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Papers by Philippe Hantraye

Alzheimers & Dementia, Jul 1, 2009

BEHAVIORAL ALTERATIONS AND NEUROPATHOLOGICAL CHANGES IN MOUSE LEMUR PRIMATES Marc Dhenain, Audrey... more BEHAVIORAL ALTERATIONS AND NEUROPATHOLOGICAL CHANGES IN MOUSE LEMUR PRIMATES Marc Dhenain, Audrey Kraska, Nadine Mestre-Frances, Olene Dorieux, Fanny Petit, Emmanuel Bourrin, Anne-Sophie Herard, Evelyne Chenu, Andreas Volk, Martine Perret, Philippe Hantraye, Fabienne Aujard, Jean-Luc Picq, URA CEA CNRS 2210, Fontenay aux Roses, France; URA CEA CNRS 2210, Orsay, France; INSERM U710EPHE, Montpellier, France; Institut Curie, Orsay, France; CNRS UMR 7179, MNHN, Brunoy, France; E.A. 2027, Université Paris 8, St Denis, France. Contact e-mail: Marc.Dhenain@cea.fr

bioRxiv (Cold Spring Harbor Laboratory), Apr 29, 2021

Lecture Notes in Computer Science, 2016

Immunohistochemistry is widely used as a gold standard to inspect tissues, characterize their str... more Immunohistochemistry is widely used as a gold standard to inspect tissues, characterize their structure and detect pathological alterations. As such, the joint analysis of histological images and other imaging modalities (MRI, PET) is of major interest to interpret these physical signals and establish their correspondence with the biological constitution of the tissues. However, it is challenging to provide a meaningful characterization of the signal specificity. In this paper, we propose an integrated method to quantitatively evaluate the discriminative power of imaging modalities. This method was validated using a macaque brain dataset containing: 3 immunohistochemically stained and 1 histochemically stained series, 1 photographic volume and 1 in vivo T2 weighted MRI. First, biological regions of interest (ROIs) were automatically delineated from histological sections stained for markers of interest and mapped on the target non-specific modalities through co-registration. These non-overlapping ROIs were considered ground truth for later classification. Voxels were evenly split in training and testing sets for a logistic regression model. The statistical significance of resulting accuracy scores was evaluated through null distribution simulations. Such an approach could be of major interest to assess relevant biological characteristics from various imaging modalities.

Alzheimers & Dementia, Jul 1, 2013

Brainstem 48.39 2.9 0.76 0.1 Cerebral cortex 166.24 1.38 9.78 0.64 Corpus callosum 12.37 0.1 3.52... more Brainstem 48.39 2.9 0.76 0.1 Cerebral cortex 166.24 1.38 9.78 0.64 Corpus callosum 12.37 0.1 3.52 0.25 Fimbria 3.92 0.06 1.47 0.26 Hippocampal region 26.97 0.46 5.6 0.39 Hypothalamus 7.66 0.4 0.33 0.18 Internal capsule 3.04 0.05 0.12 0.02 Olfactory bulbs NA NA 9.03 0.71 Striatum 32.83 0.46 0.93 0.05 Thalamus 36.72 1.02 0.86 0.14 Ventricular system 4.88 0.07 1.35 0.24 Brain 346.73 4.13 5.58 0.91 Michel Vandenberghe, Anne-Sophie H erard, Nicolas Souedet, Hantraye Philippe, Marc Dhenain, Thierry Delzescaux, Commissariat a l’Energie Atomique et aux Energies Alternatives (CEA), Fontenay-aux-Roses, France; CNRS, Fontenay-aux-Roses, France. Contact e-mail: michel.vandenberghe@cea.fr

In biomedical research, cell counting is important to assess physiological and pathophysiological... more In biomedical research, cell counting is important to assess physiological and pathophysiological information. However, the automated analysis of microscopic images of tissues remains extremely challenging. We propose an automated processing protocol for proper segmentation of individual cells in microscopic images. A Gaussian filter is applied to improve signal to noise ratio (SNR) then an original min-max method is proposed to produce an image in which information describing both cell centers (minima) and boundaries are enhanced. Finally, a contour-based model initialized from minima in the min-max cartography is carried out to achieve cell individualization. This method is evaluated on a NeuN-stained macaque brain section in sub-regions presenting various levels of fraction of neuron surface occupation. Comparison with several methods of reference demonstrates that the performances of our method are superior. A first application to the segmentation of neurons in the hippocampus illustrates the ability of our approach to deal with massive and complex data.

