RESUMEN
Magnetic resonance imaging-guided focused ultrasound combined with microbubbles injected in the bloodstream (MRIgFUS) temporarily increases the permeability of the blood-brain barrier (BBB), which facilitates the entry of intravenously administered adeno-associated viruses (AAVs) from the blood to targeted brain areas. To date, the properties of the AAVs used for MRIgFUS delivery resulted in cell transduction limited to MRIgFUS-targeted sites. Considering future clinical applications, strategies are needed to deliver genes to multiple locations and large brain volumes while creating minimal BBB modulation. Here we combine MRIgFUS with a vector that has enhanced biodistribution following brain entry, AAV2-HBKO, to mediate broad gene delivery to targeted brain regions at levels with potential therapeutic relevance. Expression of a reporter gene was achieved in 13% and 21% of all neurons present in the striatum and thalamus, respectively, while targeting only 28% of the brain regions with MRIgFUS. Compared with AAV9, MRIgFUS-mediated delivery of AAV2-HBKO showed greater diffusion in the brain and a higher percentage of the neurons expressing the transgene. MRIgFUS AAV2-HBKO gene delivery to the brain has the potential to reach levels that are functionally and clinically relevant, and this even when using relatively low intravenous AAV dosages, compared with what is currently used in clinical trials.
RESUMEN
Huntington's disease (HD) is the most common monogenic neurodegenerative disease and is fatal. CAG repeat expansions in mutant Huntingtin (mHTT) exon 1 encode for polyglutamine (polyQ) stretches and influence age of onset and disease severity, depending on their length. mHTT is more structured compared to wild-type (wt) HTT, resulting in a decreased N-terminal conformational flexibility. mHTT inflexibility may contribute to both gain of function toxicity, due to increased mHTT aggregation propensity, but also to loss of function phenotypes, due to decreased interactions with binding partners. High-throughput-screening techniques to identify mHTT flexibility states and potential flexibility modifying small molecules are currently lacking. Here, we propose a novel approach for identifying small molecules that restore mHTT's conformational flexibility in human patient fibroblasts. We have applied a well-established antibody-based time-resolved Förster resonance energy transfer (TR-FRET) immunoassay, which measures endogenous HTT flexibility using two validated HTT-specific antibodies, to a high-throughput screening platform. By performing a small-scale compound screen, we identified several small molecules that can partially rescue mHTT inflexibility, presumably by altering HTT post-translational modifications. Thus, we demonstrated that the HTT TR-FRET immunoassay can be miniaturized and applied to a compound screening workflow in patient cells. This automated assay can now be used in large screening campaigns to identify previously unknown HD drugs and drug targets.
Asunto(s)
Enfermedad de Huntington , Enfermedades Neurodegenerativas , Transferencia Resonante de Energía de Fluorescencia , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/genética , Mutación , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismoRESUMEN
Huntington's disease is a fatal neurodegenerative disorder caused by an inherited mutation in the huntingtin (HTT) gene comprising an expanded cytosine-adenine-guanine (CAG) trinucleotide repeat sequence that results in a pathogenic huntingtin protein. Adeno-associated viral (AAV) gene therapy containing a primary artificial microRNA (pri-amiRNA) specifically targeting HTT messenger RNA (mRNA) has the potential to provide long-lasting therapeutic benefit, through durable reduction of mutant HTT expression after a single administration. The efficiency and precision of processing of the pri-amiRNA precursor to the mature guide (G) strand by transduced cells are critical for specific and potent HTT mRNA lowering. The selection of the optimized pri-amiRNA comprised a series of in vitro studies followed by in vivo studies in small and then large mammals. Our studies demonstrate the predictivity of certain cell culture systems and rodent models for nonhuman primates with respect to some, but not all key features of pri-amiRNA processing. In addition, our results show that the processing of pri-amiRNAs to the mature guide strand can differ greatly across different scaffolds and sequences while providing the same levels of target lowering. Importantly, our data demonstrate that there is a combinatorial effect of guide and passenger (P) strand sequences, together with the scaffold, on pri-amiRNA processing, with different guide and passenger strand sequences within the same scaffold dramatically altering pri-amiRNA processing. Taken together, our results highlight the importance of optimizing not only target lowering but also the efficiency and precision of pri-amiRNA processing in vitro, in rodents and in large mammals to identify the most potent and selective AAV gene therapy that harnesses the endogenous microRNA (miRNA) biogenesis pathway for target lowering without perturbing the endogenous cellular miRNA profile. The optimized pri-amiRNA was selected with this focus on efficiency and precision of pri-amiRNA processing in addition to its pharmacological activity on HTT mRNA lowering and general tolerability in vivo.
