RESUMO
OBJECTIVE: Soluble CD27 is a promising cerebrospinal fluid inflammatory biomarker in multiple sclerosis. In this study, we investigate relevant immune and neuro-pathological features of soluble CD27 in multiple sclerosis. METHODS: Protein levels of soluble CD27 were correlated to inflammatory cell subpopulations and inflammatory cytokines and chemokines detected in cerebrospinal fluid of 137 patients with multiple sclerosis and 47 patients with inflammatory and non-inflammatory neurological disease from three independent cohorts. Production of soluble CD27 was investigated in cell cultures of activated T and B cells and CD27-knockout T cells. In a study including matched cerebrospinal fluid and post-mortem brain tissues of patients with multiple sclerosis and control cases, levels of soluble CD27 were correlated with perivascular and meningeal infiltrates and with neuropathological features. RESULTS: We demonstrate that soluble CD27 favours the differentiation of interferon-γ-producing T cells and is released through a secretory mechanism activated by TCR engagement and regulated by neutral sphingomyelinase. We also show that the levels of soluble CD27 correlate with the representation of inflammatory T cell subsets in the CSF of patients with relapsing-remitting multiple sclerosis and with the magnitude of perivascular and meningeal CD27 + CD4 + and CD8 + T cell infiltrates in post-mortem central nervous system tissue, defining a subgroup of patients with extensive active inflammatory lesions. INTERPRETATION: Our results demonstrate that soluble CD27 is a biomarker of disease activity, potentially informative for personalized treatment and monitoring of treatment outcomes.
Assuntos
Esclerose Múltipla Recidivante-Remitente , Esclerose Múltipla , Humanos , Linfócitos T CD8-Positivos , Sistema Nervoso Central , BiomarcadoresRESUMO
Changes to the structure of nodes of Ranvier in the normal-appearing white matter (NAWM) of multiple sclerosis (MS) brains are associated with chronic inflammation. We show that the paranodal domains in MS NAWM are longer on average than control, with Kv1.2 channels dislocated into the paranode. These pathological features are reproduced in a model of chronic meningeal inflammation generated by the injection of lentiviral vectors for the lymphotoxin-α (LTα) and interferon-γ (IFNγ) genes. We show that tumour necrosis factor (TNF), IFNγ, and glutamate can provoke paranodal elongation in cerebellar slice cultures, which could be reversed by an N-methyl-D-aspartate (NMDA) receptor blocker. When these changes were inserted into a computational model to simulate axonal conduction, a rapid decrease in velocity was observed, reaching conduction failure in small diameter axons. We suggest that glial cells activated by pro-inflammatory cytokines can produce high levels of glutamate, which triggers paranodal pathology, contributing to axonal damage and conduction deficits.
Assuntos
Esclerose Múltipla/patologia , Nós Neurofibrosos/patologia , Substância Branca/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Axônios/patologia , Encéfalo/patologia , Sinapses Elétricas/patologia , Sinapses Elétricas/efeitos da radiação , Feminino , Humanos , Inflamação/patologia , Masculino , Microglia/patologia , Pessoa de Meia-Idade , Esclerose Múltipla/diagnóstico por imagem , Bainha de Mielina/patologia , Neuroglia/patologia , Neuroimunomodulação/imunologia , Neuroimunomodulação/fisiologia , Nós Neurofibrosos/fisiologia , Substância Branca/diagnóstico por imagem , Substância Branca/imunologiaRESUMO
Organized meningeal immune cell infiltrates are suggested to play an important role in cortical grey matter pathology in the multiple sclerosis brain, but the mechanisms involved are as yet unresolved. Lymphotoxin-alpha plays a key role in lymphoid organ development and cellular cytotoxicity in the immune system and its expression is increased in the CSF of naïve and progressive multiple sclerosis patients and post-mortem meningeal tissue. Here we show that persistently increased levels of lymphotoxin-alpha in the cerebral meninges can give rise to lymphoid-like structures and underlying multiple sclerosis-like cortical pathology. Stereotaxic injections of recombinant lymphotoxin-alpha into the rat meninges led to acute meningeal inflammation and subpial demyelination that resolved after 28 days, with demyelination being dependent on prior subclinical immunization with myelin oligodendrocyte glycoprotein. Injection of a lymphotoxin-alpha lentiviral vector into the cortical meningeal space, to produce chronic localized overexpression of the cytokine, induced extensive lymphoid-like immune cell aggregates, maintained over 3 months, including T-cell rich zones containing podoplanin + fibroblastic reticular stromal cells and B-cell rich zones with a network of follicular dendritic cells, together with expression of lymphoid chemokines and their receptors. Extensive microglial and astroglial activation, subpial demyelination and marked neuronal loss occurred in the underlying cortical parenchyma. Whereas subpial demyelination was partially dependent on previous myelin oligodendrocyte glycoprotein immunization, the neuronal loss was present irrespective of immunization. Conditioned medium from LTα treated microglia was able to induce a reactive phenotype in astrocytes. Our results show that chronic lymphotoxin-alpha overexpression alone is sufficient to induce formation of meningeal lymphoid-like structures and subsequent neurodegeneration, similar to that seen in the progressive multiple sclerosis brain.
