RESUMEN
Microglia are the resident immune cells in the brain that play a key role in driving neuroinflammation, a hallmark of neurodegenerative disorders. Inducible microglia-like cells have been developed as an in vitro platform for molecular and therapeutic hypothesis generation and testing. However, there has been no systematic assessment of similarity of these cells to primary human microglia along with their responsiveness to external cues expected of primary cells in the brain. In this study, we performed transcriptional characterization of commercially available human inducible pluripotent stem cell (iPSC)-derived microglia-like (iMGL) cells by bulk and single cell RNA sequencing to assess their similarity with primary human microglia. To evaluate their stimulation responsiveness, iMGL cells were treated with Liver X Receptor (LXR) pathway agonists and their transcriptional responses characterized by bulk and single cell RNA sequencing. Bulk transcriptome analyses demonstrate that iMGL cells have a similar overall expression profile to freshly isolated human primary microglia and express many key microglial transcription factors and functional and disease-associated genes. Notably, at the single-cell level, iMGL cells exhibit distinct transcriptional subpopulations, representing both homeostatic and activated states present in normal and diseased primary microglia. Treatment of iMGL cells with LXR pathway agonists induces robust transcriptional changes in lipid metabolism and cell cycle at the bulk level. At the single cell level, we observe heterogeneity in responses between cell subpopulations in homeostatic and activated states and deconvolute bulk expression changes into their corresponding single cell states. In summary, our results demonstrate that iMGL cells exhibit a complex transcriptional profile and responsiveness, reminiscent of in vivo microglia, and thus represent a promising model system for therapeutic development in neurodegeneration.
Asunto(s)
Células Madre Pluripotentes Inducidas , Enfermedades Neurodegenerativas , Células Madre Pluripotentes , Humanos , Microglía/metabolismo , Factores de Transcripción/metabolismo , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismoRESUMEN
Expanding the arsenal of prophylactic approaches against SARS-CoV-2 is of utmost importance, specifically those strategies that are resistant to antigenic drift in Spike. Here, we conducted a screen of over 16,000 RNAi triggers against the SARS-CoV-2 genome, using a massively parallel assay to identify hyper-potent siRNAs. We selected Ten candidates for in vitro validation and found five siRNAs that exhibited hyper-potent activity (IC50 < 20 pM) and strong blockade of infectivity in live-virus experiments. We further enhanced this activity by combinatorial pairing of the siRNA candidates and identified cocktails that were active against multiple types of variants of concern (VOC). We then examined over 2,000 possible mutations in the siRNA target sites by using saturation mutagenesis and confirmed broad protection of the leading cocktail against future variants. Finally, we demonstrated that intranasal administration of this siRNA cocktail effectively attenuates clinical signs and viral measures of disease in the gold-standard Syrian hamster model. Our results pave the way for the development of an additional layer of antiviral prophylaxis that is orthogonal to vaccines and monoclonal antibodies.
Asunto(s)
COVID-19 , ARN Interferente Pequeño , SARS-CoV-2 , Animales , Cricetinae , Administración Intranasal , COVID-19/prevención & control , Mesocricetus , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/uso terapéutico , SARS-CoV-2/genéticaRESUMEN
Expanding the arsenal of prophylactic approaches against SARS-CoV-2 is of utmost importance, specifically those strategies that are resistant to antigenic drift in Spike. Here, we conducted a screen with over 16,000 RNAi triggers against the SARS-CoV-2 genome using a massively parallel assay to identify hyper-potent siRNAs. We selected 10 candidates for in vitro validation and found five siRNAs that exhibited hyper-potent activity with IC50<20pM and strong neutralisation in live virus experiments. We further enhanced the activity by combinatorial pairing of the siRNA candidates to develop siRNA cocktails and found that these cocktails are active against multiple types of variants of concern (VOC). We examined over 2,000 possible mutations to the siRNA target sites using saturation mutagenesis and identified broad protection against future variants. Finally, we demonstrated that intranasal administration of the siRNA cocktail effectively attenuates clinical signs and viral measures of disease in the Syrian hamster model. Our results pave the way to development of an additional layer of antiviral prophylaxis that is orthogonal to vaccines and monoclonal antibodies.
