RESUMEN
X-linked Charcot-Marie-Tooth disease type 1 (CMT1X) is a demyelinating neuropathy resulting from loss-of-function mutations affecting the GJB1/connexin 32 (Cx32) gene. We previously showed functional and morphological improvement in Gjb1-null mice following AAV9-mediated delivery of human Cx32 driven by the myelin protein zero (Mpz) promoter in Schwann cells. However, CMT1X mutants may interfere with virally delivered wild-type (WT) Cx32. To confirm the efficacy of this vector also in the presence of CMT1X mutants, we delivered AAV9-Mpz-GJB1 by lumbar intrathecal injection in R75W/Gjb1-null and N175D/Gjb1-null transgenic lines expressing Golgi-retained mutations, before and after the onset of the neuropathy. Widespread expression of virally delivered Cx32 was demonstrated in both genotypes. Re-establishment of WT Cx32 function resulted in improved muscle strength and increased sciatic nerve motor conduction velocities in all treated groups from both mutant lines when treated before as well as after the onset of the neuropathy. Furthermore, morphological analysis showed improvement of myelination and reduction of inflammation in lumbar motor roots and peripheral nerves. In conclusion, this study provides proof of principle for a clinically translatable gene therapy approach to treat CMT1X before and after the onset of the neuropathy, even in the presence of endogenously expressed Golgi-retained Cx32 mutants.
RESUMEN
Alzheimer's disease (AD) is a progressive neurodegenerative disease of the brain causing either familial or sporadic dementia. We have previously administered the modified C5a receptor agonist (EP67) for a short period to a transgenic mouse model of AD (5XFAD) and have observed not only reduction in ß-amyloid deposition and gliosis but also improvement in cognitive impairment. Inquiring, however, on the effects of EP67 in an already heavily burdened animal, thus representing a more realistic scenario, we treated 6-month-old 5XFAD mice for a period of 14 weeks. We recorded a significant decrease in both fibrillar and pre-fibrillar ß-amyloid as well as remarkable amelioration of cognitive impairment. Following proteomic analysis and pathway association, we postulate that these events are triggered through the upregulation of ß-adrenergic and GABAergic signaling. In summary, our results reveal how inflammatory responses can be employed in inducing tangible phenotype improvements even in advanced stages of AD.
Asunto(s)
Enfermedad de Alzheimer , Disfunción Cognitiva , Oligopéptidos , Receptor de Anafilatoxina C5a , Receptores Adrenérgicos beta , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Animales , Disfunción Cognitiva/tratamiento farmacológico , Disfunción Cognitiva/genética , Disfunción Cognitiva/metabolismo , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Oligopéptidos/farmacología , Oligopéptidos/uso terapéutico , Proteómica , Receptor de Anafilatoxina C5a/agonistas , Receptor de Anafilatoxina C5a/genética , Receptor de Anafilatoxina C5a/metabolismo , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta/metabolismoRESUMEN
Mutations in the GJB1 gene, encoding the gap junction (GJ) protein connexin32 (Cx32), cause X-linked Charcot-Marie-Tooth disease (CMT1X), an inherited demyelinating neuropathy. We developed a gene therapy approach for CMT1X using an AAV9 vector to deliver the GJB1/Cx32 gene under the myelin protein zero (Mpz) promoter for targeted expression in Schwann cells. Lumbar intrathecal injection of the AAV9-Mpz.GJB1 resulted in widespread biodistribution in the peripheral nervous system including lumbar roots, sciatic and femoral nerves, as well as in Cx32 expression in the paranodal non-compact myelin areas of myelinated fibers. A pre-, as well as post-onset treatment trial in Gjb1-null mice, demonstrated improved motor performance and sciatic nerve conduction velocities along with improved myelination and reduced inflammation in peripheral nerve tissues. Blood biomarker levels were also significantly ameliorated in treated mice. This study provides evidence that a clinically translatable AAV9-mediated gene therapy approach targeting Schwann cells could potentially treat CMT1X.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth , Células de Schwann , Animales , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/metabolismo , Enfermedad de Charcot-Marie-Tooth/terapia , Conexinas/genética , Conexinas/metabolismo , Terapia Genética/métodos , Ratones , Ratones Noqueados , Células de Schwann/metabolismo , Distribución TisularRESUMEN
