Javier Diaz-Nido | Universidad Autónoma de Madrid (original) (raw)
Papers by Javier Diaz-Nido
Gene Therapy, 2011
Novel gene-based therapies for disease will depend in many cases on long-term persistent transgen... more Novel gene-based therapies for disease will depend in many cases on long-term persistent transgene expression. To develop gene therapy strategies for Friedreich's ataxia (FRDA), we have examined the persistence of transgene expression in the brain in vivo provided by the entire 135 kb FXN genomic DNA locus delivered as an infectious bacterial artificial chromosome (iBAC) herpes simplex virus type 1 (HSV-1)-based vector injected in the adult mouse cerebellum. We constructed genomic DNA-reporter fusion vectors carrying a complete 135 kb FXN genomic locus with an insertion of the Escherichia coli lacZ gene at the ATG start codon (iBAC-FXN-lacZ). SHSY5Y human neuroblastoma cells transduced by iBAC-FXN-lacZ showed high efficiency of vector delivery and LacZ expression. Direct intracranial injection of iBAC-FXN-lacZ into the adult mouse cerebellum resulted in a large number of easily detectable transduced cells, with LacZ expression driven by the FXN genomic locus, which persisted for at least 75 days. Green fluorescent protein expression driven from the same vector but by the strong HSV-1 IE4/5 promoter was transient. Our data demonstrate for the first time sustained transgene expression in vivo by infectious delivery of a genomic DNA locus 4100 kb in size. Such an approach may be suitable for gene rescue strategies in neurological disease, such as FRDA.
Friedreich's ataxia (FA) is a recessive, predominantly neurodegenerative disorder caused in most ... more Friedreich's ataxia (FA) is a recessive, predominantly neurodegenerative disorder caused in most cases by mutations in the first intron of the frataxin (FXN) gene. This mutation drives the expansion of a homozygous GAA repeat that results in decreased levels of FXN transcription and frataxin protein. Frataxin (Fxn) is a ubiquitous mitochondrial protein involved in iron-sulfur cluster biogenesis, and a decrease in the levels of this protein is responsible for the symptoms observed in the disease. Although the pathological manifestations of FA are mainly observed in neurons of both the central and peripheral nervous system, it is not clear if changes in non-neuronal cells may also contribute to the pathogenesis of FA, as recently suggested for other neurodegenerative disorders. Therefore, the aims of this study were to generate and characterize a cell model of Fxn deficiency in human astrocytes (HAs) and to evaluate the possible involvement of non-cell autonomous processes in FA. To knockdown frataxin in vitro, we transduced HAs with a specific shRNA lentivirus (shRNA37), which produced a decrease in both frataxin mRNA and protein expression, along with mitochondrial superoxide production, and signs of p53-mediated cell cycle arrest and apoptotic cell death. To test for non-cell autonomous interactions we cultured wild-type mouse neurons in the presence of frataxin-deficient astrocyte conditioned medium, which provoked a delay in the maturation of these neurons, a decrease in neurite length and enhanced cell death. Our findings confirm a detrimental effect of frataxin silencing, not only for astrocytes, but also for neuron-glia interactions, underlining the need to take into account the role of non-cell autonomous processes in FA.
Current gene therapy, Jan 1, 2009
Neurochemistry international, Jan 1, 2007
Purkinje cells play a crucial role in sensory motor coordination since they are the only output p... more Purkinje cells play a crucial role in sensory motor coordination since they are the only output projection neurons in the cerebellar cortex and are affected in most spinocerebellar ataxias. They stand out in the central nervous system due to their large size and their profusely branched dendritic arbor. However, molecular and cellular studies on Purkinje cells are often hampered by the difficulty of maintaining these cells in culture. Here we report an easy, robust and reproducible method to obtain Purkinje-enriched mixed cerebellar cell cultures from day 16 mouse embryos using papain digestion and a semi-defined culture medium, being the composition of the culture approximately 20% Purkinje cells, 70% non-Purkinje neurons and 10% glial cells. We demonstrate that efficient gene transfer into Purkinje cells (as well as into other cerebellar populations) is possible using herpes simplex virus-1 (HSV-1)-derived vectors. Indeed, up to 50% of the Purkinje cells can be transduced and gene expression may persist for at least 14 days. As a result, this procedure permits functional gene expression studies to be carried out on cultured Purkinje neurons. To demonstrate this, we show that the expression of a dominant-negative form of glycogen synthase kinase-3 protects Purkinje neurons against cell death triggered by a chemical inhibitor of phosphatidylinositol-3 kinase. In summary, we have established reproducible and reliable cerebellar cell cultures enriched for Purkinje cells which enables gene transfer studies to be carried out using herpesviral vectors.
