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Microglial activation plays central roles in neuroinflammatory and neurodegenerative diseases. Positron emission tomography (PET) targeting 18 kDa Translocator Protein (TSPO) is widely used for localising inflammation in vivo, but its quantitative interpretation remains uncertain. We show that TSPO expression increases in activated microglia in mouse brain disease models but does not change in a non-human primate disease model or in common neurodegenerative and neuroinflammatory human diseases. We describe genetic divergence in the TSPO gene promoter, consistent with the hypothesis that the increase in TSPO expression in activated myeloid cells depends on the transcription factor AP1 and is unique to a subset of rodent species within the Muroidea superfamily. Finally, we identify LCP2 and TFEC as potential markers of microglial activation in humans. These data emphasise that TSPO expression in human myeloid cells is related to different phenomena than in mice, and that TSPO-PET signals in humans reflect the density of inflammatory cells rather than activation state.
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Microglia , Doenças Neurodegenerativas , Animais , Camundongos , Doenças Neurodegenerativas/genética , Macrófagos , Células Mieloides , Deriva GenéticaRESUMO
As a leading cause of mortality and morbidity, stroke constitutes a significant global health burden. Ischemic stroke accounts for 80% of cases and occurs due to an arterial thrombus, which impedes cerebral blood flow and rapidly leads to cell death. As the most abundant cell type within the central nervous system, astrocytes play a critical role within the injured brain. We developed a novel microphysiological platform that permits the induction of spatiotemporally controlled nutrient gradients, allowing us to study astrocytic response during and after transient nutrient deprivation. Within 24 h of inducing starvation in the platform, nutrient deprivation led to multiple changes in astrocyte response, from metabolic perturbations to gene expression changes, and cell viability. Furthermore, we observed that nutrient restoration did not reverse the functional changes in astrocyte metabolism, which mirrors reperfusion injury observed in vivo. We also identified alterations in numerous glucose metabolism-associated genes, many of which remained upregulated or downregulated even after restoration of the nutrient supply. Together, these findings suggest that astrocyte activation during and after nutrient starvation induces plastic changes that may underpin persistent stroke-induced functional impairment. Overall, our innovative device presents interesting potential to be used in the development of new therapies to improve tissue repair and even cognitive recovery after stroke.
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Astrócitos , Acidente Vascular Cerebral , Humanos , Acidente Vascular Cerebral/metabolismo , Encéfalo , Reperfusão , Dispositivos Lab-On-A-ChipRESUMO
Myelination is essential for central nervous system (CNS) formation, health, and function. Emerging evidence of oligodendrocyte heterogeneity in health and disease and divergent CNS gene expression profiles between mice and humans supports the development of experimentally tractable human myelination systems. Here, we developed human iPSC-derived myelinating organoids ("myelinoids") and quantitative tools to study myelination from oligodendrogenesis through to compact myelin formation and myelinated axon organization. Using patient-derived cells, we modeled a monogenetic disease of myelinated axons (Nfasc155 deficiency), recapitulating impaired paranodal axo-glial junction formation. We also validated the use of myelinoids for pharmacological assessment of myelination-both at the level of individual oligodendrocytes and globally across whole myelinoids-and demonstrated reduced myelination in response to suppressed synaptic vesicle release. Our study provides a platform to investigate human myelin development, disease, and adaptive myelination.
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Células-Tronco Pluripotentes Induzidas/citologia , Bainha de Mielina/fisiologia , Organoides/fisiologia , Axônios/metabolismo , Axônios/ultraestrutura , Humanos , Bainha de Mielina/ultraestrutura , Fatores de Crescimento Neural/deficiência , Fatores de Crescimento Neural/metabolismo , Organoides/ultraestrutura , Toxina Tetânica/farmacologia , Fatores de TempoRESUMO
The central nervous system hosts parenchymal macrophages, known as microglia, and non-parenchymal macrophages, collectively termed border-associated macrophages (BAMs). Microglia, but not BAMs, were reported to be absent in mice lacking a conserved Csf1r enhancer: the fms-intronic regulatory element (FIRE). However, it is unknown whether FIRE deficiency also impacts BAM arrival and/or maintenance. Here, we show that macrophages in the ventricular system of the brain, including Kolmer's epiplexus macrophages, are absent in Csf1rΔFIRE/ΔFIRE mice. Stromal choroid plexus BAMs are also considerably reduced. During normal development, we demonstrate that intracerebroventricular macrophages arrive from embryonic day 10.5, and can traverse ventricular walls in embryonic slice cultures. In Csf1rΔFIRE/ΔFIRE embryos, the arrival of both primitive microglia and intracerebroventricular macrophages was eliminated, whereas the arrival of cephalic mesenchyme and stromal choroid plexus BAMs was only partially restricted. Our results provide new insights into the development and regulation of different CNS macrophage populations.