Gap junctions (GJs) coupling oligodendrocytes to astrocytes and to other oligodendrocytes are formed mainly by connexin47 (Cx47) and a smaller portion by connexin32 (Cx32). Mutations in both connexins cause inherited demyelinating disorders, but their expression is also disrupted in multiple sclerosis (MS). To clarify whether the loss of either Cx47 or Cx32 could modify the outcome of inflammation and myelin loss, we induced experimental autoimmune encephalomyelitis (EAE) in fully backcrossed Cx32 knockout (KO) and Cx47KO mice and compared their outcome with wild type (WT, C57BI/6 N) mice. Cx47KO EAE mice developed the most severe phenotype assessed by clinical scores and behavioral testing, followed by Cx32KO and WT mice. Cx47KO more than Cx32KO EAE mice developed more microglial activation, myelin, and axonal loss than did WT mice. Oligodendrocyte apoptosis and precursor proliferation was also higher in Cx47KO than in Cx32KO or WT EAE mice. Similarly, blood-spinal cord barrier (BSCB) disruption and inflammatory infiltrates of macrophages, T- and B-cells were more severe in Cx47KO than either Cx32KO or WT EAE groups. Finally, expression profiling revealed that several proinflammatory cytokines were higher at the peak of inflammation in the Cx47KO mice and persisted at later stages of EAE in contrast to reduction of their levels in WT EAE mice. Thus, loss of oligodendrocyte GJs aggravates BSCB disruption and inflammatory myelin loss, likely due to dysregulation of proinflammatory cytokines. This mechanism may play an important role in MS brain with reduced connexin expression, as well as in patients with inherited mutations in oligodendrocyte connexins and secondary inflammation.
Asunto(s)
Citocinas/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Uniones Comunicantes/metabolismo , Regulación de la Expresión Génica/fisiología , Fuerza de la Mano/fisiología , Oligodendroglía/metabolismo , Animales , Apoptosis/genética , Astrocitos/metabolismo , Astrocitos/patología , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/fisiopatología , Proteínas de Unión al Calcio/metabolismo , Proliferación Celular/genética , Conexinas/genética , Conexinas/metabolismo , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/fisiopatología , Adyuvante de Freund/toxicidad , Uniones Comunicantes/patología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/metabolismo , Actividad Motora/efectos de los fármacos , Actividad Motora/genética , Glicoproteína Mielina-Oligodendrócito/toxicidad , Oligodendroglía/patología , Fragmentos de Péptidos/toxicidad , Proteína beta1 de Unión ComunicanteRESUMEN
Studies proposed a model for embryonic neurogenesis where the expression levels of the SOXB2 and SOXB1 factors regulate the differentiation status of the neural stem cells. However, the precise role of the SOXB2 genes remains controversial. Therefore, this study aims to investigate the effects of individual deletions of the SOX21 and SOX14 genes during the development of the dorsal midbrain. We show that SOX21 and SOX14 function distinctly during the commitment of the GABAergic lineage. More explicitly, deletion of SOX21 reduced the expression of the GABAergic precursor marker GATA3 and BHLHB5 while the expression of GAD6, which marks GABAergic terminal differentiation, was not affected. In contrast deletion of SOX14 alone was sufficient to inhibit terminal differentiation of the dorsal midbrain GABAergic neurons. Furthermore, we demonstrate through gain-of-function experiments, that despite the homology of SOX21 and SOX14, they have unique gene targets and cannot compensate for the loss of each other. Taken together, these data do not support a pan-neurogenic function for SOXB2 genes in the dorsal midbrain, but instead they influence, sequentially, the specification of GABAergic neurons.