Assuntos
Esclerose Múltipla Crônica Progressiva , Esclerose Múltipla , Ratos , Animais , Linfotoxina-alfa/metabolismo , Glicoproteína Mielina-Oligodendrócito , Inflamação/patologia , Córtex Cerebral/patologia , Meninges , Esclerose Múltipla/patologia , Esclerose Múltipla Crônica Progressiva/patologia , Tecido Linfoide/metabolismo , Tecido Linfoide/patologia , Fatores Imunológicos/metabolismoRESUMO
Sustained exposure to pro-inflammatory cytokines in the leptomeninges is thought to play a major role in the pathogenetic mechanisms leading to cortical pathology in multiple sclerosis (MS). Although the molecular mechanisms underlying neurodegeneration in the grey matter remain unclear, several lines of evidence suggest a prominent role for tumour necrosis factor (TNF). Using cortical grey matter tissue blocks from post-mortem brains from 28 secondary progressive MS subjects and ten non-neurological controls, we describe an increase in expression of multiple steps in the TNF/TNF receptor 1 signaling pathway leading to necroptosis, including the key proteins TNFR1, FADD, RIPK1, RIPK3 and MLKL. Activation of this pathway was indicated by the phosphorylation of RIPK3 and MLKL and the formation of protein oligomers characteristic of necrosomes. In contrast, caspase-8 dependent apoptotic signaling was decreased. Upregulation of necroptotic signaling occurred predominantly in macroneurons in cortical layers II-III, with little expression in other cell types. The presence of activated necroptotic proteins in neurons was increased in MS cases with prominent meningeal inflammation, with a 30-fold increase in phosphoMLKL+ neurons in layers I-III. The density of phosphoMLKL+ neurons correlated inversely with age at death, age at progression and disease duration. In vivo induction of chronically elevated TNF and INFγ levels in the CSF in a rat model via lentiviral transduction in the meninges, triggered inflammation and neurodegeneration in the underlying cortical grey matter that was associated with increased neuronal expression of TNFR1 and activated necroptotic signaling proteins. Exposure of cultured primary rat cortical neurons to TNF induced necroptosis when apoptosis was inhibited. Our data suggest that neurons in the MS cortex are dying via TNF/TNFR1 stimulated necroptosis rather than apoptosis, possibly initiated in part by chronic meningeal inflammation. Neuronal necroptosis represents a pathogenetic mechanism that is amenable to therapeutic intervention at several points in the signaling pathway.
Assuntos
Substância Cinzenta/patologia , Esclerose Múltipla Crônica Progressiva/patologia , Necroptose/fisiologia , Neurônios/patologia , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Idoso , Animais , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Feminino , Substância Cinzenta/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Ratos , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Meningeal inflammation strongly associates with demyelination and neuronal loss in the underlying cortex of progressive MS patients, thereby contributing significantly to clinical disability. However, the pathological mechanisms of meningeal inflammation-induced cortical pathology are still largely elusive. By extensive analysis of cortical microglia in post-mortem progressive MS tissue, we identified cortical areas with two MS-specific microglial populations, termed MS1 and MS2 cortex. The microglial population in MS1 cortex was characterized by a higher density and increased expression of the activation markers HLA class II and CD68, whereas microglia in MS2 cortex showed increased morphological complexity and loss of P2Y12 and TMEM119 expression. Interestingly, both populations associated with inflammation of the overlying meninges and were time-dependently replicated in an in vivo rat model for progressive MS-like chronic meningeal inflammation. In this recently developed animal model, cortical microglia at 1-month post-induction of experimental meningeal inflammation resembled microglia in MS1 cortex, and microglia at 2 months post-induction acquired a MS2-like phenotype. Furthermore, we observed that MS1 microglia in both MS cortex and the animal model were found closely apposing neuronal cell bodies and to mediate pre-synaptic displacement and phagocytosis, which coincided with a relative sparing of neurons. In contrast, microglia in MS2 cortex were not involved in these synaptic alterations, but instead associated with substantial neuronal loss. Taken together, our results show that in response to meningeal inflammation, microglia acquire two distinct phenotypes that differentially associate with neurodegeneration in the progressive MS cortex. Furthermore, our in vivo data suggests that microglia initially protect neurons from meningeal inflammation-induced cell death by removing pre-synapses from the neuronal soma, but eventually lose these protective properties contributing to neuronal loss.