RESUMEN
Real-world healthcare data hold the potential to identify therapeutic solutions for progressive diseases by efficiently pinpointing safe and efficacious repurposing drug candidates. This approach circumvents key early clinical development challenges, particularly relevant for neurological diseases, concordant with the vision of the 21st Century Cures Act. However, to-date, these data have been utilized mainly for confirmatory purposes rather than as drug discovery engines. Here, we demonstrate the usefulness of real-world data in identifying drug repurposing candidates for disease-modifying effects, specifically candidate marketed drugs that exhibit beneficial effects on Parkinson's disease (PD) progression. We performed an observational study in cohorts of ascertained PD patients extracted from two large medical databases, Explorys SuperMart (N = 88,867) and IBM MarketScan Research Databases (N = 106,395); and applied two conceptually different, well-established causal inference methods to estimate the effect of hundreds of drugs on delaying dementia onset as a proxy for slowing PD progression. Using this approach, we identified two drugs that manifested significant beneficial effects on PD progression in both datasets: rasagiline, narrowly indicated for PD motor symptoms; and zolpidem, a psycholeptic. Each confers its effects through distinct mechanisms, which we explored via a comparison of estimated effects within the drug classification ontology. We conclude that analysis of observational healthcare data, emulating otherwise costly, large, and lengthy clinical trials, can highlight promising repurposing candidates, to be validated in prospective registration trials, beneficial against common, late-onset progressive diseases for which disease-modifying therapeutic solutions are scarce.
RESUMEN
Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are emerging worldwide epidemics, projected to become the leading cause of liver transplants. The strongest genetic risk factor for NAFLD/NASH susceptibility and progression is a single-nucleotide polymorphism (SNP) in the patatin-like phospholipase domain-containing 3 gene (PNPLA3), rs738409, encoding the missense mutation I148M. This aminoacidic substitution interferes with the normal remodeling of lipid droplets in hepatocytes. It is also thought to play a key role in promoting liver fibrosis by inhibiting the release of retinol from hepatic stellate cells. Reducing PNPLA3 levels in individuals homozygous for 148M may be an effective treatment for the entire spectrum of NAFLD, based on gene dosage analysis in the human population, as well as the protective effect of another naturally occurring SNP (rs2294918) in PNPLA3 which, when co-inherited, reduces PNPLA3 mRNA levels to 50% and counteracts disease risk. By screening a clinical compound library targeting specific signaling pathways active in primary human hepatocytes, we identified momelotinib, a drug evaluated in clinical trials to treat myelofibrosis, as a potent down-regulator of PNPLA3 expression, across all genotypes. We found that momelotinib treatment yielded >80% reduction in PNPLA3 mRNA in human primary hepatocytes and stellate cells, as well as in vivo via acute and chronic treatment of WT mice. Using a human multilineage 3D spheroid model of NASH homozygous for the PNPLA3 mutant protein, we additionally show that it decreases PNPLA3 mRNA as well as intracellular lipid content. Furthermore, we show that the effects on PNPLA3 coincide with changes in chromatin accessibility within regulatory regions of the PNPLA3 locus, consistent with inhibition occurring at the level of transcription. In addition to its primary reported targets, the JAK kinases, momelotinib inhibits several non-JAK kinases, including ACVR1. Using a combination of targeted siRNA knockdowns and signaling pathway perturbations, we show that momelotinib reduces the expression of the PNPLA3 gene largely through the inhibition of BMP signaling rather than the JAK/STAT pathway. Overall, our work identified momelotinib as a potential NASH therapeutic and uncovered previously unrecognized connections between signaling pathways and PNPLA3. These pathways may be exploited by drug modalities to "tune down" the level of gene expression, and therefore offer a potential therapeutic benefit to a high at-risk subset of NAFLD/NASH patients.