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Desenvolvimento Embrionário/genética , Elementos Facilitadores Genéticos/genética , Macrófagos/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Sistema Nervoso Central/crescimento & desenvolvimento , Embrião de Mamíferos , Íntrons/genética , Camundongos , Microglia/metabolismo , Tecido Parenquimatoso/crescimento & desenvolvimento , Tecido Parenquimatoso/metabolismo , Sequências Reguladoras de Ácido NucleicoRESUMO
Progressive neuronal death during tauopathies is associated with aggregation of modified, truncated or mutant forms of tau protein. Such aggregates are neurotoxic, promote spreading of tau aggregation, and trigger release of pro-inflammatory factors by glial cells. Counteracting such pathogenic effects of tau by simultaneously inhibiting protein aggregation as well as pro-inflammatory glial cell responses would be of significant therapeutic interest. Here, we examined the use of the small heat-shock protein HspB5 for this purpose. As a molecular chaperone, HspB5 counteracts aggregation of a wide range of abnormal proteins. As a TLR2 agonist, it selectively activates protective responses by CD14-expressing myeloid cells including microglia. We show that intracerebral infusion of HspB5 in transgenic mice with selective neuronal expression of mutant human P301S tau has significant neuroprotective effects in the superficial, frontal cortical layers. Underlying these effects at least in part, HspB5 induces several potent neuroprotective mediators in both astrocytes and microglia including neurotrophic factors and increased potential for removal of glutamate. Together, these findings highlight the potentially broad therapeutic potential of HspB5 in neurodegenerative proteinopathies.
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INTRODUCTION: Neuroinflammation, which contributes to neurodegeneration, is a consistent hallmark of dementia. Emerging evidence suggests that systemic inflammation also contributes to disease progression. METHODS: The ability of systemically administered lipopolysaccharide (LPS - 500 µg/kg) to effect acute and chronic behavioural changes in C57BL/6 and P301S tauopathy mice was assessed. Markers of pathology were assessed in the brain and spinal cord. RESULTS: P301S mice display regional microgliosis. Systemic LPS treatment induced exaggerated acute sickness behaviour and motor dysfunction in P301S mice compared with wild-type controls and advanced the onset and accelerated chronic decline. LPS treatment was associated with increased tau pathology 24 hours after LPS injection and spinal cord microgliosis at the end stage. DISCUSSION: This is the first demonstration that a single systemic inflammatory episode causes exaggerated acute functional impairments and accelerates the long-term trajectory of functional decline associated with neurodegeneration in a mouse model of human tauopathy. The findings have relevance to management of human dementias.
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This corrects the article DOI: 10.1038/ncomms15132.
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The influence that neurons exert on astrocytic function is poorly understood. To investigate this, we first developed a system combining cortical neurons and astrocytes from closely related species, followed by RNA-seq and in silico species separation. This approach uncovers a wide programme of neuron-induced astrocytic gene expression, involving Notch signalling, which drives and maintains astrocytic maturity and neurotransmitter uptake function, is conserved in human development, and is disrupted by neurodegeneration. Separately, hundreds of astrocytic genes are acutely regulated by synaptic activity via mechanisms involving cAMP/PKA-dependent CREB activation. This includes the coordinated activity-dependent upregulation of major astrocytic components of the astrocyte-neuron lactate shuttle, leading to a CREB-dependent increase in astrocytic glucose metabolism and elevated lactate export. Moreover, the groups of astrocytic genes induced by neurons or neuronal activity both show age-dependent decline in humans. Thus, neurons and neuronal activity regulate the astrocytic transcriptome with the potential to shape astrocyte-neuron metabolic cooperation.