Human gene …, Jan 1, 2002
The transduction of cerebellar neurons in vivo with herpes simplex virus 1 (HSV-1) amplicon carry... more The transduction of cerebellar neurons in vivo with herpes simplex virus 1 (HSV-1) amplicon carrying the lacZ gene has been investigated after injection of the vector in the cerebellar cortex, ventricles, and inferior olive of adult rats. Injection into the cerebellar cortex resulted in transduction of Purkinje cells near the needle tract and injection into the ventricles yielded no transduced neurons. In contrast, high transduction efficiency was achieved by vector injection into the inferior olive, resulting in one of three positive Purkinje cells all over the ipsilateral and contralateral cerebellar hemispheres. Because neurons in the deep cerebellar nuclei are also transduced, we suggest that the vector is delivered from the inferior olive to the cerebellar nuclei and then to Purkinje cells by retrograde axonal transport. Expression of the lacZ gene within Purkinje cells was surprisingly persistent and was maintained at the same level for at least 40 days. Importantly, no signs of either toxicity or inflammation were observed in the cerebellum after vector injection, except for the borders of the needle tract where some reactive astrocytes were detected. Indeed, motor coordination of treated animals was entirely normal, as assessed by the rota-rod test. These results demonstrate that HSV-1 amplicon vectors can effect safe and stable transgene expression in Purkinje cells in vivo, raising the possibility of using these vectors for long-term gene therapy of human cerebellar disorders.
Journal of neuroscience …, Jan 1, 2006
Gene transfer is a powerful tool for functional gene analysis in human cells. In this respect, th... more Gene transfer is a powerful tool for functional gene analysis in human cells. In this respect, there is a need to develop experimental models that involve homogeneous cultures of human neuron-like cells susceptible to gene transduction and that are easy to handle. Here we describe an optimized and reproducible procedure to differentiate human SH-SY5Y neuroblastoma cells into a homogeneous population of neuron-like cells. The fully differentiated cells are postmitotic and resemble primary cultured neurons in terms of their cytoskeletal polarity. Notably, differentiated SH-SY5Y cells are far more susceptible to transduction by herpes simplex virus (HSV-1)-based vectors than proliferating SH-SY5Y cells. This increase in transduction efficiency after neuronal differentiation may be due to the up-regulation of cell surface receptors for herpesvirus entry. In summary, we propose that fully differentiated human neuron-like cells obtained from the SH-SY5Y neuroblastoma may constitute an excellent and versatile experimental tool for gene transfer and functional genomic studies with HSV-1 vectors.
Journal of Biological …, Jan 1, 2009
Journal of …, Jan 1, 2010
A typical feature of Parkinson&am... more A typical feature of Parkinson's disease is the progressive loss of dopaminergic neurons in the substantia nigra, in which inhibition of mitochondrial complex I activity may play an important role. Rotenone or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) inhibit the mitochondrial complex I and they cause the death of substantia nigra dopaminergic neurons, thereby providing acute murine models of Parkinson's disease. We have found that increasing mitochondrial hexokinase II activity can prevent cell death in neuronal cultures treated with rotenone. As a result, we have studied the effects of hexokinase II gene transfer in vivo using a herpes simplex virus type 1 (HSV-1) amplicon vector. The placHK2 amplicon vector was injected into substantia nigra of mice that were subsequently administered rotenone or MPTP. Overexpression of hexokinase II prevented both rotenone and MPTP-induced dopaminergic neuronal cell death, as well as reducing the associated motor defects. Our results provide the first proof-of-principle that hexokinase II protects against dopaminergic neurodegeneration in vivo, emphasizing the role of this enzyme in promoting neuronal survival. Thus, the increase of hexokinase II expression by gene transfer or other means represents a promising approach to treat Parkinson's and other neurodegenerative diseases.