Asunto(s)
Enfermedad de Huntington , MicroARNs , Animales , Terapia Genética , Vectores Genéticos/genética , Proteína Huntingtina/genética , Enfermedad de Huntington/genética , Enfermedad de Huntington/terapia , Ratones , MicroARNs/genética , Primates/genéticaRESUMEN
Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disease caused by a CAG expansion, which translates into an elongated polyglutamine (polyQ) repeat near the amino-terminus of the huntingtin protein (HTT). This results in production of a toxic mutant huntingtin protein (mHTT) that leads to neuronal dysfunction and death. Currently, no disease-modifying treatments are available; however, numerous therapeutic strategies aimed at lowering HTT levels in the brain are under development. To date, studies have not closely examined the contribution of mHTT in neurons vs astrocytes to disease pathophysiology. To better understand the role of astrocytes in HD pathophysiology and the need for cell type specific targeting of HTT lowering therapeutic strategies, AAV capsids were employed that selectively transduce neurons, or both neurons and astrocytes. These vectors carrying miRNA sequences directed against HTT were injected into the YAC128 mouse model of HD to selectively lower HTT expression in neurons alone versus neurons and astrocytes. The results suggested that HTT lowering in neurons alone was not sufficient to rescue the motor phenotype in YAC128 mice. Furthermore, HTT lowering in both cell types was required to achieve maximal functional benefit. The study suggested that astrocyte dysfunction may play a critical role in HD pathogenesis, and thus astrocytes represent an important therapeutic target.
Asunto(s)
Astrocitos/metabolismo , Proteína Huntingtina/antagonistas & inhibidores , Enfermedad de Huntington/metabolismo , Animales , Astrocitos/patología , Encéfalo/metabolismo , Encéfalo/patología , Dependovirus , Modelos Animales de Enfermedad , Vectores Genéticos , Proteína Huntingtina/genética , Enfermedad de Huntington/patología , Ratones , Ratones Transgénicos , MicroARNs , Neuronas/metabolismo , Neuronas/patología , Fenotipo , Transducción GenéticaRESUMEN
The present study was designed to characterize transduction of non-human primate brain and spinal cord with a modified adeno-associated virus serotype 2, incapable of binding to the heparan sulfate proteoglycan receptor, referred to as AAV2-HBKO. AAV2-HBKO was infused into the thalamus, intracerebroventricularly or via a combination of both intracerebroventricular and thalamic delivery. Thalamic injection of this modified vector encoding GFP resulted in widespread CNS transduction that included neurons in deep cortical layers, deep cerebellar nuclei, several subcortical regions, and motor neuron transduction in the spinal cord indicative of robust bidirectional axonal transport. Intracerebroventricular delivery similarly resulted in widespread cortical transduction, with one striking distinction that oligodendrocytes within superficial layers of the cortex were the primary cell type transduced. Robust motor neuron transduction was also observed in all levels of the spinal cord. The combination of thalamic and intracerebroventricular delivery resulted in transduction of oligodendrocytes in superficial cortical layers and neurons in deeper cortical layers. Several subcortical regions were also transduced. Our data demonstrate that AAV2-HBKO is a powerful vector for the potential treatment of a wide number of neurological disorders, and highlight that delivery route can significantly impact cellular tropism and pattern of CNS transduction.
