Understanding the role of the hematopoietic niche in Huntington's disease's phenotypic expression: in vivo evidence using a parabiosis model.

In a previous study, we have shown that parabiotic coupling of a knock-in mouse model (zQ175) of Huntington's disease (HD) to wild-type (WT) littermates resulted in a worsening of the normal phenotype as seen by detection of mutant huntingtin protein (mHTT) aggregates within peripheral organs and the cerebral cortex as well as vascular abnormalities in WT mice. In contrast, parabiosis improved disease features in the zQ175 mice such as reduction of mHTT aggregate number in the liver and cortex, decrease in blood-brain barrier (BBB) permeability and attenuation of mitochondrial impairments. While the shared circulation mediated these effects, no specific factor was identified. To better understand which blood elements were involved in the aforementioned changes, WT and zQ175 mice underwent parabiotic surgery prior to exposing one of the paired animals to irradiation. The irradiation procedure successfully eliminated the hematopoietic niche followed by repopulation with cells originating from the non-irradiated parabiont, as measured by the quantification of mHTT levels in peripheral blood mononuclear cells. Although irradiation of the WT parabiont, causing the loss of healthy hematopoietic cells, did lead to a few alterations in mitochondrial function in the muscle (TOM40 levels), and increased neuroinflammation in the striatum (GFAP levels), most of the changes observed were likely attributable to the irradiation procedure itself (e.g. mHTT aggregates in cortex and liver; cellular stress in peripheral organs). However, factors such as mHTT aggregation in the brain and periphery, and BBB leakage, which were improved in zQ175 mice when paired to WT littermates in the previous parabiosis experiment, were unaffected by perturbation of the hematopoietic niche. It would therefore appear that cells of the hematopoietic stem cell niche are largely uninvolved in the beneficial effects of parabiosis.

Passive immunization against phosphorylated tau improves features of Huntington's disease pathology.

Huntington's disease is classically described as a neurodegenerative disorder of monogenic aetiology. The disease is characterized by an abnormal polyglutamine expansion in the huntingtin gene, which drives the toxicity of the mutated form of the protein. However, accumulation of the microtubule-associated protein tau, which is involved in a number of neurological disorders, has also been observed in patients with Huntington's disease. In order to unravel the contribution of tau hyperphosphorylation to hallmark features of Huntington's disease, we administered weekly intraperitoneal injections of the anti-tau pS202 CP13 monoclonal antibody to zQ175 mice and characterized the resulting behavioral and biochemical changes. After 12 weeks of treatment, motor impairments, cognitive performance and general health were improved in zQ175 mice along with a significant reduction in hippocampal pS202 tau levels. Despite the lack of effect of CP13 on neuronal markers associated with Huntington's disease pathology, tau-targeting enzymes and gliosis, CP13 was shown to directly impact mutant huntingtin aggregation such that brain levels of amyloid fibrils and huntingtin oligomers were decreased, while larger huntingtin protein aggregates were increased. Investigation of CP13 treatment of Huntington's disease patient-derived induced pluripotent stem cells (iPSCs) revealed a reduction in pS202 levels in differentiated cortical neurons and a rescue of neurite length. Collectively, these findings suggest that attenuating tau pathology could mitigate behavioral and molecular hallmarks associated with Huntington's disease.

Shedding a new light on Huntington's disease: how blood can both propagate and ameliorate disease pathology.

Huntington's disease (HD) is a monogenic neurodegenerative disorder resulting from a mutation in the huntingtin gene. This leads to the expression of the mutant huntingtin protein (mHTT) which provokes pathological changes in both the central nervous system (CNS) and periphery. Accumulating evidence suggests that mHTT can spread between cells of the CNS but here, we explored the possibility that mHTT could also propagate and cause pathology via the bloodstream. For this, we used a parabiosis approach to join the circulatory systems of wild-type (WT) and zQ175 mice. After surgery, we observed mHTT in the plasma and circulating blood cells of WT mice and post-mortem analyses revealed the presence of mHTT aggregates in several organs including the liver, kidney, muscle and brain. The presence of mHTT in the brain was accompanied by vascular abnormalities, such as a reduction of Collagen IV signal intensity and altered vessel diameter in the striatum, and changes in expression of Glutamic acid decarboxylase 65/67 (GAD65-67) in the cortex. Conversely, we measured reduced pathology in zQ175 mice by decreased mitochondrial impairments in peripheral organs, restored vessel diameter in the cortex and improved expression of Dopamine- and cAMP-regulated phosphoprotein 32 (DARPP32) in striatal neurons. Collectively, these results demonstrate that circulating mHTT can disseminate disease, but importantly, that healthy blood can dilute pathology. These findings have significant implications for the development of therapies in HD.