Asunto(s)
Enfermedad del Hígado Graso no Alcohólico/genética , Fosfolipasas A2 Calcio-Independiente/metabolismo , Animales , Humanos , Masculino , Ratones , Transducción de Señal , TransfecciónRESUMEN
BACKGROUND: A growing number of clinical trials use various sensors and smartphone applications to collect data outside of the clinic or hospital, raising the question to what extent patients comply with the unique requirements of remote study protocols. Compliance is particularly important in conditions where patients are motorically and cognitively impaired. Here, we sought to understand patient compliance in digital trials of two such pathologies, Parkinson's disease (PD) and Huntington disease (HD). METHODS: Patient compliance was assessed in two remote, six-month clinical trials of PD (n = 51, Clinician Input Study funded by the Michael J. Fox Foundation for Parkinson's Research) and HD (n = 17, sponsored by Teva Pharmaceuticals). We monitored four compliance metrics specific to remote studies: smartphone app-based medication reporting, app-based symptoms reporting, the duration of smartwatch data streaming except while charging, and the performance of structured motor tasks at home. RESULTS: While compliance over time differed between the PD and HD studies, both studies maintained high compliance levels for their entire six month duration. None (- 1%) to a 30% reduction in compliance rate was registered for HD patients, and a reduction of 34 to 53% was registered for the PD study. Both studies exhibited marked changes in compliance rates during the initial days of enrollment. Interestingly, daily smartwatch data streaming patterns were similar, peaking around noon, dropping sharply in the late evening hours around 8 pm, and having a mean of 8.6 daily streaming hours for the PD study and 10.5 h for the HD study. Individual patients tended to have either high or low compliance across all compliance metrics as measured by pairwise correlation. Encouragingly, predefined schedules and app-based reminders fulfilled their intended effect on the timing of medication intake reporting and performance of structured motor tasks at home. CONCLUSIONS: Our findings suggest that maintaining compliance over long durations is feasible, promote the use of predefined app-based reminders, and highlight the importance of patient selection as highly compliant patients typically have a higher adherence rate across the different aspects of the protocol. Overall, these data can serve as a reference point for the design of upcoming remote digital studies. TRIAL REGISTRATION: Trials described in this study include a sub-study of the Open PRIDE-HD Huntington's disease study (TV7820-CNS-20016), which was registered on July 7th, 2015, sponsored by Teva Pharmaceuticals Ltd., and registered on Clinicaltrials.gov as NCT02494778 and EudraCT as 2015-000904-24 .
Asunto(s)
Enfermedad de Huntington/psicología , Aplicaciones Móviles , Enfermedad de Parkinson/psicología , Cooperación del Paciente , Teléfono Inteligente , Anciano , Estudios Clínicos como Asunto , Femenino , Humanos , Enfermedad de Huntington/diagnóstico , Enfermedad de Huntington/terapia , Masculino , Persona de Mediana Edad , Enfermedad de Parkinson/diagnóstico , Enfermedad de Parkinson/terapia , Proyectos de Investigación , Factores de TiempoRESUMEN
BACKGROUND: Huntington Disease (HD) is an incurable autosomal dominant neurodegenerative disorder driven by an expansion repeat giving rise to the mutant huntingtin protein (mHtt), which is known to disrupt a multitude of transcriptional pathways. Pridopidine, a small molecule in development for treatment of HD, has been shown to improve motor symptoms in HD patients. In HD animal models, pridopidine exerts neuroprotective effects and improves behavioral and motor functions. Pridopidine binds primarily to the sigma-1 receptor, (IC50 ~ 100 nM), which mediates its neuroprotective properties, such as rescue of spine density and aberrant calcium signaling in HD neuronal cultures. Pridopidine enhances brain-derived neurotrophic factor (BDNF) secretion, which is blocked by putative sigma-1 receptor antagonist NE-100, and was shown to upregulate transcription of genes in the BDNF, glucocorticoid receptor (GR), and dopamine D1 receptor (D1R) pathways in the rat striatum. The impact of different doses of pridopidine on gene expression and transcript splicing in HD across relevant brain regions was explored, utilizing the YAC128 HD mouse model, which carries the entire human mHtt gene containing 128 CAG repeats. METHODS: RNAseq was analyzed from striatum, cortex, and hippocampus of wild-type and YAC128 mice treated with vehicle, 10 mg/kg or 30 mg/kg pridopidine from the presymptomatic stage (1.5 months of age) until 11.5 months of age in which mice exhibit progressive disease phenotypes. RESULTS: The most pronounced transcriptional effect of pridopidine at both doses was observed in the striatum with minimal effects in other regions. In addition, for the first time pridopidine was found to have a dose-dependent impact on alternative exon and junction usage, a regulatory mechanism known to be impaired in HD. In the striatum of YAC128 HD mice, pridopidine treatment initiation prior to symptomatic manifestation rescues the impaired expression of the BDNF, GR, D1R and cAMP pathways. CONCLUSIONS: Pridopidine has broad effects on restoring transcriptomic disturbances in the striatum, particularly involving synaptic transmission and activating neuroprotective pathways that are disturbed in HD. Benefits of treatment initiation at early disease stages track with trends observed in the clinic.