RESUMEN
X-linked Charcot-Marie-Tooth disease (CMT1X) is a common form of inherited neuropathy resulting from different mutations affecting the gap junction (GJ) protein connexin32 (Cx32). A subset of CMT1X patients may additionally present with acute fulminant CNS dysfunction, typically triggered by conditions of systemic inflammation and metabolic stress. To clarify the underlying mechanisms of CNS phenotypes in CMT1X we studied a mouse model of systemic inflammation induced by lipopolysaccharide (LPS) injection to compare wild type (WT), connexin32 (Cx32) knockout (KO), and KO T55I mice expressing the T55I Cx32 mutation associated with CNS phenotypes. Following a single intraperitoneal LPS or saline (controls) injection at the age of 40-60 days systemic inflammatory response was documented by elevated TNF-α and IL-6 levels in peripheral blood and mice were evaluated 1 week after injection. Behavioral analysis showed graded impairment of motor performance in LPS treated mice, worse in KO T55I than in Cx32 KO and in Cx32 KO worse than WT. Iba1 immunostaining revealed widespread inflammation in LPS treated mice with diffusely activated microglia throughout the CNS. Immunostaining for the remaining major oligodendrocyte connexin Cx47 and for its astrocytic partner Cx43 revealed widely reduced expression of Cx43 and loss of Cx47 GJs in oligodendrocytes. Real-time PCR and immunoblot analysis indicated primarily a down regulation of Cx43 expression with secondary loss of Cx47 membrane localization. Inflammatory changes and connexin alterations were most severe in the KO T55I group. To examine why the presence of the T55I mutant exacerbates pathology even more than in Cx32 KO mice, we analyzed the expression of ER-stress markers BiP, Fas and CHOP by immunostaining, immunoblot and Real-time PCR. All markers were increased in LPS treated KO T55I mice more than in other genotypes. In conclusion, LPS induced neuroinflammation causes disruption of the main astrocyte-oligodendrocyte GJs, which may contribute to the increased sensitivity of Cx32 KO mice to LPS and of patients with CMT1X to various stressors. Moreover the presence of an intracellularly retained, misfolded CMT1X mutant such as T55I induces ER stress under inflammatory conditions, further exacerbating oligodendrocyte dysfunction and pathological changes in the CNS.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/inmunología , Estrés del Retículo Endoplásmico/fisiología , Retículo Endoplásmico/inmunología , Uniones Comunicantes/inmunología , Inflamación/inmunología , Oligodendroglía/inmunología , Animales , Astrocitos/inmunología , Astrocitos/patología , Proteínas de Unión al Calcio/metabolismo , Enfermedad de Charcot-Marie-Tooth/patología , Conexina 43/metabolismo , Conexinas/genética , Conexinas/metabolismo , Modelos Animales de Enfermedad , Retículo Endoplásmico/patología , Escherichia coli , Uniones Comunicantes/patología , Inflamación/patología , Interleucina-6/sangre , Lipopolisacáridos , Masculino , Ratones Noqueados , Proteínas de Microfilamentos/metabolismo , Neuroinmunomodulación/fisiología , Oligodendroglía/patología , Factor de Necrosis Tumoral alfa/sangre , Proteína beta1 de Unión ComunicanteRESUMEN
Inherited demyelinating peripheral neuropathies are progressive incurable diseases without effective treatment. To develop a gene therapy approach targeting myelinating Schwann cells that can be translatable, we delivered a lentiviral vector using a single lumbar intrathecal injection and a myelin-specific promoter. The human gene of interest, GJB1, which is mutated in X-linked Charcot-Marie-Tooth Disease (CMT1X), was delivered intrathecally into adult Gjb1-null mice, a genetically authentic model of CMT1X that develops a demyelinating peripheral neuropathy. We obtained widespread, stable, and cell-specific expression of connexin32 in up to 50% of Schwann cells in multiple lumbar spinal roots and peripheral nerves. Behavioral and electrophysiological analysis revealed significantly improved motor performance, quadriceps muscle contractility, and sciatic nerve conduction velocities. Furthermore, treated mice exhibited reduced numbers of demyelinated and remyelinated fibers and fewer inflammatory cells in lumbar motor roots, as well as in the femoral motor and sciatic nerves. This study demonstrates that a single intrathecal lentiviral gene delivery can lead to Schwann cell-specific expression in spinal roots extending to multiple peripheral nerves. This clinically relevant approach improves the phenotype of an inherited neuropathy mouse model and provides proof of principle for treating inherited demyelinating neuropathies.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Enfermedades Desmielinizantes/genética , Animales , Terapia Genética , Humanos , Vaina de Mielina/metabolismo , Células de Schwann/metabolismoRESUMEN