Beneficial effects of cysteamine in Thy1-α-Syn mice and induced pluripotent stem cells with a SNCA gene triplication.

A number of publications have reported that cysteamine has significant therapeutic effects on several aspects of Parkinson's disease (PD)-related pathology but none of these studies have evaluated its impact on pathological forms of α-Synuclein (α-Syn), one of the main hallmarks of PD. We therefore tested the efficacy of cysteamine on the Thy1-α-Syn mouse model which over-expresses full-length human wild-type α-Syn. Two-month (early stage disease) and 6-month old (late stage disease) mice and littermate controls were treated daily with cysteamine (20 mg/kg, i.p.) to assess the protective and restorative properties of this compound. After 6 weeks of treatment, animals were tested using a battery of motor tests. Cysteamine-treated transgenic mice displayed significant improvements in motor performance as compared to saline-treated transgenic littermates. Post-mortem readouts revealed a reduction in fibrillation, phosphorylation and total levels of overexpresed human α-Syn. To determine if such outcomes extended to human cells, the benefits of cysteamine were additionally tested using 6-hydroxydopamine (6-OHDA) treated neurons differentiated from induced pluripotent stem cells (iPSCs) derived from a PD patient harbouring a triplication of the SNCA gene. SNCA neurons treated with cysteamine exhibited significantly more intact/healthy neurites than cells treated with 6-OHDA alone. Additionally, SNCA neurons treated with cysteamine in the absence of 6-OHDA showed a trend towards lower total α-Syn levels. Overall, our in vivo and in vitro findings suggest that cysteamine can act as a disease-modifying molecule by enhancing -the survival of dopaminergic neurons and reducing pathological forms of α-Syn.

A novel wireless brain stimulation device for long-term use in freely moving mice.

Deep brain stimulation (DBS) has been used in clinical settings for many years despite a paucity of knowledge related to the anatomical and functional substrates that lead to benefits and/or side-effects in various disease contexts. In order to maximize the potential of this approach in humans, a better understanding of its mechanisms of action is absolutely necessary. However, the existing micro-stimulators available for pre-clinical models, are limited by the lack of relevant small size devices. This absence prevents sustained chronic stimulation and real time monitoring of animals during stimulation, parameters that are critical for comparison to clinical findings. We therefore sought to develop and refine a novel small wireless micro-stimulator as a means by which to study consequent behavioural to molecular changes in experimental animals. Building on previous work from our group, we refined our implantable micro-stimulator prototype, to be easily combined with intravital 2-photon imaging. Using our prototype we were able to replicate the well described clinical benefits on motor impairment in a mouse model of Parkinson's disease in addition to capturing microglia dynamics live during stimulation. We believe this new device represents a useful tool for performing pre-clinical studies as well as dissecting brain circuitry and function.

Demonstration of prion-like properties of mutant huntingtin fibrils in both in vitro and in vivo paradigms.

In recent years, evidence has accumulated to suggest that mutant huntingtin protein (mHTT) can spread into healthy tissue in a prion-like fashion. This theory, however, remains controversial. To fully address this concept and to understand the possible consequences of mHTT spreading to Huntington's disease pathology, we investigated the effects of exogenous human fibrillar mHTT (Q48) and huntingtin (HTT) (Q25) N-terminal fragments in three cellular models and three distinct animal paradigms. For in vitro experiments, human neuronal cells [induced pluripotent stem cell-derived GABA neurons (iGABA) and (SH-SY5Y)] as well as human THP1-derived macrophages, were incubated with recombinant mHTT fibrils. Recombinant mHTT and HTT fibrils were taken up by all cell types, inducing cell morphology changes and death. Variations in HTT aggregation were further observed following incubation with fibrils in both THP1 and SH-SY5Y cells. For in vivo experiments, adult wild-type (WT) mice received a unilateral intracerebral cortical injection and R6/2 and WT pups were administered fibrils via bilateral intraventricular injections. In both protocols, the injection of Q48 fibrils resulted in cognitive deficits and increased anxiety-like behavior. Post-mortem analysis of adult WT mice indicated that most fibrils had been degraded/cleared from the brain by 14 months post-surgery. Despite the absence of fibrils at these later time points, a change in the staining pattern of endogenous HTT was detected. A similar change was revealed in post-mortem analysis of the R6/2 mice. These effects were specific to central administration of fibrils, as mice receiving intravenous injections were not characterized by behavioral changes. In fact, peripheral administration resulted in an immune response mounting against the fibrils. Together, the in vitro and in vivo data indicate that exogenously administered mHTT is capable of both causing and exacerbating disease pathology.

