RESUMEN
Induced pluripotent stem cell (iPSC)-derived motor neurons (MNs) from patients with amyotrophic lateral sclerosis (ALS) and the C9ORF72 hexanucleotide repeat expansion (HRE) have multiple cellular phenotypes, but which of these accurately reflect the biology underlying the cell-specific vulnerability of ALS is uncertain. We therefore compared phenotypes due to the C9ORF72 HRE in MNs with sensory neurons (SNs), which are relatively spared in ALS. The iPSC models were able to partially reproduce the differential gene expression seen between adult SNs and MNs. We demonstrated that the typical hallmarks of C9ORF72-ALS, including RNA foci and dipeptide formation, as well as specific axonal transport defects, occurred equally in MNs and SNs, suggesting that these in vitro phenotypes are not sufficient to explain the cell-type selectivity of ALS in isolation.
Asunto(s)
Esclerosis Amiotrófica Lateral , Transporte Axonal , Proteína C9orf72 , Expansión de las Repeticiones de ADN , Células Madre Pluripotentes Inducidas , Neuronas Motoras , Fenotipo , Células Receptoras Sensoriales , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Humanos , Neuronas Motoras/metabolismo , Células Receptoras Sensoriales/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Expansión de las Repeticiones de ADN/genéticaRESUMEN
Dipeptide repeat proteins are a major pathogenic feature of C9orf72 amyotrophic lateral sclerosis (C9ALS)/frontotemporal dementia (FTD) pathology, but their physiological impact has yet to be fully determined. Here we generated C9orf72 dipeptide repeat knock-in mouse models characterized by expression of 400 codon-optimized polyGR or polyPR repeats, and heterozygous C9orf72 reduction. (GR)400 and (PR)400 knock-in mice recapitulate key features of C9ALS/FTD, including cortical neuronal hyperexcitability, age-dependent spinal motor neuron loss and progressive motor dysfunction. Quantitative proteomics revealed an increase in extracellular matrix (ECM) proteins in (GR)400 and (PR)400 spinal cord, with the collagen COL6A1 the most increased protein. TGF-ß1 was one of the top predicted regulators of this ECM signature and polyGR expression in human induced pluripotent stem cell neurons was sufficient to induce TGF-ß1 followed by COL6A1. Knockdown of TGF-ß1 or COL6A1 orthologues in polyGR model Drosophila exacerbated neurodegeneration, while expression of TGF-ß1 or COL6A1 in induced pluripotent stem cell-derived motor neurons of patients with C9ALS/FTD protected against glutamate-induced cell death. Altogether, our findings reveal a neuroprotective and conserved ECM signature in C9ALS/FTD.
Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Células Madre Pluripotentes Inducidas , Animales , Humanos , Ratones , Demencia Frontotemporal/patología , Esclerosis Amiotrófica Lateral/metabolismo , Factor de Crecimiento Transformador beta1 , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas Motoras/metabolismo , Drosophila , Matriz Extracelular/metabolismo , Dipéptidos/metabolismo , Expansión de las Repeticiones de ADN/genéticaRESUMEN
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive motor neuron loss, with additional pathophysiological involvement of non-neuronal cells such as microglia. The commonest ALS-associated genetic variant is a hexanucleotide repeat expansion (HRE) mutation in C9orf72. Here, we study its consequences for microglial function using human iPSC-derived microglia. By RNA-sequencing, we identify enrichment of pathways associated with immune cell activation and cyto-/chemokines in C9orf72 HRE mutant microglia versus healthy controls, most prominently after LPS priming. Specifically, LPS-primed C9orf72 HRE mutant microglia show consistently increased expression and release of matrix metalloproteinase-9 (MMP9). LPS-primed C9orf72 HRE mutant microglia are toxic to co-cultured healthy motor neurons, which is ameliorated by concomitant application of an MMP9 inhibitor. Finally, we identify release of dipeptidyl peptidase-4 (DPP4) as a marker for MMP9-dependent microglial dysregulation in co-culture. These results demonstrate cellular dysfunction of C9orf72 HRE mutant microglia, and a non-cell-autonomous role in driving C9orf72-ALS pathophysiology in motor neurons through MMP9 signaling.