Frontiers in Neuroscience, Nov 15, 2018

Recently developed techniques to visualize immunostained tissues in 3D and in large samples have ... more Recently developed techniques to visualize immunostained tissues in 3D and in large samples have expanded the scope of microscopic investigations at the level of the whole brain. Here, we propose to adapt voxel-based statistical analysis to 3D high-resolution images of the immunostained rodent brain. The proposed approach was first validated with a simulation dataset with known cluster locations. Then, it was applied to characterize the effect of ADAM30, a gene involved in the metabolism of the amyloid precursor protein, in a mouse model of Alzheimer's disease. This work introduces voxel-based analysis of 3D immunostained microscopic brain images and, therefore, opens the door to localized whole-brain exploratory investigation of pathological markers and cellular alterations.

HAL (Le Centre pour la Communication Scientifique Directe), Oct 17, 2015

International audienceDouble-cortin-like kinases (Dclks) play major roles in development and main... more International audienceDouble-cortin-like kinases (Dclks) play major roles in development and maintenance of neuronal functions. The neurobiological role of the third member of this family, the striatal-enriched Dclk3 is unknown. We characterized the cytoplasmic and nuclear localization of Dclk3 in the striatum in non-human primates and in man. A yeast-two hybrid screen identified seven Dclk3 interactors, all possessing zinc finger domains. One candidate is known to regulate many transcription activating factors (TAFs), p53 and lysine acetyl transferases. In line with this, expression of Dclk3 kinase domain in human striatal neural stem cells produces transcriptional changes that are mainly linked to chromatin remodeling. Chromatin and transcriptional perturbations have been described in 4/6/202

HAL (Le Centre pour la Communication Scientifique Directe), Oct 13, 2012

International audienceThe mechanisms underlying the preferential vulnerability of striatal neuron... more International audienceThe mechanisms underlying the preferential vulnerability of striatal neurons to mutant Huntingtin (mHtt) in Huntington's disease (HD) remain unknown. Our hypothesis, supported by recent publications, is that genes selectively expressed in the striatum may play a role in this susceptibility to mHtt. In the present study we focused on a product of the 2010001M06rik (St102) gene, previously identified based on its preferential expression in the striatum and its significantly reduced levels in the striatum of R6/2 mice (Brochier et al., Physiol Genomics, 2008). We examined whether modifying St102 expression could change the neurotoxic effects of an N-terminal fragment of mHtt expressed in the mouse striatum using the HD lentiviral model (LV-Htt171-82Q). We developed lentiviral vectors to overexpress St102 (LV-St102) or knock-down St102 using a selective shRNA (LV-shRNA-St102). RT-PCR analysis infection of the striatum of adult mice with LV-St102 or LV-shRNA-St102 led to a significant increase or decrease of St102 expression respectively, without producing overt alterations as assessed using immunohistochemistry (IHC) of NeuN and DARPP32. LV-St102 and LV-shRNA-St102 were co-injected with LV-Htt171-82Q in the striatum of adult WT mice. Six weeks after injections, LV-Htt171-82Q consistently produced striatal lesions characterized by a loss of NeuN and DARPP32. Interestingly, the co-expression of Htt171-82Q and shRNA-St102 led to a significant increase in the lesion volume. On the contrary, the co-expression of Htt171-82Q and St102 overexpression led to a significant decrease of the lesion size. These results suggest that the loss of St102 expression could play a role in striatal degeneration in HD

Brain, 2021

Deposits of different abnormal forms of tau in neurons and astrocytes represent key anatomo-patho... more Deposits of different abnormal forms of tau in neurons and astrocytes represent key anatomo-pathological features of tauopathies. Although tau protein is highly enriched in neurons and poorly expressed by astrocytes, the origin of astrocytic tau is still elusive. Here, we used innovative gene transfer tools to model tauopathies in adult mouse brains and to investigate the origin of astrocytic tau. We showed in our adeno-associated virus (AAV)-based models and in Thy-Tau22 transgenic mice that astrocytic tau pathology can emerge secondarily to neuronal pathology. By designing an in vivo reporter system, we further demonstrated bidirectional exchanges of tau species between neurons and astrocytes. We then determined the consequences of tau accumulation in astrocytes on their survival in models displaying various status of tau aggregation. Using stereological counting of astrocytes, we report that, as for neurons, soluble tau species are highly toxic to some subpopulations of astrocyte...