Outcome of cell suspension allografts in a patient with Huntington's disease.

For patients with incurable neurodegenerative disorders such as Huntington's (HD) and Parkinson's disease, cell transplantation has been explored as a potential treatment option. Here, we present the first clinicopathological study of a patient with HD in receipt of cell-suspension striatal allografts who took part in the NEST-UK multicenter clinical transplantation trial. Using various immunohistochemical techniques, we found a discrepancy in the survival of grafted projection neurons with respect to grafted interneurons as well as major ongoing inflammatory and immune responses to the grafted tissue with evidence of mutant huntingtin aggregates within the transplant area. Our results indicate that grafts can survive more than a decade post-transplantation, but show compromised survival with inflammation and mutant protein being observed within the transplant site. Ann Neurol 2018;84:950-956.

The effects of cysteamine in a mouse model of levodopa-induced dyskinesias.

Levo-dopa (L-DOPA) has shown significant and long-lasting efficacy in the treatment of motor features characteristic of Parkinson's disease (PD). However, the effects tend to wear off at a time typically when side-effects, such as L-DOPA induced dyskinesias (LIDs), start to emerge and for which the treatment options are very limited. In recent years, we have reported on the neuroprotective and neurorestorative properties of the compounds cystamine/cysteamine in ameliorating several aspects of PD. Building on these observations, we set out to further evaluate the benefits of cysteamine on LIDs. We thus treated mice displaying LIDs with single cysteamine challenges at various doses (20, 50 and 30mg/kg) or chronically for 2 weeks using cysteamine at a dose of 30mg/kg. None of the regimens nor doses ameliorated any LID-related behavioral impairments. Mice displaying LIDs did, however, respond to a single treatment of 60mg/kg of amantadine, a drug used to clinically manage LIDs. Taken together, our results suggest that cysteamine does not induce benefits on LIDs, at least at the doses and regimen tested in our study. However, the disease-modifying effects depicted by cystamine/cysteamine, which we have shown in several reports, would strongly encourage its continued evaluation in the clinical setting.

microRNA-132/212 deficiency enhances Aß production and senile plaque deposition in Alzheimer's disease triple transgenic mice.

The abnormal regulation of amyloid-ß (Aß) metabolism (e.g., production, cleavage, clearance) plays a central role in Alzheimer's disease (AD). Among endogenous factors believed to participate in AD progression are the small regulatory non-coding microRNAs (miRs). In particular, the miR-132/212 cluster is severely reduced in the AD brain. In previous studies we have shown that miR-132/212 deficiency in mice leads to impaired memory and enhanced Tau pathology as seen in AD patients. Here we demonstrate that the genetic deletion of miR-132/212 promotes Aß production and amyloid (senile) plaque formation in triple transgenic AD (3xTg-AD) mice. Using RNA-Seq and bioinformatics, we identified genes of the miR-132/212 network with documented roles in the regulation of Aß metabolism, including Tau, Mapk, and Sirt1. Consistent with these findings, we show that the modulation of miR-132, or its target Sirt1, can directly regulate Aß production in cells. Finally, both miR-132 and Sirt1 levels correlated with Aß load in humans. Overall, our results support the hypothesis that the miR-132/212 network, including Sirt1 and likely other target genes, contributes to abnormal Aß metabolism and senile plaque deposition in AD. This study strengthens the importance of miR-dependent networks in neurodegenerative disorders, and opens the door to multifactorial drug targets of AD by targeting Aß and Tau.

Partial neurorescue effects of DHA following a 6-OHDA lesion of the mouse dopaminergic system.