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Astrócitos/metabolismo , Córtex Cerebral/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Neurônios/metabolismo , Tauopatias/genética , Animais , Astrócitos/citologia , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Comunicação Celular , Córtex Cerebral/citologia , Córtex Cerebral/crescimento & desenvolvimento , Técnicas de Cocultura , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Embrião de Mamíferos , Perfilação da Expressão Gênica , Glucose/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ácido Láctico/metabolismo , Potenciais da Membrana/fisiologia , Camundongos Knockout , Neurônios/citologia , Ratos Sprague-Dawley , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais , Tauopatias/metabolismo , Tauopatias/patologiaRESUMO
BACKGROUND: [corrected] Multiple Sclerosis has two clinical phases reflecting distinct but inter-related pathological processes: focal inflammation drives the relapse-remitting stage and neurodegeneration represents the principal substrate of secondary progression. In contrast to the increasing number of effective anti-inflammatory disease modifying treatments for relapse-remitting disease, the absence of therapies for progressive disease represents a major unmet clinical need. This raises the unanswered question of whether elimination of clinical relapses will prevent subsequent progression and if so how early in the disease course should treatment be initiated. Experimental autoimmune encephalomyelitis in the Biozzi ABH mouse recapitulates the clinical and pathological features of multiple sclerosis including relapse-remitting episodes with inflammatory mediated demyelination and progressive disability with neurodegeneration. To address the relationship between inflammation and neurodegeneration we used an auto-immune tolerance strategy to eliminate clinical relapses in EAE in a manner analogous to the clinical effect of disease modifying treatments. RESULTS: By arresting clinical relapses in EAE at two distinct stages, early and late disease, we demonstrate that halting immune driven demyelination even after the first major clinical event is insufficient to prevent long-term neurodegeneration and associated gliosis. Nonetheless, early intervention is partially neuroprotective, whereas later interventions are not. Furthermore early tolerisation is also associated with increased remyelination. CONCLUSIONS: These findings are consistent with both a partial uncoupling of inflammation and neurodegeneration and that the regenerative response of remyelination is negatively correlated with inflammation. These findings strongly support the need for early combinatorial treatment of immunomodulatory therapies and neuroprotective treatments to prevent long-term neurodegeneration in multiple sclerosis.
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Encefalomielite Autoimune Experimental/fisiopatologia , Degeneração Neural/fisiopatologia , Neuroimunomodulação/fisiologia , Animais , Axônios/patologia , Axônios/fisiologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/fisiologia , Progressão da Doença , Encefalomielite Autoimune Experimental/terapia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/fisiopatologia , Gliose/terapia , Imuno-Histoquímica , Terapia de Imunossupressão , Camundongos Biozzi , Microglia/patologia , Microglia/fisiologia , Microscopia Confocal , Neurônios Motores/fisiologia , Degeneração Neural/terapia , Medula Espinal/patologia , Medula Espinal/fisiopatologiaRESUMO
In addition to being an established risk factor for neurodegenerative diseases, age is increasingly recognized as adversely influencing regeneration. Accumulating evidence also suggests that age plays important, although poorly understood, roles with respect to course and prognosis in the degenerative and untreatable later phase of multiple sclerosis. Two experimental models of multiple sclerosis have been particularly influential in modeling the different aspects of neuronal injury and regeneration: global experimental autoimmune encephalomyelitis and focal toxin-mediated injury. Against this background, we report a focal model of immune-mediated demyelinating injury that reliably generates targeted primary demyelination and axonal injury. A detailed pathologic characterization of this model, modified extensively from an earlier study, showed that aged adult animals exhibited increased vulnerability to axonal injury and reduced efficiency of remyelination compared with younger animals. More important, remyelination in aged animals was predominantly Schwann cell mediated, in contrast to the central oligodendrocyte-mediated remyelination that predominated in younger rodents. Together, these findings establish an experimental platform to further study the influence of age on injury and repair in a biologically relevant model of human demyelinating injury.
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Envelhecimento/fisiologia , Axônios/ultraestrutura , Encefalomielite Autoimune Experimental/fisiopatologia , Regeneração Nervosa/fisiologia , Envelhecimento/patologia , Animais , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Interferon gama/imunologia , Microscopia Eletrônica , Proteínas da Mielina/imunologia , Bainha de Mielina/fisiologia , Glicoproteína Mielina-Oligodendrócito , Oligodendroglia/fisiologia , Oligodendroglia/ultraestrutura , Ratos , Ratos Endogâmicos Lew , Células de Schwann/fisiologia , Células de Schwann/ultraestrutura , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Tau protein in a hyperphosphorylated state makes up the intracellular inclusions of several neurodegenerative diseases, including Alzheimer's disease and cases of frontotemporal dementia. Mutations in Tau cause familial forms of frontotemporal dementia, establishing that dysfunction of tau protein is sufficient to cause neurodegeneration and dementia. Transgenic mice expressing human mutant tau in neurons exhibit the essential features of tauopathies, including neurodegeneration and abundant filaments composed of hyperphosphorylated tau. Here we show that a previously described mouse line transgenic for human P301S tau exhibits an age-related, layer-specific loss of superficial cortical neurons, similar to what has been observed in human frontotemporal dementias. We also show that focal neural precursor cell implantation, resulting in glial cell differentiation, leads to the sustained rescue of cortical neurons. Together with evidence indicating that astrocyte transplantation may be neuroprotective, our findings suggest a beneficial role for glial cell-based repair in neurodegenerative diseases.