Asunto(s)
Esclerosis Amiotrófica Lateral , Células Madre Pluripotentes Inducidas , Enfermedades Neurodegenerativas , Humanos , Esclerosis Amiotrófica Lateral/genética , Metaloproteinasa 9 de la Matriz/genética , Proteína C9orf72/genética , Microglía , Técnicas de Cocultivo , Lipopolisacáridos , Neuronas MotorasRESUMEN
Hexanucleotide repeat expansions in the C9orf72 gene are the most prevalent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Transcripts of the expansions are translated into toxic dipeptide repeat (DPR) proteins. Most preclinical studies in cell and animal models have used protein-tagged polyDPR constructs to investigate DPR toxicity but the effects of tags on DPR toxicity have not been systematically explored. Here, we used Drosophila to assess the influence of protein tags on DPR toxicity. Tagging of 36 but not 100 arginine-rich DPRs with mCherry increased toxicity, whereas adding mCherry or GFP to GA100 completely abolished toxicity. FLAG tagging also reduced GA100 toxicity but less than the longer fluorescent tags. Expression of untagged but not GFP- or mCherry-tagged GA100 caused DNA damage and increased p62 levels. Fluorescent tags also affected GA100 stability and degradation. In summary, protein tags affect DPR toxicity in a tag- and DPR-dependent manner, and GA toxicity might be underestimated in studies using tagged GA proteins. Thus, including untagged DPRs as controls is important when assessing DPR toxicity in preclinical models.
Asunto(s)
Esclerosis Amiotrófica Lateral , Neoplasias Cutáneas , Animales , Dipéptidos , Proteína C9orf72 , Péptidos , Genes Reguladores , DrosophilaRESUMEN
OBJECTIVE: A GGGGCC repeat expansion in the C9orf72 gene is the most common cause of genetic frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). As potential therapies targeting the repeat expansion are now entering clinical trials, sensitive biomarker assays of target engagement are urgently required. Our objective was to develop such an assay. METHODS: We used the single molecule array (Simoa) platform to develop an immunoassay for measuring poly(GP) dipeptide repeat proteins (DPRs) generated by the C9orf72 repeat expansion in cerebrospinal fluid (CSF) of people with C9orf72-associated FTD/ALS. RESULTS AND CONCLUSIONS: We show the assay to be highly sensitive and robust, passing extensive qualification criteria including low intraplate and interplate variability, a high precision and accuracy in measuring both calibrators and samples, dilutional parallelism, tolerance to sample and standard freeze-thaw and no haemoglobin interference. We used this assay to measure poly(GP) in CSF samples collected through the Genetic FTD Initiative (N=40 C9orf72 and 15 controls). We found it had 100% specificity and 100% sensitivity and a large window for detecting target engagement, as the C9orf72 CSF sample with the lowest poly(GP) signal had eightfold higher signal than controls and on average values from C9orf72 samples were 38-fold higher than controls, which all fell below the lower limit of quantification of the assay. These data indicate that a Simoa-based poly(GP) DPR assay is suitable for use in clinical trials to determine target engagement of therapeutics aimed at reducing C9orf72 repeat-containing transcripts.
Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Esclerosis Amiotrófica Lateral/líquido cefalorraquídeo , Esclerosis Amiotrófica Lateral/diagnóstico , Esclerosis Amiotrófica Lateral/genética , Biomarcadores/líquido cefalorraquídeo , Proteína C9orf72/genética , Expansión de las Repeticiones de ADN/genética , Demencia Frontotemporal/diagnóstico , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , HumanosRESUMEN
BACKGROUND AND PURPOSE: Osteoarthritic pain is a chronic disabling condition lacking effective treatment. Continuous use of opioid drugs during osteoarthritic pain induces tolerance and may result in dose escalation and abuse. Sigma-1 (σ1) receptors, a chaperone expressed in key areas for pain control, modulates µ-opioid receptor activity and represents a promising target to tackle these problems. The present study investigates the efficacy of the σ1 receptor antagonist E-52862 to inhibit pain sensitization, morphine tolerance, and associated electrophysiological and molecular changes in a murine model of osteoarthritic pain. EXPERIMENTAL APPROACH: Mice received an intra-knee injection of monoiodoacetate followed by 14-day treatment with E-52862, morphine, or vehicle, and mechanical sensitivity was assessed before and after the daily doses. KEY RESULTS: Monoiodoacetate-injected mice developed persistent mechanical hypersensitivity, which was dose-dependently inhibited by E-52862. Mechanical thresholds assessed before the daily E-52862 dose showed gradual recovery, reaching complete restoration by the end of the treatment. When repeated treatment started 15 days after knee injury, E-52862 produced enhanced short-term analgesia, but recovery to baseline threshold was slower. Both a σ1 receptor agonist and a µ receptor antagonist blocked the analgesic effects of E-52862. An acute, sub-effective dose of E-52862 restored morphine analgesia in opioid-tolerant mice. Moreover, E-52862 abolished spinal sensitization in osteoarthritic mice and inhibited pain-related molecular changes. CONCLUSION AND IMPLICATIONS: These findings show dual effects of σ1 receptor antagonism alleviating both short- and long-lasting antinociception during chronic osteoarthritis pain. They identify E-52862 as a promising pharmacological agent to treat chronic pain and avoid opioid tolerance.