Pre-clinical data collected in mouse models of Parkinson's disease (PD) support the neuroprotective potential of omega-3 polyunsaturated fatty acids (n-3 PUFA)-enriched diet on the dopaminergic (DAergic) system. In this study, we investigated the effects of an n-3 PUFA-rich diet using a neurorescue/neurorestorative paradigm. C57BL/6 adult mice were submitted to a striatal stereotaxic injection of the neurotoxin 6-hydroxydopamine (6-OHDA) to induce striatal DAergic denervation and subsequent nigral DAergic cell loss. Three weeks post-lesion, mice received either a docosahexaenoic acid (DHA)-enriched or a control diet for a period of 6 weeks. HPLC analyses revealed a 111% post-lesion increase in striatal dopamine levels in the DHA-fed animals compared to controls (ctrl, P<0.05), although no improvement in the motor behavior was observed. DHA treatment led to a 89% rise in tyrosine-hydroxylase (TH)-immunoreactive terminals within the striatum (P<0.05) in lesioned animals. Despite the fact that DHA did not change the number of TH+ neurons in the substantia nigra pars compacta (SNpc), morphological analyses revealed an increased in perimeters (+7%) and areas (+21%) of DAergic cell bodies in treated animals. Collectively, our results suggest that DHA induces a partial neurorescue/neurorestoration of the DAergic system and support further studies to investigate the potential of a diet-based intervention, or at least the combination of such approach, to current treatments in PD.

Cerebrovascular and blood-brain barrier impairments in Huntington's disease: Potential implications for its pathophysiology.

OBJECTIVE: Although the underlying cause of Huntington's disease (HD) is well established, the actual pathophysiological processes involved remain to be fully elucidated. In other proteinopathies such as Alzheimer's and Parkinson's diseases, there is evidence for impairments of the cerebral vasculature as well as the blood-brain barrier (BBB), which have been suggested to contribute to their pathophysiology. We investigated whether similar changes are also present in HD. METHODS: We used 3- and 7-Tesla magnetic resonance imaging as well as postmortem tissue analyses to assess blood vessel impairments in HD patients. Our findings were further investigated in the R6/2 mouse model using in situ cerebral perfusion, histological analysis, Western blotting, as well as transmission and scanning electron microscopy. RESULTS: We found mutant huntingtin protein (mHtt) aggregates to be present in all major components of the neurovascular unit of both R6/2 mice and HD patients. This was accompanied by an increase in blood vessel density, a reduction in blood vessel diameter, as well as BBB leakage in the striatum of R6/2 mice, which correlated with a reduced expression of tight junction-associated proteins and increased numbers of transcytotic vesicles, which occasionally contained mHtt aggregates. We confirmed the existence of similar vascular and BBB changes in HD patients. INTERPRETATION: Taken together, our results provide evidence for alterations in the cerebral vasculature in HD leading to BBB leakage, both in the R6/2 mouse model and in HD patients, a phenomenon that may, in turn, have important pathophysiological implications.

A novel combinational approach of microstimulation and bioluminescence imaging to study the mechanisms of action of cerebral electrical stimulation in mice.

Deep brain stimulation (DBS) is used to treat a number of neurological conditions and is currently being tested to intervene in neuropsychiatric conditions. However, a better understanding of how it works would ensure that side effects could be minimized and benefits optimized. We have thus developed a unique device to perform brain stimulation (BS) in mice and to address fundamental issues related to this methodology in the pre-clinical setting. This new microstimulator prototype was specifically designed to allow simultaneous live bioluminescence imaging of the mouse brain, allowing real time assessment of the impact of stimulation on cerebral tissue. We validated the authenticity of this tool in vivo by analysing the expression of toll-like receptor 2 (TLR2), corresponding to the microglial response, in the stimulated brain regions of TLR2-fluc-GFP transgenic mice, which we further corroborated with post-mortem analyses in these animals as well as in human brains of patients who underwent DBS to treat their Parkinson's disease. In the present study, we report on the development of the first BS device that allows for simultaneous live in vivo imaging in mice. This tool opens up a whole new range of possibilities that allow a better understanding of BS and how to optimize its effects through its use in murine models of disease.

Toll-like receptor expression in the blood and brain of patients and a mouse model of Parkinson's disease.