Assuntos
Córtex Cerebral/patologia , Meninges/patologia , Microglia/patologia , Esclerose Múltipla/patologia , Doenças Neurodegenerativas/patologia , Doenças Neuroinflamatórias/patologia , Neurônios/patologia , Adulto , Idoso , Animais , Morte Celular , Doenças Desmielinizantes/imunologia , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Feminino , Humanos , Meninges/imunologia , Microglia/classificação , Microglia/imunologia , Microglia/metabolismo , Pessoa de Meia-Idade , Esclerose Múltipla/imunologia , Doenças Neurodegenerativas/imunologia , Fenótipo , RatosRESUMO
Cyclin-dependent kinase-like 5 disorder is a severe neurodevelopmental disorder caused by mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene. It predominantly affects females who typically present with severe early epileptic encephalopathy, global developmental delay, motor dysfunction, autistic features and sleep disturbances. To develop a gene replacement therapy, we initially characterized the human CDKL5 transcript isoforms expressed in the brain, neuroblastoma cell lines, primary astrocytes and embryonic stem cell-derived cortical interneurons. We found that the isoform 1 and to a lesser extent the isoform 2 were expressed in human brain, and both neuronal and glial cell types. These isoforms were subsequently cloned into recombinant adeno-associated viral (AAV) vector genome and high-titre viral vectors were produced. Intrajugular delivery of green fluorescence protein via AAV vector serotype PHP.B in adult wild-type male mice transduced neurons and astrocytes throughout the brain more efficiently than serotype 9. Cdkl5 knockout male mice treated with isoform 1 via intrajugular injection at age 28-30 days exhibited significant behavioural improvements compared to green fluorescence protein-treated controls (1012 vg per animal, n = 10 per group) with PHP.B vectors. Brain expression of the isoform 1 transgene was more abundant in hindbrain than forebrain and midbrain. Transgene brain expression was sporadic at the cellular level and most prominent in hippocampal neurons and cerebellar Purkinje cells. Correction of postsynaptic density protein 95 cerebellar misexpression, a major fine cerebellar structural abnormality in Cdkl5 knockout mice, was found in regions of high transgene expression within the cerebellum. AAV vector serotype DJ efficiently transduced CDKL5-mutant human induced pluripotent stem cell-derived neural progenitors, which were subsequently differentiated into mature neurons. When treating CDKL5-mutant neurons, isoform 1 expression led to an increased density of synaptic puncta, while isoform 2 ameliorated the calcium signalling defect compared to green fluorescence protein control, implying distinct functions of these isoforms in neurons. This study provides the first evidence that gene therapy mediated by AAV vectors can be used for treating CDKL5 disorder.
Assuntos
Terapia Genética , Isoformas de Proteínas/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Adenoviridae , Animais , Encéfalo/metabolismo , Cálcio/metabolismo , Células Cultivadas , Proteína 4 Homóloga a Disks-Large/biossíntese , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Isoformas de Proteínas/genética , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Sinapses/metabolismo , TransfecçãoRESUMO
Current therapies for Alzheimer's disease (AD) are symptomatic and do not target the underlying Aß pathology and other important hallmarks including neuronal loss. PPARγ-coactivator-1α (PGC-1α) is a cofactor for transcription factors including the peroxisome proliferator-activated receptor-γ (PPARγ), and it is involved in the regulation of metabolic genes, oxidative phosphorylation, and mitochondrial biogenesis. We previously reported that PGC-1α also regulates the transcription of ß-APP cleaving enzyme (BACE1), the main enzyme involved in Aß generation, and its expression is decreased in AD patients. We aimed to explore the potential therapeutic effect of PGC-1α by generating a lentiviral vector to express human PGC-1α and target it by stereotaxic delivery to hippocampus and cortex of APP23 transgenic mice at the preclinical stage of the disease. Four months after injection, APP23 mice treated with hPGC-1α showed improved spatial and recognition memory concomitant with a significant reduction in Aß deposition, associated with a decrease in BACE1 expression. hPGC-1α overexpression attenuated the levels of proinflammatory cytokines and microglial activation. This effect was accompanied by a marked preservation of pyramidal neurons in the CA3 area and increased expression of neurotrophic factors. The neuroprotective effects were secondary to a reduction in Aß pathology and neuroinflammation, because wild-type mice receiving the same treatment were unaffected. These results suggest that the selective induction of PGC-1α gene in specific areas of the brain is effective in targeting AD-related neurodegeneration and holds potential as therapeutic intervention for this disease.