Pathogenic mechanisms, 2018

Astrocytes are essential partners for neurons and their role in Huntington’s disease (HD) is emer... more Astrocytes are essential partners for neurons and their role in Huntington’s disease (HD) is emerging. In HD, astrocytes change and become reactive. Astrocyte reactivity is characterized by morphological changes but its functional impact is still unclear. To understand the roles of reactive astrocytes in HD, we have developed viral vectors that infect selectively astrocytes in vivo and either block or induce reactivity, through manipulation of the JAK2-STAT3 pathway. We used these vectors to modulate astrocyte reactivity in two complementary mouse models of HD [knock-in Hdh140 mice and lentivirus-mediated expression of a fragment of mutated Huntingtin (mHtt) in striatal neurons]. In these two models, we found that reactive astrocytes decrease the number and size of mHtt aggregates. How can reactive astrocytes reduce the aggregation of mHtt within neurons? We performed whole-genome transcriptomic analysis of acutely sorted reactive astrocytes to identify genes regulated by the JAK2-STAT3 pathway in astrocytes. We found an enrichment in genes linked to autophagy-lysosome and ubiquitin-proteasome systems, suggesting that reactive astrocytes have an enhanced capacity for protein degradation and could siphon mHtt away from neurons. Moreover, we identified several chaperones upregulated in reactive astrocytes. Chaperones prevent protein aggregation and can be released extracellularly. They could reduce mHtt aggregation within neurons themselves. Our data show that astrocytes develop a protective response in HD that involves bidirectional signaling with neurons to reduce mHtt aggregation. Reactive astrocytes are not only defective cells as usually reported, but also acquire enhanced capacities to promote mHtt clearance, which has strong therapeutic relevance for HD.

Neurobiology of Disease, 2019

In Alzheimer disease (AD), astrocytes undergo complex changes and become reactive. The consequenc... more In Alzheimer disease (AD), astrocytes undergo complex changes and become reactive. The consequences of this reaction are still unclear. To evaluate the net impact of reactive astrocytes in AD, we recently developed viral vectors targeting astrocytes that either activate or inhibit the JAK2-STAT3 pathway, a central cascade controlling astrocyte reaction. We aimed to evaluate whether reactive astrocytes contribute to Tau as well as amyloid pathologies in the hippocampus of 3xTg-AD mice, an AD model that develops Tau hyperphosphorylation and aggregation in addition to amyloid deposition. JAK2-STAT3 pathway-mediated modulation of reactive astrocytes in the hippocampus of 3xTg-AD mice, did not significantly influence Tau phosphorylation or amyloid processing and deposition, at early, advanced and terminal stage of the disease. Interestingly, inhibition of the JAK2-STAT3 pathway in hippocampal astrocytes did not improve short-term spatial memory in the Y maze but it reduced anxiety in the...