Asunto(s)
Expresión Génica/efectos de los fármacos , Enfermedad de Huntington , Neuroprotección/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Piperidinas/farmacología , Animales , Encéfalo/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Transmisión Sináptica/efectos de los fármacosRESUMEN
Pridopidine is currently under clinical development for Huntington disease (HD), with on-going studies to better characterize its therapeutic benefit and mode of action. Pridopidine was administered either prior to the appearance of disease phenotypes or in advanced stages of disease in the YAC128 mouse model of HD. In the early treatment cohort, animals received 0, 10, or 30 mg/kg pridopidine for a period of 10.5 months. In the late treatment cohort, animals were treated for 8 weeks with 0 mg/kg or an escalating dose of pridopidine (10 to 30 mg/kg over 3 weeks). Early treatment improved motor coordination and reduced anxiety- and depressive-like phenotypes in YAC128 mice, but it did not rescue striatal and corpus callosum atrophy. Late treatment, conversely, only improved depressive-like symptoms. RNA-seq analysis revealed that early pridopidine treatment reversed striatal transcriptional deficits, upregulating disease-specific genes that are known to be downregulated during HD, a finding that is experimentally confirmed herein. This suggests that pridopidine exerts beneficial effects at the transcriptional level. Taken together, our findings support continued clinical development of pridopidine for HD, particularly in the early stages of disease, and provide valuable insight into the potential therapeutic mode of action of pridopidine.
Asunto(s)
Enfermedad de Huntington/tratamiento farmacológico , Fármacos Neuroprotectores/administración & dosificación , Piperidinas/administración & dosificación , Animales , Ansiedad/tratamiento farmacológico , Ansiedad/etiología , Conducta Animal/efectos de los fármacos , Cuerpo Calloso/patología , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Depresión/tratamiento farmacológico , Depresión/etiología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Evaluación Preclínica de Medicamentos/métodos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Enfermedad de Huntington/patología , Enfermedad de Huntington/fisiopatología , Enfermedad de Huntington/psicología , Masculino , Ratones Transgénicos , Actividad Motora/efectos de los fármacos , Fármacos Neuroprotectores/uso terapéutico , Piperidinas/uso terapéutico , Prevención Secundaria/métodos , Transcripción Genética/efectos de los fármacosRESUMEN
Copaxone (glatiramer acetate, GA), a structurally and compositionally complex polypeptide nonbiological drug, is an effective treatment for multiple sclerosis, with a well-established favorable safety profile. The short antigenic polypeptide sequences comprising therapeutically active epitopes in GA cannot be deciphered with state-of-the-art methods; and GA has no measurable pharmacokinetic profile and no validated pharmacodynamic markers. The study reported herein describes the use of orthogonal standard and high-resolution physicochemical and biological tests to characterize GA and a U.S. Food and Drug Administration-approved generic version of GA, Glatopa (USA-FoGA). While similarities were observed with low-resolution or destructive tests, differences between GA and USA-FoGA were measured with high-resolution methods applied to an intact mixture, including variations in surface charge and a unique, high-molecular-weight, hydrophobic polypeptide population observed only in some USA-FoGA lots. Consistent with published reports that modifications in physicochemical attributes alter immune-related processes, genome-wide expression profiles of ex vivo activated splenocytes from mice immunized with either GA or USA-FoGA showed that 7-11% of modulated genes were differentially expressed and enriched for immune-related pathways. Thus, differences between USA-FoGA and GA may include variations in antigenic epitopes that differentially activate immune responses. We propose that the assays reported herein should be considered during the regulatory assessment process for nonbiological complex drugs such as GA.
Asunto(s)
Medicamentos Genéricos/farmacología , Expresión Génica/efectos de los fármacos , Acetato de Glatiramer/farmacología , Fenómenos del Sistema Inmunológico/efectos de los fármacos , Animales , Células Cultivadas , Fenómenos Químicos , Medicamentos Genéricos/química , Medicamentos Genéricos/farmacocinética , Femenino , Perfilación de la Expresión Génica/métodos , Acetato de Glatiramer/química , Acetato de Glatiramer/farmacocinética , Humanos , Fenómenos del Sistema Inmunológico/genética , Inmunosupresores/química , Inmunosupresores/farmacocinética , Inmunosupresores/uso terapéutico , Ratones Endogámicos BALB C , Microscopía de Fuerza Atómica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunología , Bazo/citología , Bazo/efectos de los fármacos , Bazo/metabolismo , Equivalencia TerapéuticaRESUMEN
BACKGROUND: As part of the preparations to establish a population-based biobank in a large Israeli health organization, we aimed to investigate through focus groups the knowledge, perceptions and attitudes of insured Israelis, toward biobanking, and then, after input from focus groups' participants, to empirically assess the impact of a revised recruitment process on recruitment rates. METHODS: 1) Six Focus group discussions were conducted (n = 10 per group) with individuals who had routine blood laboratory tests taken in the last 2 years. 2) After addressing the issues raised in the focus groups and revising the recruitment process, individuals undergoing routine blood tests in phlebotomy clinics (N = 10,262) were invited to participate in the future biobank. RESULTS: At the outset of the focus groups there was an overall positive response to the prospect of a population-based biobank. Concerns revolved around infringement on privacy, fears of the "big brother"(e.g. insurance companies), and anxiety about inequality. Reaction to the language of the informed consent document revolved around concerns over ability to maintain anonymity, to withdraw consent, involvement of commercial entities, and the general tenor of the informed consent, which was perceived as legalistic and unilateral. In general, the longer participants were exposed to discussion about the biobank, the less likely they were to consent to sign in. Overall, only 20% (12) of the 60 participants stated they would agree to sign in by the end of the 2 hour group session. The feedback obtained from the focus groups was used in the second stage ("real life") of the study. A team of recruiters received extensive training to enable fruitful discussion and a detailed explanation to questions and concerns raised during the recruitment process. During the second stage of the study, after revising the consent form and training recruiters, a 53% consent rate was observed among 10,262 participants, more than 4 fold higher than estimated at the focus group stage. CONCLUSIONS: The qualitative focus group research helped identify important perceptions and concerns, which were subsequently addressed in the revised consent form and in the discussion the recruiters had with potential biobank donors.