Asunto(s)
Terapia Genética , Vectores Genéticos/efectos adversos , Neuronas/efectos de los fármacos , Parvovirinae/genética , Médula Espinal/efectos de los fármacos , Animales , Transporte Axonal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/patología , Proteínas de la Cápside/administración & dosificación , Proteínas de la Cápside/genética , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/patología , Dependovirus , Modelos Animales de Enfermedad , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Proteoglicanos de Heparán Sulfato/administración & dosificación , Proteoglicanos de Heparán Sulfato/genética , Humanos , Infusiones Intraventriculares , Neuronas Motoras/efectos de los fármacos , Neuronas/patología , Primates , Médula Espinal/patología , Tálamo/efectos de los fármacosRESUMEN
The successful application of adeno-associated virus (AAV) gene delivery vectors as a therapeutic paradigm will require efficient gene delivery to the appropriate cells in affected organs. In this study, we utilized a rational design approach to introduce modifications to the AAV2 and AAVrh8R capsids and the resulting variants were evaluated for transduction activity in the retina and brain. The modifications disrupted either capsid/receptor binding or altered capsid surface charge. Specifically, we mutated AAV2 amino acids R585A and R588A, which are required for binding to its receptor, heparan sulfate proteoglycans, to generate a variant referred to as AAV2-HBKO. In contrast to parental AAV2, the AAV2-HBKO vector displayed low-transduction activity following intravitreal delivery to the mouse eye; however, following its subretinal delivery, AAV2-HBKO resulted in significantly greater photoreceptor transduction. Intrastriatal delivery of AAV2-HBKO to mice facilitated widespread striatal and cortical expression, in contrast to the restricted transduction pattern of the parental AAV2 vector. Furthermore, we found that altering the surface charge on the AAVrh8R capsid by modifying the number of arginine residues on the capsid surface had a profound impact on subretinal transduction. The data further validate the potential of capsid engineering to improve AAV gene therapy vectors for clinical applications.
Asunto(s)
Terapia Genética/métodos , Parvovirinae/crecimiento & desarrollo , Parvovirinae/inmunología , Animales , Encéfalo/metabolismo , Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Dependovirus/inmunología , Técnicas de Transferencia de Gen , Vectores Genéticos , Células HeLa , Heparitina Sulfato , Humanos , Ratones , Ratones Endogámicos C57BL , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Transducción Genética/métodosRESUMEN
Huntington's disease (HD) is caused by a toxic gain-of-function associated with the expression of the mutant huntingtin (htt) protein. Therefore, the use of RNA interference to inhibit Htt expression could represent a disease-modifying therapy. The potential of two recombinant adeno-associated viral vectors (AAV), AAV1 and AAV2, to transduce the cortico-striatal tissues that are predominantly affected in HD was explored. Green fluorescent protein was used as a reporter in each vector to show that both serotypes were broadly distributed in medium spiny neurons in the striatum and cortico-striatal neurons after infusion into the putamen and caudate nucleus of nonhuman primates (NHP), with AAV1-directed expression being slightly more robust than AAV2-driven expression. This study suggests that both serotypes are capable of targeting neurons that degenerate in HD, and it sets the stage for the advanced preclinical evaluation of an RNAi-based therapy for this disease.
RESUMEN
Huntington Disease (HD) is a progressive neurodegenerative disease caused by an elongated CAG repeat in the huntingtin (HTT) gene that encodes a polyglutamine tract in the HTT protein. Proteolysis of the mutant HTT protein (mHTT) has been detected in human and murine HD brains and is implicated in the pathogenesis of HD. Of particular importance is the site at amino acid (aa) 586 that contains a caspase-6 (Casp6) recognition motif. Activation of Casp6 occurs presymptomatically in human HD patients and the inhibition of mHTT proteolysis at aa586 in the YAC128 mouse model results in the full rescue of HD-like phenotypes. Surprisingly, Casp6 ablation in two different HD mouse models did not completely prevent the generation of this fragment, and therapeutic benefits were limited, questioning the role of Casp6 in the disease. We have evaluated the impact of the loss of Casp6 in the YAC128 mouse model of HD. Levels of the mHTT-586 fragment are reduced but not absent in the absence of Casp6 and we identify caspase 8 as an alternate enzyme that can generate this fragment. In vivo, the ablation of Casp6 results in a partial rescue of body weight gain, normalized IGF-1 levels, a reversal of the depression-like phenotype and decreased HTT levels. In the YAC128/Casp6-/- striatum there is a concomitant reduction in p62 levels, a marker of autophagic activity, suggesting increased autophagic clearance. These results implicate the HTT-586 fragment as a key contributor to certain features of HD, irrespective of the enzyme involved in its generation.