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Transplante de Células , Terapia Baseada em Transplante de Células e Tecidos , Modelos Animais de Doenças , Neurônios/fisiologia , Tauopatias/terapia , Fatores Etários , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Córtex Cerebral/patologia , Feminino , Regulação da Expressão Gênica/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fator de Transcrição 2 de Oligodendrócitos , Prolina/genética , RNA Mensageiro/metabolismo , Serina/genética , Células-Tronco/fisiologia , Ácido gama-Aminobutírico/metabolismo , Proteínas tau/genéticaRESUMO
Multiple sclerosis (MS) represents a considerable challenge to experimentally model due to its twin pathologies of inflammatory demyelination and neurodegeneration along with its multifocal and multiphasic nature. Experimental autoimmune encephalomyelitis (EAE) in Biozzi ABH mice has previously been shown to reproduce many clinical features also found in secondary progressive MS. In this study we sought to characterise the pathology of chronic EAE in ABH mice. In addition to marked gliosis, we report substantial demyelination, remyelination and axonal and neuronal loss. Together with the clinical pattern, our findings identify chronic EAE as an excellent model of secondary progressive multiple sclerosis.
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Axônios/patologia , Gliose/etiologia , Esclerose Múltipla Crônica Progressiva/patologia , Esclerose Múltipla Crônica Progressiva/fisiopatologia , Bainha de Mielina/patologia , Neurônios/patologia , Animais , Antígenos/metabolismo , Modelos Animais de Doenças , Camundongos , Camundongos Biozzi , Bainha de Mielina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteoglicanas/metabolismo , Medula Espinal/metabolismo , Medula Espinal/patologiaRESUMO
Bone morphogenetic proteins (BMPs) and their endogenous inhibitors, including noggin, chordin and follistatin, have roles in pattern formation and fate specification of neuronal and glial cells during nervous system development. We have examined their influence on glial reactions in the injured central nervous system (CNS). We show that penetrating injuries to the brain and spinal cord resulted in the upregulation of BMP-2/4, BMP-7, and noggin, with the latter being expressed almost exclusively by reactive astrocytes at the injury site, and we show that astrocytes in vitro produce noggin. As BMPs have been shown to drive cultured NG2-positive oligodendrocyte precursors (OPCs) towards a multipotential phenotype (type II astrocytes), we investigated the effects of inhibiting noggin with a function-blocking antibody (noggin-FbAb). In vitro, BMP-driven conversion of OPCs to type 2 astrocytes was inhibited by noggin, an effect that was reversed by noggin-FbAb. Noggin-FbAb also increased the number of type 2 astrocytes generated from cultured OPCs exposed to an astrocyte feeder layer, consistent with astrocytes producing both BMPs and noggin. In knife cut injuries in vivo, noggin-FbAb treatment resulted in an increase in the number of NG2-positive cells and small GFAP-positive cells in the injury site, and the appearance of glial cells with the morphological and antigenic characteristics of type 2 astrocytes (as generated in vitro), with coexpression of both GFAP and NG2. This potential conversion of inhibitory OPCs to type 2 astrocyte-like cells in vivo suggests that endogenous BMPs, unmasked by noggin antagonism, might be exploited to manipulate cell fate following CNS trauma.