Asunto(s)
Modelos Animales de Enfermedad , Hiperalgesia/metabolismo , Inflamación/metabolismo , Osteoartritis/metabolismo , Dolor/metabolismo , Receptores sigma/metabolismo , Analgésicos Opioides/farmacología , Animales , Relación Dosis-Respuesta a Droga , Tolerancia a Medicamentos , Hiperalgesia/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Inyecciones Intraarticulares , Ácido Yodoacético/administración & dosificación , Masculino , Ratones , Morfina/farmacología , Morfolinas/farmacología , Osteoartritis/inducido químicamente , Osteoartritis/tratamiento farmacológico , Dolor/inducido químicamente , Dolor/tratamiento farmacológico , Pirazoles/farmacología , Receptores sigma/antagonistas & inhibidores , Receptor Sigma-1RESUMEN
Osteoarthritis is the most common musculoskeletal disease worldwide, often characterized by degradation of the articular cartilage, chronic joint pain and disability. Cognitive dysfunction, anxiety and depression are common comorbidities that impact the quality of life of these patients. In this study, we evaluated the involvement of sigma-1 receptor (σ1R) on the nociceptive, cognitive and emotional alterations associated with chronic osteoarthritis pain. Monosodium iodoacetate (MIA) was injected into the knee of Swiss-albino CD1 mice to induce osteoarthritis pain, which then received a repeated treatment with the σ1R antagonist E-52862 or its vehicle. Nociceptive responses and motor performance were assessed with the von Frey and the Catwalk gait tests. Cognitive alterations were evaluated using the novel object recognition task, anxiety-like behavior with the elevated plus maze and the zero-maze tests, whereas depressive-like responses were determined using the forced swimming test. We also studied the local effect of the σ1R antagonist on cartilage degradation, and its central effects on microglial reactivity in the medial prefrontal cortex. MIA induced mechanical allodynia and gait abnormalities that were prevented by the chronic treatment with the σ1R antagonist. E-52862 also reduced the memory impairment and the depressive-like behavior associated to osteoarthritis pain. Interestingly, the effect of E-52862 on depressive-like behavior was not accompanied by a modification of anxiety-like behavior. The pain-relieving effects of the σ1R antagonist were not due to a local effect on the articular cartilage, since E-52862 treatment did not modify the histological alterations of the knee joints. However, E-52862 induced central effects revealed by a reduction of the cortical microgliosis observed in mice with osteoarthritis pain. These findings show that σ1R antagonism inhibits mechanical hypersensitivity, cognitive deficits and depressive-like states associated with osteoarthritis pain in mice. These effects are associated with central modulation of glial activity but are unrelated to changes in cartilage degradation. Therefore, targeting the σ1R with E-52862 represents a promising pharmacological approach with effects on multiple aspects of chronic osteoarthritis pain that may go beyond the strict inhibition of nociception.