Assuntos
Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/genética , Ácido Aspártico Endopeptidases/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Agregação Patológica de Proteínas/genética , Doença de Alzheimer/patologia , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/metabolismo , Animais , Regulação da Expressão Gênica/genética , Vetores Genéticos/uso terapêutico , Humanos , Lentivirus/genética , Memória/fisiologia , Camundongos Transgênicos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/administração & dosagem , Agregação Patológica de Proteínas/terapia , Células Piramidais/metabolismo , Células Piramidais/patologiaRESUMO
IκBα resides in the cytosol where it retains the inducible transcription factor NF-κB. We show that IκBα also localises to the outer mitochondrial membrane (OMM) to inhibit apoptosis. This effect is especially pronounced in tumour cells with constitutively active NF-κB that accumulate high amounts of mitochondrial IκBα as a NF-κB target gene. 3T3 IκBα(-/-) cells also become protected from apoptosis when IκBα is specifically reconstituted at the OMM. Using various IκBα mutants, we demonstrate that apoptosis inhibition and NF-κB inhibition can be functionally and structurally separated. At mitochondria, IκBα stabilises the complex of VDAC1 and hexokinase II (HKII), thereby preventing Bax recruitment to VDAC1 and the release of cytochrome c for apoptosis induction. When IκBα is reduced in tumour cells with constitutively active NF-κB, they show an enhanced response to anticancer treatment in an in vivo xenograft tumour model. Our results reveal the unexpected activity of IκBα in guarding the integrity of the OMM against apoptosis induction and open possibilities for more specific interference in tumours with deregulated NF-κB.
Assuntos
Apoptose/fisiologia , Proteínas I-kappa B/metabolismo , Membranas Mitocondriais/fisiologia , Modelos Biológicos , NF-kappa B/metabolismo , Animais , Western Blotting , Linhagem Celular , Citocromos c/metabolismo , Feminino , Citometria de Fluxo , Hexoquinase/metabolismo , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Membranas Mitocondriais/metabolismo , Inibidor de NF-kappaB alfa , Oligonucleotídeos/genética , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Rabies pseudotyped lentiviral vectors have great potential in gene therapy, not least because of their ability to transduce neurons following their distal axonal application. However, very little is known about the molecular processes that underlie their retrograde transport and cell transduction. Using multiple labeling techniques and confocal microscopy, we demonstrated that pseudotyping with rabies virus envelope glycoprotein (RV-G) enabled the axonal retrograde transport of two distinct subtypes of lentiviral vector in motor neuron cultures. Analysis of this process revealed that these vectors trafficked through Rab5-positive endosomes and accumulated within a non-acidic Rab7 compartment. RV-G pseudotyped vectors were co-transported with both the tetanus neurotoxin-binding fragment and the membrane proteins thought to mediate rabies virus endocytosis (neural cell adhesion molecule, nicotinic acetylcholine receptor, and p75 neurotrophin receptor), thus demonstrating that pseudotyping with RV-G targets lentiviral vectors for transport along the same pathway exploited by several toxins and viruses. Using motor neurons cultured in compartmentalized chambers, we demonstrated that axonal retrograde transport of these vectors was rapid and efficient; however, it was not able to transduce the targeted neurons efficiently, suggesting that impairment in processes occurring after arrival of the viral vector in the soma is responsible for the low transduction efficiency seen in vivo, which suggests a novel area for improvement of gene therapy vectors.