The Journal of …, Jan 1, 2006
Neurochemistry …, Jan 1, 2007
Journal of …, Jan 1, 2003
Molecular …, Jan 1, 2006
Immortalized cell lines of olfactory ensheathing glia (OEG) that maintain the proregenerative pro... more Immortalized cell lines of olfactory ensheathing glia (OEG) that maintain the proregenerative properties of primary cultures provide an unlimited source of OEG for both basic and applied studies. Indeed, one specific immortalized rat OEG clonal line (TEG3) proved to be as good as primary OEG in promoting neuritogenesis and axon regeneration in culture models. Thus, we examined the capacity of TEG3 to promote axonal repair in an animal model of spinal cord injury, dorsal column crush. TEG3 cells can acquire astrocyte-like or Schwann cell-like morphology depending on the conditions under which they are cultured. In the injured spinal cord, prelabeled TEG3 survived for at least 10 weeks after grafting and they integrated into the spinal cord, adopting Schwann cell-like, astrocyte-like, or intermediate morphologies. In TEG3-transplanted animals, sensory projection axons grow into the lesion site and there was robust sprouting/axonal growth of the corticospinal tract, both into and beyond the lesion site, after crushing of the spinal cord-dorsal columns. TEG3-transplanted animals also recovered sensory and motor function in tape removal and beam walking behavioral tests. These data indicate that certain immortalized cell lines derived from a single cell can maintain the regenerative properties of primary OEG.
Journal of …, Jan 1, 2003
… of Biomedicine and …, Jan 1, 2002
Olfactory ensheathing glia (OEG) accompany olfactory growing axons in their entry to the adult ma... more Olfactory ensheathing glia (OEG) accompany olfactory growing axons in their entry to the adult mammalian central nervous system (CNS). Due to this special characteristic, considerable attention has been focused on the possibility of using OEG for CNS regeneration. OEG present a large heterogeneity in culture with respect to their cellular morphology and expressed molecules. The specific characteristics of OEG responsible for their regenerative properties have to be defined. These properties probably result from the combination of several factors: molecular composition of the membrane (expressing adhesion molecules as PSA-NCAM, L1 and/or others) combined with their ability to reduce glial scarring and to accompany new growing axons into the host CNS. Their capacity to produce some neurotrophic factors might also account for their ability to produce CNS regeneration.
Journal of Biological …, Jan 1, 2003
FEBS letters, Jan 1, 1999
The deposition of beta-amyloid peptide (A beta), the hyperphosphorylation of tau protein and the ... more The deposition of beta-amyloid peptide (A beta), the hyperphosphorylation of tau protein and the death of neurons in certain brain regions are characteristic features of Alzheimer's disease. It has been proposed that the accumulation of aggregates of A beta is the trigger of neurodegeneration in this disease. In support of this view, several studies have demonstrated that the treatment of cultured neurons with A beta leads to the hyperphosphorylation of tau protein and neuronal cell death. Here we report that lithium prevents the enhanced phosphorylation of tau protein at the sites recognized by antibodies Tau-1 and PHF-1 which occurs when cultured rat cortical neurons are incubated with A beta. Interestingly, lithium also significantly protects cultured neurons from A beta-induced cell death. These results raise the possibility of using chronic lithium treatment for the therapy of Alzheimer's disease.