RESUMEN
RATIONALE: The activation of cannabinoid 2 receptors (CB2R) attenuates chronic pain, but the role played by carbon monoxide synthesized by the inducible heme oxygenase 1 (HO-1) on the anti-nociceptive effects produced by a selective CB2R agonist, JWH-015, during painful diabetic neuropathy remains unknown. OBJECTIVES AND METHODS: In streptozotocin (STZ)-induced diabetic mice, the anti-allodynic and anti-hyperalgesic effects of the subcutaneous administration of JWH-015 alone or combined with the intraperitoneal administration of a carbon monoxide-releasing molecule (tricarbonyldichlororuthenium(II) dimer (CORM-2)) or an HO-1 inducer compound (cobalt protoporphyrin IX (CoPP)) at 10 mg/kg were evaluated. Reversion of JWH-015 anti-nociceptive effects by the administration of an HO-1 inhibitor (tin protoporphyrin IX (SnPP)) and a CB2R antagonist (AM630) was also evaluated. Furthermore, the protein levels of HO-1, neuronal nitric oxide synthase (NOS1), and CB2R in diabetic mice treated with CORM-2 and CoPP alone or combined with JWH-015 were also assessed. RESULTS: The administration of JWH-015 dose dependently inhibited hypersensitivity induced by diabetes. The effects of JWH-015 were enhanced by their coadministration with CORM-2 or CoPP and reversed by SnPP or AM630. The increased protein levels of HO-1 induced by CORM-2 and CoPP treatments were further enhanced in JWH-015-treated mice. All treatments similarly enhanced the peripheral expression of CB2R and avoided the spinal cord over-expression of NOS1 induced by diabetes. CONCLUSIONS: The activation of HO-1 enhanced the anti-nociceptive effects of JWH-015 in diabetic mice, suggesting that coadministration of JWH-015 with CORM-2 or CoPP might be an interesting approach for the treatment of painful diabetic neuropathy in mice.
Asunto(s)
Analgésicos/farmacología , Monóxido de Carbono/fisiología , Neuropatías Diabéticas/fisiopatología , Receptor Cannabinoide CB2/biosíntesis , Receptor Cannabinoide CB2/efectos de los fármacos , Animales , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/fisiopatología , Inducción Enzimática , Hemo-Oxigenasa 1/biosíntesis , Hiperalgesia/tratamiento farmacológico , Indoles/administración & dosificación , Indoles/farmacología , Masculino , Metaloporfirinas/farmacología , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo I/biosíntesis , Óxido Nítrico Sintasa de Tipo I/genética , Compuestos Organometálicos/farmacología , Protoporfirinas/farmacología , Receptor Cannabinoide CB2/agonistas , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismoRESUMEN
Diabetic neuropathy is poorly controlled by classical analgesics and the research of new therapeutic alternatives is indispensable. Our aim is to investigate if treatment with a carbon monoxide-releasing molecule (tricarbonyldichlororuthenium(II) dimer; CORM-2) or an inducible heme oxygenase (HO-1) inducer (cobalt protoporphyrin IX; CoPP) could enhance the antinociceptive effects produced by a δ-opioid receptor (DOR) agonist in mice with painful diabetic neuropathy. In diabetic mice induced by streptozotocin (STZ) injection, the antiallodynic and antihyperalgesic effects produced by the subcutaneous administration of a DOR agonist ([d-Pen(2),d-Pen(5)]-Enkephalin; DPDPE) and the reversion of its effects with the administration of an HO-1 inhibitor (tin protoporphyrin IX; SnPP) were evaluated. Moreover, the antinociceptive effects produced by the intraperitoneal administration of 10mg/kg of CORM-2 or CoPP, alone or combined, with a subanalgesic dose of DPDPE were also assessed. Our results demonstrated that the subcutaneous administration of DPDPE inhibited the mechanical and thermal allodynia as well as the thermal hyperalgesia induced by diabetes in a dose-dependent manner. Moreover, while the antinociceptive effects produced by a low dose of DPDPE were enhanced by CORM-2 or CoPP co-treatments, the inhibitory effects produced by a high dose of DPDPE were completely reversed by the administration of an HO-1 inhibitor, SnPP, indicating the involvement of HO-1 in the antinociceptive effects produced by this DOR agonist during diabetic neuropathic pain in mice. In conclusion, this study shows that the administration of CORM-2 or CoPP combined with a DOR agonist could be an interesting strategy for the treatment of painful diabetic neuropathy.