Asunto(s)
Actitud Frente a la Salud , Bancos de Muestras Biológicas , Conducta de Elección , Consentimiento Informado , Selección de Paciente , Adulto , Anciano , Confidencialidad , Formularios de Consentimiento , Femenino , Grupos Focales , Humanos , Israel , Masculino , Persona de Mediana Edad , Percepción , Privacidad , Investigación CualitativaRESUMEN
BACKGROUND: Copaxone is an efficacious and safe therapy that has demonstrated clinical benefit for over two decades in patients with relapsing forms of multiple sclerosis (MS). On an individual level, patients show variability in their response to Copaxone, with some achieving significantly higher response levels. The involvement of genes (e.g., HLA-DRB1*1501) with high inter-individual variability in Copaxone's mechanism of action (MoA) suggests the potential contribution of genetics to treatment response. This study aimed to identify genetic variants associated with Copaxone response in patient cohorts from late-phase clinical trials. METHODS: Single nucleotide polymorphisms (SNPs) associated with high and low levels of response to Copaxone were identified using genome-wide SNP data in a discovery cohort of 580 patients from two phase III clinical trials of Copaxone. Multivariable Bayesian modeling on the resulting SNPs in an expanded discovery cohort with 1171 patients identified a multi-SNP signature of Copaxone response. This signature was examined in 941 Copaxone-treated MS patients from seven independent late-phase trials of Copaxone and assessed for specificity to Copaxone in 310 Avonex-treated and 311 placebo-treated patients, also from late-phase trials. RESULTS: A four-SNP signature consisting of rs80191572 (in UVRAG), rs28724893 (in HLA-DQB2), rs1789084 (in MBP), and rs139890339 (in ZAK(CDCA7)) was identified as significantly associated with Copaxone response. Copaxone-treated signature-positive patients had a greater reduction in annualized relapse rate (ARR) compared to signature-negative patients in both discovery and independent cohorts, an effect not observed in Avonex-treated patients. Additionally, signature-positive placebo-treated cohorts did not show a reduction in ARR, demonstrating the predictive as opposed to prognostic nature of the signature. A 10% subset of patients, delineated by the signature, showed marked improvements across multiple clinical parameters, including ARR, MRI measures, and higher proportion with no evidence of disease activity (NEDA). CONCLUSIONS: This study is the largest pharmacogenetic study in MS reported to date. Gene regions underlying the four-SNP signature have been linked with pathways associated with either Copaxone's MoA or the pathophysiology of MS. The pronounced association of the four-SNP signature with clinical improvements in a ~10% subset of the MS patient population demonstrates the complex interplay of immune mechanisms and the individualized nature of response to Copaxone.