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Proteínas Morfogenéticas Ósseas/fisiologia , Neuroglia/fisiologia , Transdução de Sinais/fisiologia , Traumatismos do Sistema Nervoso/metabolismo , Traumatismos do Sistema Nervoso/patologia , Células-Tronco Adultas/efeitos dos fármacos , Células-Tronco Adultas/fisiologia , Animais , Animais Recém-Nascidos , Anticorpos/farmacologia , Proteínas Morfogenéticas Ósseas/farmacologia , Bromodesoxiuridina/metabolismo , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Interações Medicamentosas , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Proteína Glial Fibrilar Ácida/metabolismo , Camundongos , Neuroglia/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/fisiologia , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
Bone morphogenetic proteins (BMPs) and their antagonists, including noggin, are required for nervous system development, but their potential roles in the reactions of the adult central nervous system to injury are unknown. Here we have examined the expression of noggin and BMPs in the spinal cord following dorsal rhizotomy. Through the use of a function-blocking antibody, we have also investigated the role of endogenous noggin in the neuritic plasticity which follows rhizotomy. Dorsal rhizotomy resulted in the upregulation of BMPs 2/4, 7 and noggin in the superficial white matter and in the dorsal neuropil of the spinal cord. These co-localized with glial fibrillary acidic protein, indicating their expression by astrocytes. Because BMPs induce dendritic sprouting and synaptogenesis in some neuronal populations in vitro, we hypothesized that administration of a noggin function-blocking antibody (FbAb) in vivo would augment rhizotomy-induced sprouting in the spinal cord. Topical application of noggin-FbAb to the dorsal surface of the spinal cord following rhizotomy resulted in significant increases in the density of microtubule-associated protein 2 (MAP-2) and substance P (SP)-positive processes within the lateral spinal nucleus. In the deafferented dorsal horn, noggin-FbAb treatment induced significant increases in the density of SP, calcitonin gene-related peptide (CGRP)- and 5-hydroxytryptamine (5-HT)-positive axons. These results suggest a novel mechanism by which endogenous plasticity of spared axons is suppressed following dorsal rhizotomy, and which might be exploited to improve the outcome of spinal cord injury and other CNS trauma.
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Axônios/fisiologia , Proteínas de Transporte/metabolismo , Dendritos/fisiologia , Plasticidade Neuronal , Rizotomia , Medula Espinal/metabolismo , Animais , Anticorpos/farmacologia , Proteínas Morfogenéticas Ósseas/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/imunologia , Células Cultivadas , Proteínas Associadas aos Microtúbulos/metabolismo , Período Pós-Operatório , Ratos , Ratos Wistar , Serotonina/metabolismo , Substância P/metabolismo , Regulação para CimaRESUMO
Lentiviral vectors have been demonstrated as efficient tools for gene delivery to the CNS. We describe a novel approach for vector delivery using the thermoresponsive Gel, Pluronic F127 as a carrier. A HIV-1 lentiviral vector expressing GFP was contained in various concentrations of gel (15, 30 and 40%) and applied to cultures of 293T cells. FACS analysis of cells transduced with 8ng of lentiviral vector revealed a similar transduction efficiency for each Gel concentration compared to vector added to cells without PF127. Primary Rat CNS mixed glial cultures were also transduced with lentiviral vector in 15% Pluronic F127 and results demonstrated a similar transduction efficiency of astrocytes compared to virus without gel and no evidence of cell toxicity or death. Stereotaxic delivery of viral vector in 15% PF127 to the rat brain resulted in transduction of cells, predominantly astrocytes close to the injection site. Pluronic F127 gel delivery of viral vectors to the CNS may provide a platform for localised release particularly in areas of brain or spinal cord injury.
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Encéfalo/metabolismo , Técnicas de Transferência de Genes , Lentivirus/genética , Neuroglia/metabolismo , Poloxâmero/administração & dosagem , Animais , Células Cultivadas , Géis , Vetores Genéticos , Ratos , Técnicas Estereotáxicas , Transdução GenéticaRESUMO
BACKGROUND: Lentiviral vectors have shown immense promise as vehicles for gene delivery to non-dividing cells particularly to cells of the central nervous system (CNS). Improvements in the biosafety of viral vectors are paramount as lentiviral vectors move into human clinical trials. This study investigates the packaging relationship between gene transfer (vector) and Gag-Pol expression constructs of HIV-1, HIV-2 and SIV. Cross-packaged vectors expressing GFP were assessed for RNA packaging, viral vector titre and their ability to transduce rat primary glial cell cultures and human neural stem cells. RESULTS: HIV-1 Gag-Pol demonstrated the ability to cross package both HIV-2 and SIV gene transfer vectors. However both HIV-2 and SIV Gag-Pol showed a reduced ability to package HIV-1 vector RNA with no significant gene transfer to target cells. An unexpected packaging relationship was found to exist between HIV-2 and SIV with SIV Gag-Pol able to package HIV-2 vector RNA and transduce dividing SV2T cells and CNS cell cultures with an efficiency equivalent to the homologous HIV-1 vector however HIV-2 was unable to deliver SIV based vectors. CONCLUSION: This new non-reciprocal cross packaging relationship between SIV and HIV-2 provides a novel way of significantly increasing bio-safety with a reduced sequence homology between the HIV-2 gene transfer vector and the SIV Gag-Pol construct thus ensuring that vector RNA packaging is unidirectional.