Asunto(s)
Caspasa 6/metabolismo , Enfermedad de Huntington/enzimología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Animales , Peso Corporal , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Caspasa 6/genética , Cuerpo Estriado/metabolismo , Depresión/metabolismo , Modelos Animales de Enfermedad , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones Transgénicos , Actividad MotoraRESUMEN
Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disease caused by an increase in the number of polyglutamine residues in the huntingtin (Htt) protein. With the identification of the underlying basis of HD, therapies are being developed that reduce expression of the causative mutant Htt. RNA interference (RNAi) that seeks to selectively reduce the expression of such disease-causing agents is emerging as a potential therapeutic strategy for this and similar disorders. This study examines the merits of administering a recombinant adeno-associated viral (AAV) vector designed to deliver small interfering RNA (siRNA) that targets the degradation of the Htt transcript. The aim was to lower Htt levels and to correct the behavioral, biochemical, and neuropathological deficits shown to be associated with the YAC128 mouse model of HD. Our data demonstrate that AAV-mediated RNAi is effective at transducing greater than 80% of the cells in the striatum and partially reducing the levels (~40%) of both wild-type and mutant Htt in this region. Concomitant with these reductions are significant improvements in behavioral deficits, reduction of striatal Htt aggregates, and partial correction of the aberrant striatal transcriptional profile observed in YAC128 mice. Importantly, a partial reduction of both the mutant and wild-type Htt levels is not associated with any notable overt neurotoxicity. Collectively, these results support the continued development of AAV-mediated RNAi as a therapeutic strategy for HD.
Asunto(s)
Dependovirus/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Proteínas Mutantes/genética , Proteínas del Tejido Nervioso/genética , Interferencia de ARN , Animales , Conducta Animal , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Proteína Huntingtina , Ratones , Ratones Transgénicos , MicroARNs/metabolismo , Neostriado/metabolismo , Neostriado/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción GenéticaRESUMEN
BACKGROUND: Huntington's disease (HD) is a neurological disorder caused by mutations in the huntingtin (HTT) gene, the product of which leads to selective and progressive neuronal cell death in the striatum and cortex. Transcriptional dysregulation has emerged as a core pathologic feature in the CNS of human and animal models of HD. It is still unclear whether perturbations in gene expression are a consequence of the disease or importantly, contribute to the pathogenesis of HD. OBJECTIVE: To examine if transcriptional dysregulation can be ameliorated with antisense oligonucleotides that reduce levels of mutant Htt and provide therapeutic benefit in the YAC128 mouse model of HD. METHODS: Quantitative real-time PCR analysis was used to evaluate dysregulation of a subset of striatal genes in the YAC128 mouse model. Transcripts were then evaluated following ICV delivery of antisense oligonucleotides (ASO). Rota rod and Porsolt swim tests were used to evaluate phenotypic deficits in these mice following ASO treatment. RESULTS: Transcriptional dysregulation was detected in the YAC128 mouse model and appears to progress with age. ICV delivery of ASOs directed against mutant Htt resulted in reduction in mutant Htt levels and amelioration in behavioral deficits in the YAC128 mouse model. These improvements were correlated with improvements in the levels of several dysregulated striatal transcripts. CONCLUSIONS: The role of transcriptional dysregulation in the pathogenesis of Huntington's disease is not well understood, however, a wealth of evidence now strongly suggests that changes in transcriptional signatures are a prominent feature in the brains of both HD patients and animal models of the disease. Our study is the first to show that a therapeutic agent capable of improving an HD disease phenotype is concomitantly correlated with normalization of a subset of dysregulated striatal transcripts. Our data suggests that correction of these disease-altered transcripts may underlie, at least in part, the therapeutic efficacy shown associated with ASO-mediated correction of HD phenotypes and may provide a novel set of early biomarkers for evaluating future therapeutic concepts for HD.
Asunto(s)
Conducta Animal/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Enfermedad de Huntington/genética , Destreza Motora/efectos de los fármacos , Neostriado/efectos de los fármacos , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas Nucleares/efectos de los fármacos , Oligonucleótidos Antisentido/farmacología , Animales , Modelos Animales de Enfermedad , Fosfoproteína 32 Regulada por Dopamina y AMPc/efectos de los fármacos , Fosfoproteína 32 Regulada por Dopamina y AMPc/genética , Encefalinas/efectos de los fármacos , Encefalinas/genética , Proteína Huntingtina , Hipoxantina Fosforribosiltransferasa/efectos de los fármacos , Hipoxantina Fosforribosiltransferasa/genética , Infusiones Intraventriculares , Ratones , Neostriado/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Cannabinoide CB1/efectos de los fármacos , Receptor Cannabinoide CB1/genética , Receptores de Dopamina D1/efectos de los fármacos , Receptores de Dopamina D1/genética , Receptores de Dopamina D2/efectos de los fármacos , Receptores de Dopamina D2/genéticaRESUMEN
The primary cause of Huntington's disease (HD) is expression of huntingtin with a polyglutamine expansion. Despite an absence of consensus on the mechanism(s) of toxicity, diminishing the synthesis of mutant huntingtin will abate toxicity if delivered to the key affected cells. With antisense oligonucleotides (ASOs) that catalyze RNase H-mediated degradation of huntingtin mRNA, we demonstrate that transient infusion into the cerebrospinal fluid of symptomatic HD mouse models not only delays disease progression but mediates a sustained reversal of disease phenotype that persists longer than the huntingtin knockdown. Reduction of wild-type huntingtin, along with mutant huntingtin, produces the same sustained disease reversal. Similar ASO infusion into nonhuman primates is shown to effectively lower huntingtin in many brain regions targeted by HD pathology. Rather than requiring continuous treatment, our findings establish a therapeutic strategy for sustained HD disease reversal produced by transient ASO-mediated diminution of huntingtin synthesis.