BACKGROUND: Accumulating evidence supports a role for the immune system in the pathogenesis of Parkinson's disease. Importantly, recent preclinical studies are now suggesting a specific contribution of inflammation to the α-synuclein-induced pathology seen in this condition. METHODS: We used flow cytometry and western blots to detect toll-like receptor 2 and 4 expression in blood and brain samples of Parkinson's disease patients and mice overexpressing human α-synuclein. To further assess the effects of α-synuclein overexpression on the innate immune system, we performed a longitudinal study using Thy1.2-α-synuclein mice that expressed a bicistronic DNA construct (reporter genes luciferase and green fluorescent protein) under the transcriptional control of the murine toll-like receptor 2 promoter. RESULTS: Here, we report increases in toll-like receptors 2 and 4 expression in circulating monocytes and of toll-like receptor 4 in B cells and in the caudate/putamen of Parkinson's disease patients. Monthly bioluminescence imaging of Thy1.2-α-synuclein mice showed increasing toll-like receptor 2 expression from 10 months of age, although no change in toll-like receptor 2 and 4 expression was observed in the blood and brain of these mice at 12 months of age. Dexamethasone treatment starting at 5 months of age for 1 month significantly decreased the microglial response in the brain of these mice and promoted functional recovery as observed using a wheel-running activity test. CONCLUSION: Our results show that toll-like receptors 2 and 4 are modulated in the blood and brain of Parkinson's disease patients and that overexpression of α-synuclein leads to a progressive microglial response, the inhibition of which has a beneficial impact on some motor phenotypes of an animal model of α-synucleinopathy.

Mutant huntingtin is present in neuronal grafts in Huntington disease patients.

OBJECTIVE: Huntington disease (HD) is caused by a genetically encoded pathological protein (mutant huntingtin [mHtt]), which is thought to exert its effects in a cell-autonomous manner. Here, we tested the hypothesis that mHtt is capable of spreading within cerebral tissue by examining genetically unrelated fetal neural allografts within the brains of patients with advancing HD. METHODS: The presence of mHtt aggregates within the grafted tissue was confirmed using 3 different types of microscopy (bright-field, fluorescence, and electron), 2 additional techniques consisting of Western immunoblotting and infrared spectroscopy, and 4 distinct antibodies targeting different epitopes of mHtt aggregates. RESULTS: We describe the presence of mHtt aggregates within intracerebral allografts of striatal tissue in 3 HD patients who received their transplants approximately 1 decade earlier and then died secondary to the progression of their disease. The mHtt(+) aggregates were observed in the extracellular matrix of the transplanted tissue, whereas in the host brain they were seen in neurons, neuropil, extracellular matrix, and blood vessels. INTERPRETATION: This is the first demonstration of the presence of mHtt in genetically normal and unrelated allografted neural tissue transplanted into the brain of affected HD patients. These observations raise questions on protein spread in monogenic neurodegenerative disorders of the central nervous system characterized by the formation of mutant protein oligomers/aggregates.

Striatal allografts in patients with Huntington's disease: impact of diminished astrocytes and vascularization on graft viability.

Neuronal transplantation has been proposed as a potential therapy to replace lost neurons in Huntington's disease. Transplant vascularization and trophic support are important for graft survival. However, very few studies have specifically addressed graft vascularization in patients with neurological disorders. In the present study, we analysed the vasculature of the host putamen and solid grafts of foetal striatal tissue transplanted into patients with Huntington's disease 9 and 12 years previously. Grafts were characterized by a significantly reduced number of large calibre blood vessels in comparison with the host brain. There were also significantly fewer astrocytes and gap junctions, suggesting a lack of functional blood-brain barrier components within the grafted tissue. Additionally, grafts demonstrated a nearly complete absence of pericytes (compared with the striatum) that are considered important for vascular stabilization and angiogenesis. Finally, the host striatum had a marked increase in atrophic astrocytes in comparison with controls and grafts. The extent to which the lower number of large calibre vessels and astrocytes within the transplants contributed to suboptimal graft survival is unknown. The marked increase in atrophic astrocytes in the host brain surrounding the grafts suggests that reduced host trophic support may also contribute to poor graft survival in Huntington's disease. A better understanding of the way in which these components support allografted tissue is critical to the future development of cell-based therapies for the treatment of Huntington's disease.