Biochemical …, Jan 1, 2003
Gene Therapy, 2011
Novel gene-based therapies for disease will depend in many cases on long-term persistent transgen... more Novel gene-based therapies for disease will depend in many cases on long-term persistent transgene expression. To develop gene therapy strategies for Friedreich's ataxia (FRDA), we have examined the persistence of transgene expression in the brain in vivo provided by the entire 135 kb FXN genomic DNA locus delivered as an infectious bacterial artificial chromosome (iBAC) herpes simplex virus type 1 (HSV-1)-based vector injected in the adult mouse cerebellum. We constructed genomic DNA-reporter fusion vectors carrying a complete 135 kb FXN genomic locus with an insertion of the Escherichia coli lacZ gene at the ATG start codon (iBAC-FXN-lacZ). SHSY5Y human neuroblastoma cells transduced by iBAC-FXN-lacZ showed high efficiency of vector delivery and LacZ expression. Direct intracranial injection of iBAC-FXN-lacZ into the adult mouse cerebellum resulted in a large number of easily detectable transduced cells, with LacZ expression driven by the FXN genomic locus, which persisted for at least 75 days. Green fluorescent protein expression driven from the same vector but by the strong HSV-1 IE4/5 promoter was transient. Our data demonstrate for the first time sustained transgene expression in vivo by infectious delivery of a genomic DNA locus 4100 kb in size. Such an approach may be suitable for gene rescue strategies in neurological disease, such as FRDA.
Friedreich's ataxia (FA) is a recessive, predominantly neurodegenerative disorder caused in most ... more Friedreich's ataxia (FA) is a recessive, predominantly neurodegenerative disorder caused in most cases by mutations in the first intron of the frataxin (FXN) gene. This mutation drives the expansion of a homozygous GAA repeat that results in decreased levels of FXN transcription and frataxin protein. Frataxin (Fxn) is a ubiquitous mitochondrial protein involved in iron-sulfur cluster biogenesis, and a decrease in the levels of this protein is responsible for the symptoms observed in the disease. Although the pathological manifestations of FA are mainly observed in neurons of both the central and peripheral nervous system, it is not clear if changes in non-neuronal cells may also contribute to the pathogenesis of FA, as recently suggested for other neurodegenerative disorders. Therefore, the aims of this study were to generate and characterize a cell model of Fxn deficiency in human astrocytes (HAs) and to evaluate the possible involvement of non-cell autonomous processes in FA. To knockdown frataxin in vitro, we transduced HAs with a specific shRNA lentivirus (shRNA37), which produced a decrease in both frataxin mRNA and protein expression, along with mitochondrial superoxide production, and signs of p53-mediated cell cycle arrest and apoptotic cell death. To test for non-cell autonomous interactions we cultured wild-type mouse neurons in the presence of frataxin-deficient astrocyte conditioned medium, which provoked a delay in the maturation of these neurons, a decrease in neurite length and enhanced cell death. Our findings confirm a detrimental effect of frataxin silencing, not only for astrocytes, but also for neuron-glia interactions, underlining the need to take into account the role of non-cell autonomous processes in FA.
Current gene therapy, Jan 1, 2009
Neurochemistry international, Jan 1, 2007
Purkinje cells play a crucial role in sensory motor coordination since they are the only output p... more Purkinje cells play a crucial role in sensory motor coordination since they are the only output projection neurons in the cerebellar cortex and are affected in most spinocerebellar ataxias. They stand out in the central nervous system due to their large size and their profusely branched dendritic arbor. However, molecular and cellular studies on Purkinje cells are often hampered by the difficulty of maintaining these cells in culture. Here we report an easy, robust and reproducible method to obtain Purkinje-enriched mixed cerebellar cell cultures from day 16 mouse embryos using papain digestion and a semi-defined culture medium, being the composition of the culture approximately 20% Purkinje cells, 70% non-Purkinje neurons and 10% glial cells. We demonstrate that efficient gene transfer into Purkinje cells (as well as into other cerebellar populations) is possible using herpes simplex virus-1 (HSV-1)-derived vectors. Indeed, up to 50% of the Purkinje cells can be transduced and gene expression may persist for at least 14 days. As a result, this procedure permits functional gene expression studies to be carried out on cultured Purkinje neurons. To demonstrate this, we show that the expression of a dominant-negative form of glycogen synthase kinase-3 protects Purkinje neurons against cell death triggered by a chemical inhibitor of phosphatidylinositol-3 kinase. In summary, we have established reproducible and reliable cerebellar cell cultures enriched for Purkinje cells which enables gene transfer studies to be carried out using herpesviral vectors.