Asunto(s)
Acetato de Glatiramer/uso terapéutico , Esclerosis Múltiple/tratamiento farmacológico , Variantes Farmacogenómicas , Polimorfismo de Nucleótido Simple , Adulto , Teorema de Bayes , Ensayos Clínicos Fase III como Asunto , Ensayos Clínicos Fase IV como Asunto , Femenino , Acetato de Glatiramer/genética , Humanos , Masculino , Persona de Mediana Edad , Modelos Genéticos , Modelos Estadísticos , Esclerosis Múltiple/genética , Medicina de Precisión , Adulto JovenRESUMEN
Multiple sclerosis (MS) is a chronic, progressive, disabling disorder characterized by immune-mediated demyelination, inflammation, and neurodegenerative tissue damage in the central nervous system (CNS), associated with frequent exacerbations and remissions of neurologic symptoms and eventual permanent neurologic disability. While there are several MS therapies that are successful in reducing MS relapses, none have been effective in treating all patients. The specific response of an individual patient to any one of the MS therapies remains largely unpredictable, and physicians and patients are forced to use a trial and error approach when deciding on treatment regimens. A priori markers to predict the optimal benefit-to-risk profile of an individual MS patient would greatly facilitate the decision-making process, thereby helping the patient receive the most optimal treatment early on in the disease process. Pharmacogenomic methods evaluate how a person's genetic and genomic makeup affects their response to therapeutics. This review focuses on how pharmacogenomics studies are being used to identify biologically relevant differences in MS treatments and provide characterization of the predictive clinical response patterns. As pharmacogenomics research is dependent on the availability of longitudinal clinical research, studies concerning glatiramer acetate and the interferon beta products which have the majority of published long term data to date are described in detail. These studies have provided considerable insight in the prognostic markers associated with MS disease and potential predictive markers of safety and beneficial response.
Asunto(s)
Investigación Biomédica/tendencias , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/genética , Farmacogenética/tendencias , Pruebas de Farmacogenómica/tendencias , Medicina de Precisión/tendencias , Medicina Basada en la Evidencia/tendencias , Humanos , Resultado del TratamientoRESUMEN
The tri-nucleotide repeat expansion underlying Huntington disease (HD) results in corticostriatal synaptic dysfunction and subsequent neurodegeneration of striatal medium spiny neurons (MSNs). HD is a devastating autosomal dominant disease with no disease-modifying treatments. Pridopidine, a postulated "dopamine stabilizer", has been shown to improve motor symptoms in clinical trials of HD. However, the target(s) and mechanism of action of pridopidine remain to be fully elucidated. As binding studies identified sigma-1 receptor (S1R) as a high-affinity receptor for pridopidine, we evaluated the relevance of S1R as a therapeutic target of pridopidine in HD. S1R is an endoplasmic reticulum - (ER) resident transmembrane protein and is regulated by ER calcium homeostasis, which is perturbed in HD. Consistent with ER calcium dysregulation, we observed striatal upregulation of S1R in aged YAC128 transgenic HD mice and HD patients. We previously demonstrated that dendritic MSN spines are lost in aged corticostriatal co-cultures from YAC128 mice. We report here that pridopidine and the chemically similar S1R agonist 3-PPP prevent MSN spine loss in aging YAC128 co-cultures. Spine protection was blocked by neuronal deletion of S1R. Pridopidine treatment suppressed supranormal ER Ca2+ release, restored ER calcium levels and reduced excessive store-operated calcium (SOC) entry in spines, which may account for its synaptoprotective effects. Normalization of ER Ca2+ levels by pridopidine was prevented by S1R deletion. To evaluate long-term effects of pridopidine, we analyzed expression profiles of calcium signaling genes. Pridopidine elevated striatal expression of calbindin and homer1a, whereas their striatal expression was reduced in aged Q175KI and YAC128 HD mouse models compared to WT. Pridopidine and 3-PPP are proposed to prevent calcium dysregulation and synaptic loss in a YAC128 corticostriatal co-culture model of HD. The actions of pridopidine were mediated by S1R and led to normalization of ER Ca2+ release, ER Ca2+ levels and spine SOC entry in YAC128 MSNs. This is a new potential mechanism of action for pridopidine, highlighting S1R as a potential target for HD therapy. Upregulation of striatal proteins that regulate calcium, including calbindin and homer1a, upon chronic therapy with pridopidine, may further contribute to long-term beneficial effects of pridopidine in HD.