SSRN Electronic Journal, 2019

Molecular Therapy, 2016

Almost 90 years ago, Canavan Disease (CD) was described for the first time. Since then, the scien... more Almost 90 years ago, Canavan Disease (CD) was described for the first time. Since then, the scientific community has unsuccessfully sought to cure this devastating leukodystrophy and understand its pathomechanism. Early hope for gene therapy was fueled with the cloning of the disease causing gene, Aspartoacylase, in 1993. Unfortunately, the only clinical trial for CD gene therapy failed to show significant clinical improvements in Canavan patients. At that time, animal models for CD were just engineered and comprehensive pre-clinical evaluation of CD gene therapy was missing. Earlier, we reported that our 1 st generation IV delivered pre-clinical gene therapy was able to rescue early lethality and partially restored motor function in a mouse model of CD. Now in its 3 rd generation, our gene therapy cures the disease in Canavan mice by a single intravenous injection, shown by behavioral, cognitive, and neuropathology tests. Taking advantage of this complete reversal of the disease, we used whole brain neurometabolome profiling to closely monitor the molecular efficacy and mechanism(s) in curing Canavan disease in mouse. Hierarchical cluster analysis (HCA) shows complete restoration of the disease associated metabolic derangements, including the array of detected myelin lipids. In the next step, we hypothesized that the metabolic nature of Canavan Disease mandates the origin of its pathomechanism in the metabolic regulation. We identified a specific dysregulation in the energy metabolism in vitro and in vivo that suggests the self-digestion of myelin for energetic purposes, calling current hypotheses about the Canavan disease pathomechanism into question. Currently, we are intensifying our insight into this mechanism by mircoRNAome, transcriptome analyses, a series of in vitro cell culture models, as well as supplementary and alternative strategies for the treatment of Canavan Disease. In summary, our data demonstrates strong evidence that rAAV mediated pre-clinical gene therapy not only cures the Canavan phenotype but also corrects the extensive neurometabolome, which provides meticulous evidence for gene therapy's high efficacy. Furthermore, we revealed a new pathomechanism that supports a paradigm shift in our perception of the function of ASPA and NAA in Canavan disease and their potential implications in other CNS and metabolic disorders in general.

Alzheimers & Dementia, Jul 1, 2009

BEHAVIORAL ALTERATIONS AND NEUROPATHOLOGICAL CHANGES IN MOUSE LEMUR PRIMATES Marc Dhenain, Audrey... more BEHAVIORAL ALTERATIONS AND NEUROPATHOLOGICAL CHANGES IN MOUSE LEMUR PRIMATES Marc Dhenain, Audrey Kraska, Nadine Mestre-Frances, Olene Dorieux, Fanny Petit, Emmanuel Bourrin, Anne-Sophie Herard, Evelyne Chenu, Andreas Volk, Martine Perret, Philippe Hantraye, Fabienne Aujard, Jean-Luc Picq, URA CEA CNRS 2210, Fontenay aux Roses, France; URA CEA CNRS 2210, Orsay, France; INSERM U710EPHE, Montpellier, France; Institut Curie, Orsay, France; CNRS UMR 7179, MNHN, Brunoy, France; E.A. 2027, Université Paris 8, St Denis, France. Contact e-mail: Marc.Dhenain@cea.fr

bioRxiv (Cold Spring Harbor Laboratory), Apr 29, 2021

Lecture Notes in Computer Science, 2016

Immunohistochemistry is widely used as a gold standard to inspect tissues, characterize their str... more Immunohistochemistry is widely used as a gold standard to inspect tissues, characterize their structure and detect pathological alterations. As such, the joint analysis of histological images and other imaging modalities (MRI, PET) is of major interest to interpret these physical signals and establish their correspondence with the biological constitution of the tissues. However, it is challenging to provide a meaningful characterization of the signal specificity. In this paper, we propose an integrated method to quantitatively evaluate the discriminative power of imaging modalities. This method was validated using a macaque brain dataset containing: 3 immunohistochemically stained and 1 histochemically stained series, 1 photographic volume and 1 in vivo T2 weighted MRI. First, biological regions of interest (ROIs) were automatically delineated from histological sections stained for markers of interest and mapped on the target non-specific modalities through co-registration. These non-overlapping ROIs were considered ground truth for later classification. Voxels were evenly split in training and testing sets for a logistic regression model. The statistical significance of resulting accuracy scores was evaluated through null distribution simulations. Such an approach could be of major interest to assess relevant biological characteristics from various imaging modalities.