Neuroinflammation is associated with changes in glial mGluR5 expression and the development of neonatal excitotoxic lesions.

It has been hypothesized that neuroinflammation triggered during brain development can alter brain functions later in life. We investigated the contribution of inflammation to the alteration of normal brain circuitries in the context of neuroexcitotoxicity following neonatal ventral hippocampal lesions in rats with ibotenic acid, an NMDA glutamate receptor agonist. Excitotoxic ibotenic acid lesions led to a significant and persistent astrogliosis and microglial activation, associated with the production of inflammatory mediators. This response was accompanied by a significant increase in metabotropic glutamate receptor type 5 (mGluR5) expression within two distinct neuroinflammatory cell types; astrocytes and microglia. The participation of inflammation to the neurotoxin-induced lesion was further supported by the prevention of hippocampal neuronal loss, glial mGluR5 expression and some of the behavioral perturbations associated to the excitotoxic lesion by concurrent anti-inflammatory treatment with minocycline. These results indicate that neuroinflammation significantly contributes to long-lasting excitotoxic effects of the neurotoxin and to some behavioral phenotypes associated with this model. Thus, the control of the inflammatory response may prevent the deleterious effects of excitotoxic processes that are triggered during brain development, limiting the risk to develop some of the behavioral manifestations related to these processes in adulthood.

Differences between subacute and chronic MPTP mice models: investigation of dopaminergic neuronal degeneration and alpha-synuclein inclusions.

Animal models are invaluable tools to study neurodegenerative disorders but a general consensus on the most accurate rodent model of Parkinson's disease has not been reached. Here, we examined how different methods of MPTP administration influence the degeneration of the dopaminergic (DA) system. Adult male C57BL/6 mice were treated with the same cumulative dose of MPTP following four distinct procedures: (i) subacute i.p. injections; (ii) 28-day chronic s.c. infusion; (iii) 28-day chronic i.p. infusion; and (iv) 14-day chronic i.p. infusion. Subacute MPTP treatment significantly affected all aspects of the DA system within the nigral and striatal territories. In contrast, the 28-day chronic s.c. infusion did not significantly alter any components of the DA system. The 28- and 14-day chronic i.p. infusions induced loss of tyrosine hydroxylase (TH)-positive cells correlated with a decrease in Nurr1 mRNA levels, but no significant decrease in the density of TH striatal fibers. Importantly, however, only the 14-day chronic MPTP i.p. infusion protocol promoted the formation of neuronal inclusions as noted by the expression of alpha-synuclein protein within the cytoplasm of TH nigral neurons. Overall, we found that the 14-day chronic MPTP i.p. infusion reproduces more accurately the pathological characteristics of early stage Parkinson's disease.

Dual-modality in vivo monitoring of subventricular zone stem cell migration and metabolism.

Rat subventricular zone (SVZ) stem cells were labeled with superparamagnetic iron oxide particles (SPIO) to follow their fate and migratory potential with magnetic resonance imaging (MRI) and positron emission tomography (PET). Labeled cells were transplanted into either the right rostral migratory stream (RMS) or striatum of normal adult Sprague-Dawley rats and serially followed for 3 months. Minimal migration of the cells implanted into the striatum was observed after 3 weeks whereas SVZ cells implanted into the RMS migrated toward the olfactory bulb at 1 week post-transplantation. PET studies of glucose metabolism using (18)F-FDG demonstrated enhanced glucose utilization in the striatum of transplanted animals. PET studies conducted 3 months after transplantation showed elevated accumulation of (11)C-raclopride (dopamine receptor type 2) and (11)C-CFT (dopamine transporter) binding in the striatal grafts. Implanted SVZ cells did not induce significant inflammation as identified by PET using (11)C-PK11195, a ligand detecting activated microglia. Histological analysis identified viable SPIO-labeled cells (some of which were nestin-positive) 7 weeks post-transplantation, suggesting a prolonged presence of undifferentiated neural stem cells within transplants. In addition, double immunostaining for neuronal and astrocytic markers (NeuN and GFAP) indicated that differentiation into neuronal and astrocytic phenotypes also occurred. Thus, combining MRI and PET enables monitoring of cell migration and metabolism non-invasively in vivo for extended periods of time.