Asunto(s)
Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/metabolismo , Fármacos Neuroprotectores/farmacología , Piperidinas/farmacología , Receptores sigma/metabolismo , Envejecimiento/efectos de los fármacos , Envejecimiento/metabolismo , Animales , Calbindinas/metabolismo , Calcio/metabolismo , Cationes Bivalentes/metabolismo , Técnicas de Cocultivo , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Espinas Dendríticas/efectos de los fármacos , Espinas Dendríticas/metabolismo , Modelos Animales de Enfermedad , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Humanos , Ratones , Ratones Transgénicos , Fármacos Neuroprotectores/química , Piperidinas/química , Ratas Endogámicas SHR , Receptores sigma/genética , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Receptor Sigma-1RESUMEN
Laquinimod is an oral drug currently being evaluated for the treatment of relapsing, remitting, and primary progressive multiple sclerosis and Huntington's disease. Laquinimod exerts beneficial activities on both the peripheral immune system and the CNS with distinctive changes in CNS resident cell populations, especially astrocytes and microglia. Analysis of genome-wide expression data revealed activation of the aryl hydrocarbon receptor (AhR) pathway in laquinimod-treated mice. The AhR pathway modulates the differentiation and function of several cell populations, many of which play an important role in neuroinflammation. We therefore tested the consequences of AhR activation in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) using AhR knockout mice. We demonstrate that the pronounced effect of laquinimod on clinical score, CNS inflammation, and demyelination in EAE was abolished in AhR-/- mice. Furthermore, using bone marrow chimeras we show that deletion of AhR in the immune system fully abrogates, whereas deletion within the CNS partially abrogates the effect of laquinimod in EAE. These data strongly support the idea that AhR is necessary for the efficacy of laquinimod in EAE and that laquinimod may represent a first-in-class drug targeting AhR for the treatment of multiple sclerosis and other neurodegenerative diseases.
Asunto(s)
Encefalomielitis Autoinmune Experimental/etiología , Encefalomielitis Autoinmune Experimental/metabolismo , Quinolonas/farmacología , Receptores de Hidrocarburo de Aril/agonistas , Receptores de Hidrocarburo de Aril/metabolismo , Animales , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/patología , Femenino , Eliminación de Gen , Expresión Génica , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Sistema Inmunológico/inmunología , Sistema Inmunológico/metabolismo , Ratones , Ratones Noqueados , Receptores de Hidrocarburo de Aril/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , TranscriptomaRESUMEN
Pridopidine has demonstrated improvement in Huntington Disease (HD) motor symptoms as measured by secondary endpoints in clinical trials. Originally described as a dopamine stabilizer, this mechanism is insufficient to explain the clinical and preclinical effects of pridopidine. This study therefore explored pridopidine's potential mechanisms of action. The effect of pridopidine versus sham treatment on genome-wide expression profiling in the rat striatum was analysed and compared to the pathological expression profile in Q175 knock-in (Q175 KI) vs Q25 WT mouse models. A broad, unbiased pathway analysis was conducted, followed by testing the enrichment of relevant pathways. Pridopidine upregulated the BDNF pathway (P = 1.73E-10), and its effect on BDNF secretion was sigma 1 receptor (S1R) dependent. Many of the same genes were independently found to be downregulated in Q175 KI mice compared to WT (5.2e-7 < P < 0.04). In addition, pridopidine treatment upregulated the glucocorticoid receptor (GR) response, D1R-associated genes and the AKT/PI3K pathway (P = 1E-10, P = 0.001, P = 0.004, respectively). Pridopidine upregulates expression of BDNF, D1R, GR and AKT/PI3K pathways, known to promote neuronal plasticity and survival, as well as reported to demonstrate therapeutic benefit in HD animal models. Activation of S1R, necessary for its effect on the BDNF pathway, represents a core component of the mode of action of pridopidine. Since the newly identified pathways are downregulated in neurodegenerative diseases, including HD, these findings suggest that pridopidine may exert neuroprotective effects beyond its role in alleviating some symptoms of HD.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Cuerpo Estriado/metabolismo , Enfermedad de Huntington/tratamiento farmacológico , Fármacos Neuroprotectores/administración & dosificación , Piperidinas/administración & dosificación , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Genoma , Humanos , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Ratones , Fármacos Neuroprotectores/metabolismo , Ratas , Receptores de Dopamina D5/biosíntesis , Receptores de Dopamina D5/genética , Receptores de Glucocorticoides/biosíntesis , Receptores de Glucocorticoides/genética , Transducción de Señal/efectos de los fármacosRESUMEN