Alzheimers & Dementia, Jul 1, 2013

Brainstem 48.39 2.9 0.76 0.1 Cerebral cortex 166.24 1.38 9.78 0.64 Corpus callosum 12.37 0.1 3.52... more Brainstem 48.39 2.9 0.76 0.1 Cerebral cortex 166.24 1.38 9.78 0.64 Corpus callosum 12.37 0.1 3.52 0.25 Fimbria 3.92 0.06 1.47 0.26 Hippocampal region 26.97 0.46 5.6 0.39 Hypothalamus 7.66 0.4 0.33 0.18 Internal capsule 3.04 0.05 0.12 0.02 Olfactory bulbs NA NA 9.03 0.71 Striatum 32.83 0.46 0.93 0.05 Thalamus 36.72 1.02 0.86 0.14 Ventricular system 4.88 0.07 1.35 0.24 Brain 346.73 4.13 5.58 0.91 Michel Vandenberghe, Anne-Sophie H erard, Nicolas Souedet, Hantraye Philippe, Marc Dhenain, Thierry Delzescaux, Commissariat a l’Energie Atomique et aux Energies Alternatives (CEA), Fontenay-aux-Roses, France; CNRS, Fontenay-aux-Roses, France. Contact e-mail: michel.vandenberghe@cea.fr

In biomedical research, cell counting is important to assess physiological and pathophysiological... more In biomedical research, cell counting is important to assess physiological and pathophysiological information. However, the automated analysis of microscopic images of tissues remains extremely challenging. We propose an automated processing protocol for proper segmentation of individual cells in microscopic images. A Gaussian filter is applied to improve signal to noise ratio (SNR) then an original min-max method is proposed to produce an image in which information describing both cell centers (minima) and boundaries are enhanced. Finally, a contour-based model initialized from minima in the min-max cartography is carried out to achieve cell individualization. This method is evaluated on a NeuN-stained macaque brain section in sub-regions presenting various levels of fraction of neuron surface occupation. Comparison with several methods of reference demonstrates that the performances of our method are superior. A first application to the segmentation of neurons in the hippocampus illustrates the ability of our approach to deal with massive and complex data.

Frontiers in Neuroscience, Nov 15, 2018

Recently developed techniques to visualize immunostained tissues in 3D and in large samples have ... more Recently developed techniques to visualize immunostained tissues in 3D and in large samples have expanded the scope of microscopic investigations at the level of the whole brain. Here, we propose to adapt voxel-based statistical analysis to 3D high-resolution images of the immunostained rodent brain. The proposed approach was first validated with a simulation dataset with known cluster locations. Then, it was applied to characterize the effect of ADAM30, a gene involved in the metabolism of the amyloid precursor protein, in a mouse model of Alzheimer's disease. This work introduces voxel-based analysis of 3D immunostained microscopic brain images and, therefore, opens the door to localized whole-brain exploratory investigation of pathological markers and cellular alterations.

HAL (Le Centre pour la Communication Scientifique Directe), Oct 17, 2015

International audienceDouble-cortin-like kinases (Dclks) play major roles in development and main... more International audienceDouble-cortin-like kinases (Dclks) play major roles in development and maintenance of neuronal functions. The neurobiological role of the third member of this family, the striatal-enriched Dclk3 is unknown. We characterized the cytoplasmic and nuclear localization of Dclk3 in the striatum in non-human primates and in man. A yeast-two hybrid screen identified seven Dclk3 interactors, all possessing zinc finger domains. One candidate is known to regulate many transcription activating factors (TAFs), p53 and lysine acetyl transferases. In line with this, expression of Dclk3 kinase domain in human striatal neural stem cells produces transcriptional changes that are mainly linked to chromatin remodeling. Chromatin and transcriptional perturbations have been described in 4/6/202

HAL (Le Centre pour la Communication Scientifique Directe), Oct 13, 2012

International audienceThe mechanisms underlying the preferential vulnerability of striatal neuron... more International audienceThe mechanisms underlying the preferential vulnerability of striatal neurons to mutant Huntingtin (mHtt) in Huntington's disease (HD) remain unknown. Our hypothesis, supported by recent publications, is that genes selectively expressed in the striatum may play a role in this susceptibility to mHtt. In the present study we focused on a product of the 2010001M06rik (St102) gene, previously identified based on its preferential expression in the striatum and its significantly reduced levels in the striatum of R6/2 mice (Brochier et al., Physiol Genomics, 2008). We examined whether modifying St102 expression could change the neurotoxic effects of an N-terminal fragment of mHtt expressed in the mouse striatum using the HD lentiviral model (LV-Htt171-82Q). We developed lentiviral vectors to overexpress St102 (LV-St102) or knock-down St102 using a selective shRNA (LV-shRNA-St102). RT-PCR analysis infection of the striatum of adult mice with LV-St102 or LV-shRNA-St102 led to a significant increase or decrease of St102 expression respectively, without producing overt alterations as assessed using immunohistochemistry (IHC) of NeuN and DARPP32. LV-St102 and LV-shRNA-St102 were co-injected with LV-Htt171-82Q in the striatum of adult WT mice. Six weeks after injections, LV-Htt171-82Q consistently produced striatal lesions characterized by a loss of NeuN and DARPP32. Interestingly, the co-expression of Htt171-82Q and shRNA-St102 led to a significant increase in the lesion volume. On the contrary, the co-expression of Htt171-82Q and St102 overexpression led to a significant decrease of the lesion size. These results suggest that the loss of St102 expression could play a role in striatal degeneration in HD