The popularity of vascular stents continues to increase for a variety of applications, including coronary, lower limb, renal, carotid, and neurovascular disorders. However, their clinical effectiveness is hindered by numerous postdeployment complications, which may stimulate inflammatory and fibrotic reactions. The purpose of this study was to evaluate the vessel inflammatory response via in vivo imaging in a mouse stent implantation model. Corroded and noncorroded self-expanding miniature nitinol stents were implanted in mice abdominal aortas, and novel in vivo imaging techniques were used to assess trafficking and accumulation of fluorescent donor monocytes as well as cellular proliferation at the implantation site. Monocytes were quantitatively tracked in vivo and found to rapidly clear from circulation within hours after injection. Differences were found between the test groups with respect to the numbers of recruited monocytes and the intensity of the resulting fluorescent signal. Image analysis also revealed a subtle increase in matrix metalloproteinase activity in corroded compared with the normal stented aortas. In conclusion, this study has been successful in developing a murine stent inflammation model and applying novel in vivo imaging tools and methods to monitor the complex biological processes of the host vascular wall response.
Asunto(s)
Aorta Abdominal/patología , Inflamación/patología , Monitoreo Fisiológico , Stents , Aleaciones/farmacología , Animales , Aorta Abdominal/efectos de los fármacos , Aorta Abdominal/enzimología , Separación Celular , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/patología , Corrosión , Modelos Animales de Enfermedad , Fluorescencia , Masculino , Metaloproteinasas de la Matriz/metabolismo , Metales/sangre , Ratones , Monocitos/citología , Monocitos/efectos de los fármacosRESUMEN
Oligodendrocytes are coupled by gap junctions (GJs) formed mainly by connexin47 (Cx47) and Cx32. Recessive GJC2/Cx47 mutations cause Pelizaeus-Merzbacher-like disease, a hypomyelinating leukodystrophy, while GJB1/Cx32 mutations cause neuropathy and chronic or acute-transient encephalopathy syndromes. Cx32/Cx47 double knockout (Cx32/Cx47dKO) mice develop severe CNS demyelination beginning at 1 month of age leading to death within weeks, offering a relevant model to study disease mechanisms. In order to clarify whether the loss of oligodendrocyte connexins has cell autonomous effects, we generated transgenic mice expressing the wild-type human Cx32 under the control of the mouse proteolipid protein promoter, obtaining exogenous hCx32 expression in oligodendrocytes. By crossing these mice with Cx32KO mice, we obtained expression of hCx32 on Cx32KO background. Immunohistochemical and immunoblot analysis confirmed strong CNS expression of hCx32 specifically in oligodendrocytes and correct localization forming GJs at cell bodies and along the myelin sheath. TG(+)Cx32/Cx47dKO mice generated by further crossing with Cx47KO mice showed that transgenic expression of hCx32 rescued the severe early phenotype of CNS demyelination in Cx32/Cx47dKO mice, resulting in marked improvement of behavioral abnormalities at 1 month of age, and preventing the early mortality. Furthermore, TG(+)Cx32/Cx47dKO mice showed significant improvement of myelination compared with Cx32/Cx47dKO CNS at 1 month of age, while the inflammatory and astrogliotic changes were fully reversed. Our study confirms that loss of oligodendrocyte GJs has cell autonomous effects and that re-establishment of GJ connectivity by replacement of least one GJ protein provides correction of the leukodystrophy phenotype.