Asunto(s)
Enfermedad de Huntington/terapia , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas Nucleares/antagonistas & inhibidores , Oligodesoxirribonucleótidos Antisentido/uso terapéutico , Animales , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Proteína Huntingtina , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Infusión Espinal , Macaca mulatta , Ratones , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Neuronas/patología , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Oligodesoxirribonucleótidos Antisentido/administración & dosificación , Tiempo , Resultado del TratamientoRESUMEN
The development of animal models of Huntington disease (HD) has enabled studies that help define the molecular aberrations underlying the disease. The BACHD and YAC128 transgenic mouse models of HD harbor a full-length mutant huntingtin (mHTT) and recapitulate many of the behavioural and neuropathological features of the human condition. Here, we demonstrate that while BACHD and YAC128 animals exhibit similar deficits in motor learning and coordination, depressive-like symptoms, striatal volume loss and forebrain weight loss, they show obvious differences in key features characteristic of HD. While YAC128 mice exhibit significant and widespread accumulation of mHTT striatal aggregates, these mHTT aggregates are absent in BACHD mice. Furthermore, the levels of several striatally enriched mRNA for genes, such as DARPP-32, enkephalin, dopamine receptors D1 and D2 and cannabinoid receptor 1, are significantly decreased in YAC128 but not BACHD mice. These findings may reflect sequence differences in the human mHTT transgenes harboured by the BACHD and YAC128 mice, including both single nucleotide polymorphisms as well as differences in the nature of CAA interruptions of the CAG tract. Our findings highlight a similar profile of HD-like behavioural and neuropathological deficits and illuminate differences that inform the use of distinct endpoints in trials of therapeutic agents in the YAC128 and BACHD mice.
Asunto(s)
Enfermedad de Huntington/genética , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Proteína Huntingtina , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Masculino , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Neuronas/patología , TransgenesRESUMEN
Syrian hamsters readily form dominant-subordinate relationships under laboratory conditions. Winning or losing in agonistic encounters can have striking, long-term effects on social behavior, but the mechanisms underlying this experience-induced behavioral plasticity are unclear. The present study tested the hypothesis that changes in brain-derived neurotrophic factor (BDNF) may at least in part mediate this plasticity. Male hamsters were paired for 15-min using a resident-intruder model, and individuals were identified as winners or losers on the basis of their behavior. BDNF was examined with in situ hybridization 2 hr after treatment during the consolidation period of emotional learning. Losing animals had significantly more BDNF mRNA in the basolateral (BLA) and medial (MeA) nuclei of the amygdala when compared with winning animals as well as novel cage and home cage controls. Interestingly, winning animals had significantly more BDNF mRNA in the dentate gyrus of the dorsal hippocampus than did losing animals, novel, and home cage controls. No conflict-related changes in BDNF mRNA were observed in several other regions including the bed nucleus of the stria terminalis and central amygdala. Next, we demonstrated that K252a, a Trk receptor antagonist, significantly reduced the acquisition of conditioned defeat when administered within the BLA. These data support a model in which BDNF-mediated plasticity within the BLA supports learning of submission or subordinate social status in losing animals, whereas BDNF-mediated plasticity within the hippocampus may instantiate aspects of winning such as control of a territory in dominant animals.