Assuntos
Transporte Axonal , Vetores Genéticos , Lentivirus/genética , Neurônios Motores/metabolismo , Vírus da Raiva/metabolismo , Proteínas do Envelope Viral/metabolismo , Animais , Endocitose , Células HEK293 , Humanos , Neurônios Motores/virologia , Ratos , Proteínas do Envelope Viral/genéticaRESUMO
BACKGROUND: Parkinson's disease is typically treated with oral dopamine replacement therapies; however, long-term treatment leads to motor complications and, occasionally, impulse control disorders caused by intermittent stimulation of dopamine receptors and off-target effects, respectively. We aimed to assess the safety, tolerability, and efficacy of bilateral, intrastriatal delivery of ProSavin, a lentiviral vector-based gene therapy aimed at restoring local and continuous dopamine production in patients with advanced Parkinson's disease. METHODS: We undertook a phase 1/2 open-label trial with 12-month follow-up at two study sites (France and UK) to assess the safety and efficacy of ProSavin after bilateral injection into the putamen of patients with Parkinson's disease. All patients were then enrolled in a separate open-label follow-up study of long-term safety. Three doses were assessed in separate cohorts: low dose (1·9×10(7) transducing units [TU]); mid dose (4·0×10(7) TU); and high dose (1×10(8) TU). Inclusion criteria were age 48-65 years, disease duration 5 years or longer, motor fluctuations, and 50% or higher motor response to oral dopaminergic therapy. The primary endpoints of the phase 1/2 study were the number and severity of adverse events associated with ProSavin and motor responses as assessed with Unified Parkinson's Disease Rating Scale (UPDRS) part III (off medication) scores, at 6 months after vector administration. Both trials are registered at ClinicalTrials.gov, NCT00627588 and NCT01856439. FINDINGS: 15 patients received ProSavin and were followed up (three at low dose, six mid dose, six high dose). During the first 12 months of follow-up, 54 drug-related adverse events were reported (51 mild, three moderate). Most common were increased on-medication dyskinesias (20 events, 11 patients) and on-off phenomena (12 events, nine patients). No serious adverse events related to the study drug or surgical procedure were reported. A significant improvement in mean UPDRS part III motor scores off medication was recorded in all patients at 6 months (mean score 38 [SD 9] vs 26 [8], n=15, p=0·0001) and 12 months (38 vs 27 [8]; n=15, p=0·0001) compared with baseline. INTERPRETATION: ProSavin was safe and well tolerated in patients with advanced Parkinson's disease. Improvement in motor behaviour was observed in all patients. FUNDING: Oxford BioMedica.
Assuntos
Antiparkinsonianos/administração & dosagem , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vírus da Anemia Infecciosa Equina/genética , Doença de Parkinson/terapia , Transfecção/métodos , Idoso , Antiparkinsonianos/efeitos adversos , Dopa Descarboxilase/genética , Dopamina/biossíntese , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/virologia , Seguimentos , GTP Cicloidrolase/administração & dosagem , GTP Cicloidrolase/efeitos adversos , GTP Cicloidrolase/genética , Terapia Genética/efeitos adversos , Vetores Genéticos/efeitos adversos , Humanos , Injeções Intralesionais , Masculino , Pessoa de Meia-Idade , Putamen , Transgenes/genética , Tirosina 3-Mono-Oxigenase/administração & dosagem , Tirosina 3-Mono-Oxigenase/efeitos adversos , Tirosina 3-Mono-Oxigenase/genéticaRESUMO
UNLABELLED: To investigate the potential benefits which may arise from pseudotyping the HIV-1 lentiviral vector with its homologous gp41 envelope glycoprotein (GP) cytoplasmic tail (CT), we created chimeric RVG/HIV-1gp41 GPs composed of the extracellular and transmembrane sequences of RVG and either the full-length gp41 CT or C terminus gp41 truncations sequentially removing existing conserved motifs. Lentiviruses (LVs) pseudotyped with the chimeric GPs were evaluated in terms of particle release (physical titer), biological titers, infectivity, and in vivo central nervous system (CNS) transduction. We report here that LVs carrying shorter CTs expressed higher levels of envelope GP and showed a higher average infectivity than those bearing full-length GPs. Interestingly, complete removal of GP CT led to vectors with the highest transduction efficiency. Removal of all C-terminal gp41 CT conserved motifs, leaving just 17 amino acids (aa), appeared to preserve infectivity and resulted in a significantly increased physical titer. Furthermore, incorporation of these 17 aa in the RVG CT notably enhanced the physical titer. In vivo stereotaxic delivery of LV vectors exhibiting the best in vitro titers into rodent striatum facilitated efficient transduction of the CNS at the site of injection. A particular observation was the improved retrograde transduction of neurons in connected distal sites that resulted from the chimeric envelope R5 which included the "Kennedy" sequence (Ken) and lentivirus lytic peptide 2 (LLP2) conserved motifs in the CT, and although it did not exhibit a comparable high titer upon pseudotyping, it led to a significant increase in distal retrograde transduction of neurons. IMPORTANCE: In this study, we have produced novel chimeric envelopes bearing the extracellular domain of rabies fused to the cytoplasmic tail (CT) of gp41 and pseudotyped lentiviral vectors with them. Here we report novel effects on the transduction efficiency and physical titer of these vectors, depending on CT length and context. We also managed to achieve increased neuronal transduction in vivo in the rodent CNS, thus demonstrating that the efficiency of these vectors can be enhanced following merely CT manipulation. We believe that this paper is a novel contribution to the field and opens the way for further attempts to surface engineer lentiviral vectors and make them more amenable for applications in human disease.