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Conexinas/metabolismo , Enfermedades Desmielinizantes/metabolismo , Uniones Comunicantes/metabolismo , Oligodendroglía/metabolismo , Animales , Animales Modificados Genéticamente , Conexinas/genética , Enfermedades Desmielinizantes/genética , Femenino , Uniones Comunicantes/genética , Humanos , Masculino , Ratones , Ratones Transgénicos , Fenotipo , Proteína beta1 de Unión ComunicanteRESUMEN
Thin-basement-membrane nephropathy (TBMN) and Alport syndrome (AS) are progressive collagen IV nephropathies caused by mutations in COL4A3/A4/A5 genes. These nephropathies invariably present with microscopic hematuria and frequently progress to proteinuria and CKD or ESRD during long-term follow-up. Nonetheless, the exact molecular mechanisms by which these mutations exert their deleterious effects on the glomerulus remain elusive. We hypothesized that defective trafficking of the COL4A3 chain causes a strong intracellular effect on the cell responsible for COL4A3 expression, the podocyte. To this end, we overexpressed normal and mutant COL4A3 chains (G1334E mutation) in human undifferentiated podocytes and tested their effects in various intracellular pathways using a microarray approach. COL4A3 overexpression in the podocyte caused chain retention in the endoplasmic reticulum (ER) that was associated with activation of unfolded protein response (UPR)-related markers of ER stress. Notably, the overexpression of normal or mutant COL4A3 chains differentially activated the UPR pathway. Similar results were observed in a novel knockin mouse carrying the Col4a3-G1332E mutation, which produced a phenotype consistent with AS, and in biopsy specimens from patients with TBMN carrying a heterozygous COL4A3-G1334E mutation. These results suggest that ER stress arising from defective localization of collagen IV chains in human podocytes contributes to the pathogenesis of TBMN and AS through activation of the UPR, a finding that may pave the way for novel therapeutic interventions for a variety of collagenopathies.
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Colágeno Tipo IV/deficiencia , Estrés del Retículo Endoplásmico/fisiología , Membrana Basal Glomerular/metabolismo , Nefritis Hereditaria/metabolismo , Podocitos/metabolismo , Respuesta de Proteína Desplegada/fisiología , Animales , Autoantígenos/genética , Autoantígenos/fisiología , Biopsia , Células Cultivadas , Colágeno Tipo IV/genética , Colágeno Tipo IV/fisiología , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Chaperón BiP del Retículo Endoplásmico , Perfilación de la Expresión Génica , Técnicas de Sustitución del Gen , Membrana Basal Glomerular/patología , Proteínas de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/genética , Heterocigoto , Humanos , Riñón/metabolismo , Riñón/patología , Ratones , Mutación Missense , Nefritis Hereditaria/genética , Nefritis Hereditaria/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Podocitos/patología , Mutación Puntual , Análisis por Matrices de Proteínas , Transporte de Proteínas , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Proteínas Recombinantes de Fusión , Factores de Transcripción del Factor Regulador X , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
Catalytic hydrogenation of 3,5-bis-arylidenetetramic acids, known for their biological activity, has been developed. The chemoselective ruthenium-catalyzed reduction of the exocyclic carbon-carbon double bonds on pyrrolidine-2,4-dione ring system, containing other reducible functions, has been investigated. Depending on the substrate the yield of the hydrogenation process can reach up to 95%. The structural elucidation has been established using NMR and HRMS spectral data.