflecting reduced amyloid pathology. Amyloid plaque load was particularly decreased in the retrosplenial cortex of stressed TASTPM mice, which also showed an overall reduction in the number of diffuse plaques. These findings support the hypothesis that repeated mild levels of stress induced by novel activities can delay the progression of pathological changes relevant to Alzheimer's disease.

The occurrence of a deficit in contextual fear extinction in adult amyloid-over-expressing TASTPM mice is independent of the strength of conditioning but can be prevented by mild novel cage stress.

In the amyloid over-expressing TASTPM mouse model of Alzheimer's disease, impaired contextual fear memory occurs early, and is preceded, at 4 months of age, by a deficit in extinction of contextual fear that is resistant to improvement by repeated mild novel cage stress. The first aim of this study was thus to establish whether the extinction deficit could be prevented if the novel cage procedure was applied prior to its onset. The second aim was to establish whether the occurrence of the extinction deficit was dependent on the robustness of the conditioning protocol. We first compared 3-month-old wild-type and TASTPM mice for acquisition, retention and extinction of contextual fear and then, looked at the impact of 5 weeks of novel cage stress (4 x 1 h/week) applied from 3 months onwards, on age-related changes in these behaviours evaluated at 4.5 months of age. In another experiment, we compared 4-month-old TASTPM and wild-type mice for the impact of a 2 and 5-pairing conditioning procedure on the three phases of contextual fear conditioning. In 4.5-month-old TASTPM mice, the deficit in extinction was alleviated by repeated novel cage stress, applied from prior to its onset at 3 months. At 4 months of age, the occurrence of an extinction deficit was independent of the strength of the conditioning procedure, in TASTPM mice, which even showed an increase in aversive memory under the 2-pairing condition. The robust early impairment in the extinction of contextual fear seen in adult TASTPM mice suggests that a deficit in cognitive flexibility is the first sign of behavioural pathology in this model of Alzheimer's disease.

Repeated novel cage exposure-induced improvement of early Alzheimer's-like cognitive and amyloid changes in TASTPM mice is unrelated to changes in brain endocannabinoids levels.

Environmental factors (e.g. stress, exercise, enrichment) are thought to play a role in the development of Alzheimer's disease later in life. We investigated the influence of repeated novel cage exposure on the development of early Alzheimer's-like pathology in adult (4 months old) double transgenic mice over-expressing the amyloid precursor protein and presenilin-1 genes (TASTPM mouse line). The procedure involves the repeated placement of the animal into a novel clean cage, a manipulation which induces a stress response and exploratory activity and, as such, can also be seen as a mild form of enrichment. Before and after exposure to the novel cage procedure, separate groups of mice were evaluated for locomotor performance and short-term contextual memory in the fear-conditioning test. Repeated novel cage exposure prevented the onset of a short-term memory deficit that was apparent in 5.5- but not 4-month-old TASTPM mice, without reversing the deficit in extinction already evident at 4 months of age. Brain regional levels of soluble and insoluble amyloid and of endocannabinoids were quantified. Novel cage exposure attenuated soluble and insoluble amyloid accumulation in the hippocampus and frontal cortex, without affecting the age-related increases in regional brain endocannabinoids levels. These beneficial effects are likely to be the consequence of the increase in physical and exploratory activity induced by novel cage exposure and suggest that the impact of environmental factors on Alzheimer's-like changes may be dependent on the degree of activation of stress pathways.