Human gene …, Jan 1, 2002
The transduction of cerebellar neurons in vivo with herpes simplex virus 1 (HSV-1) amplicon carry... more The transduction of cerebellar neurons in vivo with herpes simplex virus 1 (HSV-1) amplicon carrying the lacZ gene has been investigated after injection of the vector in the cerebellar cortex, ventricles, and inferior olive of adult rats. Injection into the cerebellar cortex resulted in transduction of Purkinje cells near the needle tract and injection into the ventricles yielded no transduced neurons. In contrast, high transduction efficiency was achieved by vector injection into the inferior olive, resulting in one of three positive Purkinje cells all over the ipsilateral and contralateral cerebellar hemispheres. Because neurons in the deep cerebellar nuclei are also transduced, we suggest that the vector is delivered from the inferior olive to the cerebellar nuclei and then to Purkinje cells by retrograde axonal transport. Expression of the lacZ gene within Purkinje cells was surprisingly persistent and was maintained at the same level for at least 40 days. Importantly, no signs of either toxicity or inflammation were observed in the cerebellum after vector injection, except for the borders of the needle tract where some reactive astrocytes were detected. Indeed, motor coordination of treated animals was entirely normal, as assessed by the rota-rod test. These results demonstrate that HSV-1 amplicon vectors can effect safe and stable transgene expression in Purkinje cells in vivo, raising the possibility of using these vectors for long-term gene therapy of human cerebellar disorders.
Journal of neuroscience …, Jan 1, 2006
Gene transfer is a powerful tool for functional gene analysis in human cells. In this respect, th... more Gene transfer is a powerful tool for functional gene analysis in human cells. In this respect, there is a need to develop experimental models that involve homogeneous cultures of human neuron-like cells susceptible to gene transduction and that are easy to handle. Here we describe an optimized and reproducible procedure to differentiate human SH-SY5Y neuroblastoma cells into a homogeneous population of neuron-like cells. The fully differentiated cells are postmitotic and resemble primary cultured neurons in terms of their cytoskeletal polarity. Notably, differentiated SH-SY5Y cells are far more susceptible to transduction by herpes simplex virus (HSV-1)-based vectors than proliferating SH-SY5Y cells. This increase in transduction efficiency after neuronal differentiation may be due to the up-regulation of cell surface receptors for herpesvirus entry. In summary, we propose that fully differentiated human neuron-like cells obtained from the SH-SY5Y neuroblastoma may constitute an excellent and versatile experimental tool for gene transfer and functional genomic studies with HSV-1 vectors.
Journal of Biological …, Jan 1, 2009
Journal of …, Jan 1, 2010
A typical feature of Parkinson&am... more A typical feature of Parkinson's disease is the progressive loss of dopaminergic neurons in the substantia nigra, in which inhibition of mitochondrial complex I activity may play an important role. Rotenone or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) inhibit the mitochondrial complex I and they cause the death of substantia nigra dopaminergic neurons, thereby providing acute murine models of Parkinson's disease. We have found that increasing mitochondrial hexokinase II activity can prevent cell death in neuronal cultures treated with rotenone. As a result, we have studied the effects of hexokinase II gene transfer in vivo using a herpes simplex virus type 1 (HSV-1) amplicon vector. The placHK2 amplicon vector was injected into substantia nigra of mice that were subsequently administered rotenone or MPTP. Overexpression of hexokinase II prevented both rotenone and MPTP-induced dopaminergic neuronal cell death, as well as reducing the associated motor defects. Our results provide the first proof-of-principle that hexokinase II protects against dopaminergic neurodegeneration in vivo, emphasizing the role of this enzyme in promoting neuronal survival. Thus, the increase of hexokinase II expression by gene transfer or other means represents a promising approach to treat Parkinson's and other neurodegenerative diseases.