Brain, 2021

Deposits of different abnormal forms of tau in neurons and astrocytes represent key anatomo-patho... more Deposits of different abnormal forms of tau in neurons and astrocytes represent key anatomo-pathological features of tauopathies. Although tau protein is highly enriched in neurons and poorly expressed by astrocytes, the origin of astrocytic tau is still elusive. Here, we used innovative gene transfer tools to model tauopathies in adult mouse brains and to investigate the origin of astrocytic tau. We showed in our adeno-associated virus (AAV)-based models and in Thy-Tau22 transgenic mice that astrocytic tau pathology can emerge secondarily to neuronal pathology. By designing an in vivo reporter system, we further demonstrated bidirectional exchanges of tau species between neurons and astrocytes. We then determined the consequences of tau accumulation in astrocytes on their survival in models displaying various status of tau aggregation. Using stereological counting of astrocytes, we report that, as for neurons, soluble tau species are highly toxic to some subpopulations of astrocyte...

Pathogenic mechanisms, 2018

Astrocytes are essential partners for neurons and their role in Huntington’s disease (HD) is emer... more Astrocytes are essential partners for neurons and their role in Huntington’s disease (HD) is emerging. In HD, astrocytes change and become reactive. Astrocyte reactivity is characterized by morphological changes but its functional impact is still unclear. To understand the roles of reactive astrocytes in HD, we have developed viral vectors that infect selectively astrocytes in vivo and either block or induce reactivity, through manipulation of the JAK2-STAT3 pathway. We used these vectors to modulate astrocyte reactivity in two complementary mouse models of HD [knock-in Hdh140 mice and lentivirus-mediated expression of a fragment of mutated Huntingtin (mHtt) in striatal neurons]. In these two models, we found that reactive astrocytes decrease the number and size of mHtt aggregates. How can reactive astrocytes reduce the aggregation of mHtt within neurons? We performed whole-genome transcriptomic analysis of acutely sorted reactive astrocytes to identify genes regulated by the JAK2-STAT3 pathway in astrocytes. We found an enrichment in genes linked to autophagy-lysosome and ubiquitin-proteasome systems, suggesting that reactive astrocytes have an enhanced capacity for protein degradation and could siphon mHtt away from neurons. Moreover, we identified several chaperones upregulated in reactive astrocytes. Chaperones prevent protein aggregation and can be released extracellularly. They could reduce mHtt aggregation within neurons themselves. Our data show that astrocytes develop a protective response in HD that involves bidirectional signaling with neurons to reduce mHtt aggregation. Reactive astrocytes are not only defective cells as usually reported, but also acquire enhanced capacities to promote mHtt clearance, which has strong therapeutic relevance for HD.

Neurobiology of Disease, 2019

In Alzheimer disease (AD), astrocytes undergo complex changes and become reactive. The consequenc... more In Alzheimer disease (AD), astrocytes undergo complex changes and become reactive. The consequences of this reaction are still unclear. To evaluate the net impact of reactive astrocytes in AD, we recently developed viral vectors targeting astrocytes that either activate or inhibit the JAK2-STAT3 pathway, a central cascade controlling astrocyte reaction. We aimed to evaluate whether reactive astrocytes contribute to Tau as well as amyloid pathologies in the hippocampus of 3xTg-AD mice, an AD model that develops Tau hyperphosphorylation and aggregation in addition to amyloid deposition. JAK2-STAT3 pathway-mediated modulation of reactive astrocytes in the hippocampus of 3xTg-AD mice, did not significantly influence Tau phosphorylation or amyloid processing and deposition, at early, advanced and terminal stage of the disease. Interestingly, inhibition of the JAK2-STAT3 pathway in hippocampal astrocytes did not improve short-term spatial memory in the Y maze but it reduced anxiety in the...