Assuntos
Sistema Nervoso Central/metabolismo , Vetores Genéticos/genética , Proteína gp41 do Envelope de HIV/genética , HIV-1/genética , Proteínas Recombinantes de Fusão/genética , Transdução Genética , Proteínas do Envelope Viral/genética , Encéfalo/metabolismo , Linhagem Celular , Neurônios Dopaminérgicos/metabolismo , Expressão Gênica , Vetores Genéticos/administração & dosagem , Células HEK293 , Proteína gp41 do Envelope de HIV/metabolismo , Humanos , Lentivirus/genética , Plasmídeos/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas do Envelope Viral/metabolismo , Carga ViralRESUMO
Huntington's disease (HD) is a devastating neurodegenerative disorder caused by abnormal polyglutamine expansion in the huntingtin protein (Exp-Htt). Currently, there are no effective treatments for HD. We used bidirectional lentiviral transfer vectors to generate in vitro and in vivo models of HD and to test the therapeutic potential of vascular endothelial growth factor 165 (VEGF165). Lentiviral-mediated expression of Exp-Htt caused cell death and aggregate formation in human neuroblastoma SH-SY5Y and rat primary striatal cultures. Lentiviral-mediated VEGF165 expression was found to be neuroprotective in both of these models. Unilateral stereotaxic vector delivery of Exp-Htt vector in adult rat striatum led to progressive inclusion formation and striatal neuron loss at 10 weeks post-transduction. Coinjection of a lower dose VEGF165 significantly attenuated DARPP-32(+) neuronal loss, enhanced NeuN staining and reduced Exp-Htt aggregation. A tenfold higher dose VEGF165 led to overt neuronal toxicity marked by tissue damage, neovascularization, extensive astrogliosis, vascular leakage, chronic inflammation and distal neuronal loss. No overt behavioral phenotype was observed in these animals. Expression of VEGF165 at this higher dose in the brain of wild-type rats led to early mortality with global neuronal loss. This report raises important safety concerns about unregulated VEGF165 CNS applications.
Assuntos
Corpo Estriado/patologia , Terapia Genética , Doença de Huntington/patologia , Degeneração Neural/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Morte Celular , Linhagem Celular Tumoral , Células Cultivadas , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Vetores Genéticos , Células HEK293 , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Lentivirus/genética , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores , Ratos , Ratos Sprague-Dawley , Transdução GenéticaRESUMO
Gene therapy vectors are among the treatments currently used to treat malignant tumors. Gene therapy vectors use a specific therapeutic transgene that causes death in cancer cells. In early attempts at gene therapy, therapeutic transgenes were driven by non-specific vectors which induced toxicity to normal cells in addition to the cancer cells. Recently, novel cancer specific viral vectors have been developed that target cancer cells leaving normal cells unharmed. Here we review such cancer specific gene therapy systems currently used in the treatment of cancer and discuss the major challenges and future directions in this field.
Assuntos
Terapia Genética/métodos , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos , Transgenes , Animais , Humanos , Neoplasias/genéticaRESUMO
We report a unique mutation in the D-amino acid oxidase gene (R199W DAO) associated with classical adult onset familial amyotrophic lateral sclerosis (FALS) in a three generational FALS kindred, after candidate gene screening in a 14.52 cM region on chromosome 12q22-23 linked to disease. Neuronal cell lines expressing R199W DAO showed decreased viability and increased ubiquitinated aggregates compared with cells expressing the wild-type protein. Similarly, lentiviral-mediated expression of R199W DAO in primary motor neuron cultures caused increased TUNEL labeling. This effect was also observed when motor neurons were cocultured on transduced astrocytes expressing R199W, indicating that the motor neuron cell death induced by this mutation is mediated by both cell autonomous and noncell autonomous processes. DAO controls the level of D-serine, which accumulates in the spinal cord in cases of sporadic ALS and in a mouse model of ALS, indicating that this abnormality may represent a fundamental component of ALS pathogenesis.
Assuntos
Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/genética , D-Aminoácido Oxidase/genética , D-Aminoácido Oxidase/fisiologia , Mutação , Animais , Apoptose , Células COS , Linhagem Celular , Chlorocebus aethiops , Feminino , Ligação Genética , Masculino , Camundongos , Repetições de Microssatélites , Neurônios Motores/metabolismo , Doenças Neurodegenerativas/genética , Neurônios/metabolismo , RatosRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease resulting in the selective death of motor neurons in the brain and spinal cord. Some familial cases of ALS are caused by dominant mutations in the gene encoding superoxide dismutase (SOD1). The emergence of interfering RNA (RNAi) for specific gene silencing could be therapeutically beneficial for the treatment of such dominantly inherited diseases. We generated a lentiviral vector to mediate expression of RNAi molecules specifically targeting the human SOD1 gene (SOD1). Injection of this vector into various muscle groups of mice engineered to overexpress a mutated form of human SOD1 (SOD1(G93A)) resulted in an efficient and specific reduction of SOD1 expression and improved survival of vulnerable motor neurons in the brainstem and spinal cord. Furthermore, SOD1 silencing mediated an improved motor performance in these animals, resulting in a considerable delay in the onset of ALS symptoms by more than 100% and an extension in survival by nearly 80% of their normal life span. These data are the first to show a substantial extension of survival in an animal model of a fatal, dominantly inherited neurodegenerative condition using RNAi and provide the highest therapeutic efficacy observed in this field to date.