The Journal of …, Jan 1, 2006
Neurochemistry …, Jan 1, 2007
Journal of …, Jan 1, 2003
Molecular …, Jan 1, 2006
Immortalized cell lines of olfactory ensheathing glia (OEG) that maintain the proregenerative pro... more Immortalized cell lines of olfactory ensheathing glia (OEG) that maintain the proregenerative properties of primary cultures provide an unlimited source of OEG for both basic and applied studies. Indeed, one specific immortalized rat OEG clonal line (TEG3) proved to be as good as primary OEG in promoting neuritogenesis and axon regeneration in culture models. Thus, we examined the capacity of TEG3 to promote axonal repair in an animal model of spinal cord injury, dorsal column crush. TEG3 cells can acquire astrocyte-like or Schwann cell-like morphology depending on the conditions under which they are cultured. In the injured spinal cord, prelabeled TEG3 survived for at least 10 weeks after grafting and they integrated into the spinal cord, adopting Schwann cell-like, astrocyte-like, or intermediate morphologies. In TEG3-transplanted animals, sensory projection axons grow into the lesion site and there was robust sprouting/axonal growth of the corticospinal tract, both into and beyond the lesion site, after crushing of the spinal cord-dorsal columns. TEG3-transplanted animals also recovered sensory and motor function in tape removal and beam walking behavioral tests. These data indicate that certain immortalized cell lines derived from a single cell can maintain the regenerative properties of primary OEG.
Journal of …, Jan 1, 2003
… of Biomedicine and …, Jan 1, 2002
Olfactory ensheathing glia (OEG) accompany olfactory growing axons in their entry to the adult ma... more Olfactory ensheathing glia (OEG) accompany olfactory growing axons in their entry to the adult mammalian central nervous system (CNS). Due to this special characteristic, considerable attention has been focused on the possibility of using OEG for CNS regeneration. OEG present a large heterogeneity in culture with respect to their cellular morphology and expressed molecules. The specific characteristics of OEG responsible for their regenerative properties have to be defined. These properties probably result from the combination of several factors: molecular composition of the membrane (expressing adhesion molecules as PSA-NCAM, L1 and/or others) combined with their ability to reduce glial scarring and to accompany new growing axons into the host CNS. Their capacity to produce some neurotrophic factors might also account for their ability to produce CNS regeneration.
Journal of Biological …, Jan 1, 2003
FEBS letters, Jan 1, 1999
The deposition of beta-amyloid peptide (A beta), the hyperphosphorylation of tau protein and the ... more The deposition of beta-amyloid peptide (A beta), the hyperphosphorylation of tau protein and the death of neurons in certain brain regions are characteristic features of Alzheimer's disease. It has been proposed that the accumulation of aggregates of A beta is the trigger of neurodegeneration in this disease. In support of this view, several studies have demonstrated that the treatment of cultured neurons with A beta leads to the hyperphosphorylation of tau protein and neuronal cell death. Here we report that lithium prevents the enhanced phosphorylation of tau protein at the sites recognized by antibodies Tau-1 and PHF-1 which occurs when cultured rat cortical neurons are incubated with A beta. Interestingly, lithium also significantly protects cultured neurons from A beta-induced cell death. These results raise the possibility of using chronic lithium treatment for the therapy of Alzheimer's disease.
Biochemical …, Jan 1, 2003
Since 2005, and as part of their adaptation to the framework of the European Higher Education Are... more Since 2005, and as part of their adaptation to the framework of the European Higher Education Area, Spanish universities have undertaken a profound remodelling of their postgraduate programmes. As a result of this process, new Master courses have been created and Doctorate programmes updated. In this context, the "Universidad Autónoma of Madrid (UAM)" has launched a new postgraduate programme in Molecular Biosciences.