SSRN Electronic Journal, 2019

Molecular Therapy, 2016

Almost 90 years ago, Canavan Disease (CD) was described for the first time. Since then, the scien... more Almost 90 years ago, Canavan Disease (CD) was described for the first time. Since then, the scientific community has unsuccessfully sought to cure this devastating leukodystrophy and understand its pathomechanism. Early hope for gene therapy was fueled with the cloning of the disease causing gene, Aspartoacylase, in 1993. Unfortunately, the only clinical trial for CD gene therapy failed to show significant clinical improvements in Canavan patients. At that time, animal models for CD were just engineered and comprehensive pre-clinical evaluation of CD gene therapy was missing. Earlier, we reported that our 1 st generation IV delivered pre-clinical gene therapy was able to rescue early lethality and partially restored motor function in a mouse model of CD. Now in its 3 rd generation, our gene therapy cures the disease in Canavan mice by a single intravenous injection, shown by behavioral, cognitive, and neuropathology tests. Taking advantage of this complete reversal of the disease, we used whole brain neurometabolome profiling to closely monitor the molecular efficacy and mechanism(s) in curing Canavan disease in mouse. Hierarchical cluster analysis (HCA) shows complete restoration of the disease associated metabolic derangements, including the array of detected myelin lipids. In the next step, we hypothesized that the metabolic nature of Canavan Disease mandates the origin of its pathomechanism in the metabolic regulation. We identified a specific dysregulation in the energy metabolism in vitro and in vivo that suggests the self-digestion of myelin for energetic purposes, calling current hypotheses about the Canavan disease pathomechanism into question. Currently, we are intensifying our insight into this mechanism by mircoRNAome, transcriptome analyses, a series of in vitro cell culture models, as well as supplementary and alternative strategies for the treatment of Canavan Disease. In summary, our data demonstrates strong evidence that rAAV mediated pre-clinical gene therapy not only cures the Canavan phenotype but also corrects the extensive neurometabolome, which provides meticulous evidence for gene therapy's high efficacy. Furthermore, we revealed a new pathomechanism that supports a paradigm shift in our perception of the function of ASPA and NAA in Canavan disease and their potential implications in other CNS and metabolic disorders in general.

Alzheimer's & Dementia, 2011

disease (AD) and neurodegeneration. Vitamin D has been shown to down-regulate the L-type voltage-... more disease (AD) and neurodegeneration. Vitamin D has been shown to down-regulate the L-type voltage-sensitive calcium channels, LVSCC-A1C and LVSCC-A1D, and up-regulate (nerve growth factor) NGF. However, expression of these proteins when VDR is repressed is unknown. The aim of this study is to investigate LVSCC-A1C, LVSCC-A1D, calbindin-D28k, inducible nitric oxide synthase (iNOS) expressions and NGF release in VDR-silenced primary cortical neurons. Methods: qRT-PCR and western blots were performed to determine VDR, LVSCC-A1C, LVSCC-A1D, calbindin-D28k and iNOS expression levels. NGF and cytotoxicity levels were determined by ELISA. Apoptosis was determined by TUNEL. Results: Our findings illustrate that LVSCC-A1C and iNOS expressions increased rapidly in cortical neurons when VDR is down-regulated, whereas, LVSCC-A1D levels did not change, NGF release calbindin-D28k expression decreased in response to VDR down-regulation. Although vitamin D regulates LVSCC-A1C through VDR, it may not regulate LVSCC-A1D through VDR. Conclusions: Our results indicate that suppression of VDR or vitamin D-VDR pathway disruption disrupts LVSCC-A1C, calbindin-D28k and NGF production and increase oxidative stress. Thus it can make neurons vulnerable to aging and neurodegeneration, and when combined with Aß toxicity, it is possible to explain some of the events that occur during neurodegeneration.