Assuntos
Esclerose Lateral Amiotrófica/genética , Degeneração Neural , Interferência de RNA , Superóxido Dismutase/genética , Esclerose Lateral Amiotrófica/terapia , Animais , Modelos Animais de Doenças , Terapia Genética , Vetores Genéticos , Humanos , Lentivirus/genética , Camundongos , Camundongos Transgênicos , Mutação , RNA Interferente Pequeno , Taxa de Sobrevida , TransfecçãoRESUMO
Background and Objectives: CDKL5 deficiency disorder (CDD) is a neurodevelopmental encephalopathy characterized by early-onset epilepsy and impaired psychomotor development. Variations in the X-linked CDKL5 gene coding for a kinase cause CDD. Molecular genetics has proved that almost all pathogenic missense substitutions localize in the N-terminal catalytic domain, therefore underlining the importance for brain development and functioning of the kinase activity. CDKL5 also features a long C-terminal domain that acts as negative regulator of the enzymatic activity and modulates its subcellular distribution. CDD is generally attributed to loss-of-function variations, whereas the clinical consequences of increased CDKL5 activity remain uncertain. We have identified a female patient characterized by mild epilepsy and neurologic symptoms, harboring a novel c.2873C>G nucleotide substitution, leading to the missense variant p.(Thr958Arg). To increase our comprehension of genetic variants in CDKL5-associated neurologic disorders, we have characterized the molecular consequences of the identified substitution. Methods: MRI and video EEG telemetry were used to describe brain activity and capture seizure. The Bayley III test was used to evaluate the patient development. Reverse transcriptase PCR was used to analyze whether the identified nucleotide variant affects messenger RNA stability and/or splicing. The X chromosome inactivation pattern was analyzed determining the DNA methylation status of the androgen receptor (AR) gene and by sequencing of expressed alleles. Western blotting was used to investigate whether the novel Thr958Arg substitution affects the stability and/or enzymatic activity of CDKL5. Immunofluorescence was used to define whether CDKL5 subcellular distribution is affected by the Thr958Arg substitution. Results: Our data suggested that the proband tends toward a skewed X chromosome inactivation pattern in favor of the novel variant. The molecular investigation revealed that the p.(Thr958Arg) substitution leads to a significant increase in the autophosphorylation of both the TEY motif and residue Tyr171 of CDKL5, as well as in the phosphorylation of the target protein MAP1S, indicating an hyperactivation of CDKL5. This occurs without evidently affecting the kinase subcellular distribution. Discussion: Our data provide a strong indication that the c.2873C>G nucleotide substitution represents an hypermorphic pathogenic variation of CDKL5, therefore highlighting the importance of a tight control of CDKL5 activity in the brain.
RESUMO
Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are genetically modified G-protein-coupled receptors (GPCRs), that can be activated by a synthetic ligand which is otherwise inert at endogenous receptors. DREADDs can be expressed in cells in the central nervous system (CNS) and subsequently offer the opportunity for remote and reversible silencing or activation of the target cells when the synthetic ligand is systemically administered. In neuroscience, DREADDs have thus far shown to be useful tools for several areas of research and offer considerable potential for the development of gene therapy strategies for neurological disorders. However, in order to design a DREADD-based gene therapy, it is necessary to first evaluate the viral vector delivery methods utilised in the literature to deliver these chemogenetic tools. This review evaluates each of the prominent strategies currently utilised for DREADD delivery, discussing their respective advantages and limitations. We focus on adeno-associated virus (AAV)-based and lentivirus-based systems, and the manipulation of these through cell-type specific promoters and pseudotyping. Furthermore, we address how virally mediated DREADD delivery could be improved in order to make it a viable gene therapy strategy and thus expand its translational potential.