The treatment of early Parkinson's disease with dopaminergic agents remains the mainstay of symptomatic therapy for this incurable neurodegenerative disorder. However, clinical responses to dopaminergic drugs vary substantially from person to person due to individual-, drug- and disease-related factors that may in part be genetically determined. Using clinical data and DNA samples ascertained through the largest placebo-controlled clinical trial of the monoamine oxidase B inhibitor, rasagiline (ClinicalTrials.gov number, NCT00256204), we examined how polymorphisms in candidate genes associate with the clinical response to rasagiline in early Parkinson's disease. Variants in genes that express proteins involved in the pharmacokinetics and pharmacodynamics of rasagiline, and genes previously associated with the risk to develop Parkinson's disease were genotyped. The LifeTechnologies OpenArray NT genotyping platform and polymerase chain reaction-based methods were used to analyse 204 single nucleotide polymorphisms and five variable number tandem repeats from 30 candidate genes in 692 available DNA samples from this clinical trial. The peak symptomatic response to rasagiline, the rate of symptom progression, and their relation to genetic variation were examined controlling for placebo effects using general linear and mixed effects models, respectively. Single nucleotide polymorphisms, rs2283265 and rs1076560, in the dopamine D2 receptor gene (DRD2) were found to be significantly associated with a favourable peak response to rasagiline at 12 weeks in early Parkinson's disease after controlling for multiple testing. From a linear regression, the betas were 2.5 and 2.38, respectively, with false discovery rate-corrected P-values of 0.032. These polymorphisms were in high linkage disequilibrium with each other (r(2) = 0.96) meaning that the same clinical response signal was identified by each of them. No polymorphisms were associated with slowing the rate of worsening in Parkinson symptoms from Weeks 12 to 36 after correction for multiple testing. This is the largest and most comprehensive pharmacogenetics study to date examining clinical response to an anti-parkinsonian drug and the first to be conducted in patients with early stage Parkinson's disease receiving monotherapy. The results indicate a clinically meaningful benefit to rasagiline in terms of the magnitude of improvement in parkinsonian symptoms for those with the favourable response genotypes. Future work is needed to elucidate the specific mechanisms through which these DRD2 variants operate in modulating the function of the nigrostriatal dopaminergic system.media-1vid110.1093/brain/aww109_video_abstractaww109_video_abstract.
Asunto(s)
Indanos/farmacología , Inhibidores de la Monoaminooxidasa/farmacología , Evaluación de Resultado en la Atención de Salud , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/genética , Pruebas de Farmacogenómica/métodos , Receptores de Dopamina D2/genética , Anciano , Femenino , Estudios de Seguimiento , Humanos , Indanos/administración & dosificación , Masculino , Persona de Mediana Edad , Inhibidores de la Monoaminooxidasa/administración & dosificación , Polimorfismo de Nucleótido Simple , Índice de Severidad de la Enfermedad , Secuencias Repetidas en TándemRESUMEN
The secreted disulfide catalyst Quiescin sulfhydryl oxidase-1 (QSOX1) affects extracellular matrix organization and is overexpressed in various adenocarcinomas and associated stroma. Inhibition of extracellular human QSOX1 by a monoclonal antibody decreased tumor cell migration in a cell co-culture model and hence may have therapeutic potential. However, the species specificity of the QSOX1 monoclonal antibody has been a setback in assessing its utility as an anti-metastatic agent in vivo, a common problem in the antibody therapy industry. We therefore used structurally guided engineering to expand the antibody species specificity, improving its affinity toward mouse QSOX1 by at least four orders of magnitude. A crystal structure of the re-engineered variant, complexed with its mouse antigen, revealed that the antibody accomplishes dual-species targeting through altered contacts between its heavy and light chains, plus replacement of bulky aromatics by flexible side chains and versatile water-bridged polar interactions. In parallel, we produced a surrogate antibody targeting mouse QSOX1 that exhibits a new QSOX1 inhibition mode. This set of three QSOX1 inhibitory antibodies is compatible with various mouse models for pre-clinical trials and biotechnological applications. In this study we provide insights into structural blocks to cross-reactivity and set up guideposts for successful antibody design and re-engineering.
Asunto(s)
Anticuerpos Monoclonales/química , Antineoplásicos/química , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/antagonistas & inhibidores , Ingeniería de Proteínas/métodos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Antineoplásicos/metabolismo , Células Cultivadas , Descubrimiento de Drogas , Humanos , Laminina , Ratones , Modelos Moleculares , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/química , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Especificidad de la EspecieRESUMEN
Glatiramer acetate (Copaxone®; GA) is a non-biological complex drug for multiple sclerosis. GA modulated thousands of genes in genome-wide expression studies conducted in THP-1 cells and mouse splenocytes. Comparing GA with differently-manufactured glatiramoid Polimunol (Synthon) in mice yielded hundreds of differentially expressed probesets, including biologically-relevant genes (e.g. Il18, adj p<9e-6) and pathways. In human monocytes, 700+ probesets differed between Polimunol and GA, enriching for 130+ pathways including response to lipopolysaccharide (adj. p<0.006). Key differences were confirmed by qRT-PCR (splenocytes) or proteomics (THP-1). These studies demonstrate the complexity of GA's mechanisms of action, and may help inform therapeutic equivalence assessment.