Asunto(s)
Agresión/fisiología , Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Regulación de la Expresión Génica , Sistema Límbico/metabolismo , Conducta Social , Estrés Psicológico/metabolismo , Territorialidad , Agresión/psicología , Amígdala del Cerebelo/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/fisiología , Cricetinae , Masculino , Mesocricetus , Plasticidad Neuronal/fisiología , Estrés Psicológico/psicologíaRESUMEN
Emerging genetic and clinical evidence suggests a link between Gaucher disease and the synucleinopathies Parkinson disease and dementia with Lewy bodies. Here, we provide evidence that a mouse model of Gaucher disease (Gba1(D409V/D409V)) exhibits characteristics of synucleinopathies, including progressive accumulation of proteinase K-resistant α-synuclein/ubiquitin aggregates in hippocampal neurons and a coincident memory deficit. Analysis of homozygous (Gba1(D409V/D409V)) and heterozygous (Gba1(D409V/+) and Gba1(+/-)) Gaucher mice indicated that these pathologies are a result of the combination of a loss of glucocerebrosidase activity and a toxic gain-of-function resulting from expression of the mutant enzyme. Importantly, adeno-associated virus-mediated expression of exogenous glucocerebrosidase injected into the hippocampus of Gba1(D409V/D409V) mice ameliorated both the histopathological and memory aberrations. The data support the contention that mutations in GBA1 can cause Parkinson disease-like α-synuclein pathology, and that rescuing brain glucocerebrosidase activity might represent a therapeutic strategy for GBA1-associated synucleinopathies.
Asunto(s)
Enfermedad de Gaucher/patología , Glucosilceramidasa/metabolismo , Hipocampo/enzimología , alfa-Sinucleína/metabolismo , Análisis de Varianza , Animales , Western Blotting , Dependovirus , Endopeptidasa K/metabolismo , Enfermedad de Gaucher/metabolismo , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Glucosilceramidasa/genética , Hipocampo/citología , Inmunohistoquímica , RatonesRESUMEN
Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations in the SMN1 gene that result in a deficiency of SMN protein. One approach to treat SMA is to use antisense oligonucleotides (ASOs) to redirect the splicing of a paralogous gene, SMN2, to boost production of functional SMN. Injection of a 2'-O-2-methoxyethyl-modified ASO (ASO-10-27) into the cerebral lateral ventricles of mice with a severe form of SMA resulted in splice-mediated increases in SMN protein and in the number of motor neurons in the spinal cord, which led to improvements in muscle physiology, motor function and survival. Intrathecal infusion of ASO-10-27 into cynomolgus monkeys delivered putative therapeutic levels of the oligonucleotide to all regions of the spinal cord. These data demonstrate that central nervous system-directed ASO therapy is efficacious and that intrathecal infusion may represent a practical route for delivering this therapeutic in the clinic.
Asunto(s)
Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patología , Atrofia Muscular Espinal/terapia , Oligonucleótidos Antisentido/uso terapéutico , Médula Espinal/patología , Animales , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Humanos , Macaca fascicularis , Ratones , Neuronas Motoras/fisiología , Atrofia Muscular Espinal/fisiopatología , Unión Neuromuscular/ultraestructura , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacocinética , Empalme del ARN , Médula Espinal/fisiopatología , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína 2 para la Supervivencia de la Neurona Motora/genéticaRESUMEN
CART is a neuropeptide that appears to play an important role in a variety of physiological processes. The major research focus into the function of CART peptide has been on feeding behavior, modulation of mesolimbic dopamine, and actions of psychostimulant drugs. The neuroanatomic expression profile of CART does however suggest other functions as well, and its presence within the limbic system points to a possible role in emotionality. There are now several published reports which describe a new role for CART as a mediator of anxiety-like behaviors in rodents. This review will summarize these findings and speculate on the mechanisms by which CART might be involved in the modulation of these behaviors. We will also consider what future studies need to be done to further clarify the role of this peptide in anxiety.
Asunto(s)
Ansiedad/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Ansiedad/fisiopatología , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Encéfalo/anatomía & histología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Dopamina/metabolismo , Modelos Biológicos , Proteínas del Tejido Nervioso/farmacología , Proteínas del Tejido Nervioso/fisiologíaRESUMEN
CART (cocaine- and amphetamine-regulated transcript) peptides are neuropeptides expressed throughout the central nervous system and have been implicated in a variety of physiological processes. Research on the many physiological processes involving CART peptide have been somewhat limited by the lack of an identified CART antagonist. Development of CART peptide deficient mice has allowed scientists to further explore the many functions of CART peptide. This review briefly summarizes recent findings in the literature characterizing CART peptide deficient mice.