Assuntos
Sistema Nervoso Central/efeitos dos fármacos , Dependovirus/genética , Desenho de Fármacos , Vetores Genéticos/uso terapêutico , Lentivirus/genética , Terapia de Alvo Molecular/métodos , Receptores Acoplados a Proteínas G/genética , Animais , Encéfalo/efeitos dos fármacos , Drogas Desenhadas/farmacologia , Terapia Genética/métodos , Humanos , Ligantes , Neurônios/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/biossíntese , Transdução de Sinais/efeitos dos fármacosRESUMO
Amyotrophic lateral sclerosis (ALS) causes adult-onset, progressive motor neuron degeneration in the brain and spinal cord, resulting in paralysis and death three to five years after onset in most patients. ALS is still incurable, in part because its complex aetiology remains insufficiently understood. Recent reports have indicated that reduced levels of vascular endothelial growth factor (VEGF), which is essential in angiogenesis and has also been implicated in neuroprotection, predispose mice and humans to ALS. However, the therapeutic potential of VEGF for the treatment of ALS has not previously been assessed. Here we report that a single injection of a VEGF-expressing lentiviral vector into various muscles delayed onset and slowed progression of ALS in mice engineered to overexpress the gene coding for the mutated G93A form of the superoxide dismutase-1 (SOD1(G93A)) (refs 7-10), even when treatment was only initiated at the onset of paralysis. VEGF treatment increased the life expectancy of ALS mice by 30 per cent without causing toxic side effects, thereby achieving one of the most effective therapies reported in the field so far.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/terapia , Transporte Axonal , Modelos Animais de Doenças , Vírus da Anemia Infecciosa Equina/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/uso terapêutico , Esclerose Lateral Amiotrófica/patologia , Animais , Tronco Encefálico/patologia , Progressão da Doença , Expressão Gênica , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Injeções Intramusculares , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Mutação Puntual/genética , Medula Espinal/patologia , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Taxa de Sobrevida , Fatores de Tempo , Transgenes/genética , Fator A de Crescimento do Endotélio Vascular/efeitos adversos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The embryonic CNS readily undergoes regeneration, unlike the adult CNS, which has limited axonal repair after injury. Here we tested the hypothesis that retinoic acid receptor beta2 (RARbeta2), critical in development for neuronal growth, may enable adult neurons to grow in an inhibitory environment. Overexpression of RARbeta2 in adult rat dorsal root ganglion cultures increased intracellular levels of cyclic AMP and stimulated neurite outgrowth. Stable RARbeta2 expression in DRG neurons in vitro and in vivo enabled their axons to regenerate across the inhibitory dorsal root entry zone and project into the gray matter of the spinal cord. The regenerated neurons enhanced second-order neuronal activity in the spinal cord, and RARbeta2-treated rats showed highly significant improvement in sensorimotor tasks. These findings show that RARbeta2 induces axonal regeneration programs within injured neurons and may thus offer new therapeutic opportunities for CNS regeneration.
Assuntos
Regeneração Nervosa/fisiologia , Neurônios Aferentes/metabolismo , Receptores do Ácido Retinoico/metabolismo , Medula Espinal/metabolismo , Animais , Células Cultivadas , Gânglios Espinais/lesões , Gânglios Espinais/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Masculino , Ratos , Ratos Wistar , Recuperação de Função Fisiológica/fisiologiaRESUMO
Analysis of isolated meninges and cerebrospinal fluid (CSF) of post-mortem MS cases has shown increased gene and protein expression for the pro-inflammatory cytokines: tumour necrosis factor (TNF) and interferon-γ (IFNγ). Here we tested the hypothesis that persistent production of these cytokines in the meningeal compartment and diffusion into underlying GM can drive chronic MS-like GM pathology. Lentiviral transfer vectors were injected into the sagittal sulcus of DA rats to deliver continuous expression of TNF + IFNγ transgenes in the meninges and the resulting neuropathology analysed after 1 and 2 months. Injection of TNF + IFNγ viral vectors, with or without prior MOG immunisation, induced extensive immune cell infiltration (CD4+ and CD8+ T-cells, CD79a + B-cells and macrophages) in the meninges by 28 dpi, which remained at 2 months. Control GFP viral vector did not induce infiltration. Subpial demyelination was seen underlying these infiltrates, which was partly dependant on prior myelin oligodendrocyte glycoprotein (MOG) immunisation. A significant decrease in neuronal numbers was seen at 28 and 56 days in cortical layers II-V that was independent of MOG immunisation. RNA analysis at 28 dpi showed an increase in expression of necroptotic pathway genes, including RIP3, MLKL, cIAP2 and Nox2. PhosphoRIP3+ and phosphoMLKL+ neurons were present in TNF + IFNγ vector injected animals, indicating activation of necroptosis. Our results suggest that persistent expression of TNF in the presence of IFNγ is a potent inducer of meningeal inflammation and can activate TNF signalling pathways in cortical cells leading to neuronal death and subpial demyelination and thus may contribute to clinical progression in MS.