Asunto(s)
Analgésicos Opioides/uso terapéutico , Neuropatías Diabéticas/tratamiento farmacológico , Encefalina D-Penicilamina (2,5)/uso terapéutico , Hemo-Oxigenasa 1/metabolismo , Receptores Opioides delta/agonistas , Animales , Neuropatías Diabéticas/enzimología , Neuropatías Diabéticas/fisiopatología , Relación Dosis-Respuesta a Droga , Inducción Enzimática , Hemo-Oxigenasa 1/antagonistas & inhibidores , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/fisiopatología , Inyecciones Subcutáneas , Masculino , Ratones Endogámicos C57BL , Compuestos Organometálicos/farmacología , Estimulación Física , Pirazinas/farmacología , Pirroles/farmacología , RutenioRESUMEN
Painful diabetic neuropathy is a common complication of diabetes mellitus which is poorly controlled by conventional analgesics. This study investigates if treatment with an heme oxygenase 1 (HO-1) inducer, cobalt protoporphyrin IX (CoPP), could modulate the allodynia and hyperalgesia induced by diabetes and enhanced the antinociceptive effects of morphine. In a diabetic mice model induced by the injection of streptozotocin (STZ), we evaluated the antiallodynic and antihyperalgesic effects produced by the intraperitoneal administration of 5 and 10 mg/kg of CoPP at several days after its administration. The antinociceptive actions produced by the systemic administration of morphine alone or combined with CoPP were also evaluated. In addition, the effects of CoPP treatment on the expression of HO-1, the microglial activation marker (CD11b/c), the inducible nitric oxide synthase (NOS2) and µ-opioid receptors (MOR), were also assessed. Our results showed that the administration of 10 mg/kg of CoPP during 5 consecutive days completely blocked the mechanical and thermal hypersensitivity induced by diabetes. These effects are accompanied by the increased spinal cord, dorsal root ganglia and sciatic nerve protein levels of HO-1. In addition, the STZ-induced activation of microglia and overexpression of NOS2 in the spinal cord were inhibited by CoPP treatment. Furthermore, the antinociceptive effects of morphine were enhanced by CoPP treatment and reversed by the administration of an HO-1 inhibitor, tin protoporphyrin IX (SnPP). The spinal cord expression of MOR was also increased by CoPP treatment in diabetic mice. In conclusion, our data provide the first evidence that the induction of HO-1 attenuated STZ-induced painful diabetic neuropathy and enhanced the antinociceptive effects of morphine via inhibition of microglia activation and NOS2 overexpression as well as by increasing the spinal cord levels of MOR. This study proposes the administration of CoPP alone or combined with morphine as an interesting therapeutic approach for the treatment of painful diabetic neuropathy.
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Neuropatías Diabéticas/prevención & control , Hemo-Oxigenasa 1/biosíntesis , Morfina/farmacología , Protoporfirinas/farmacología , Analgésicos/farmacología , Análisis de Varianza , Animales , Western Blotting , Antígeno CD11b/metabolismo , Antígeno CD11c/metabolismo , Diabetes Mellitus Experimental/complicaciones , Neuropatías Diabéticas/etiología , Neuropatías Diabéticas/fisiopatología , Inducción Enzimática/efectos de los fármacos , Hemo-Oxigenasa 1/antagonistas & inhibidores , Hiperalgesia/etiología , Hiperalgesia/fisiopatología , Hiperalgesia/prevención & control , Masculino , Metaloporfirinas/farmacología , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo II/metabolismo , Nervio Ciático/efectos de los fármacos , Nervio Ciático/enzimologíaRESUMEN
The administration of µ-opioid receptor (MOR), δ-opioid receptor (DOR), and cannabinoid 2 receptor (CB2R) agonists attenuates inflammatory pain. We investigated whether treatment with the heme oxygenase 1 (HO-1) inducer, cobalt protoporphyrin IX (CoPP), could modulate the local effects and expression of MOR, DOR, or CB2R during chronic inflammatory pain. In mice with inflammatory pain induced by the subplantar administration of complete Freund's adjuvant, we evaluated the effects of the intraperitoneal administration of 10 mg/kg CoPP on the antiallodynic and antihyperalgesic actions of locally administered MOR (morphine), DOR (DPDPE {[d-Pen(2),d-Pen(5)]-enkephalin}), or CB2R [JWH-015 {(2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone}] agonists and its reversion with the HO-1 inhibitor, tin protoporphyrin IX (SnPP). The effect of CoPP treatment on the dorsal root ganglia expression of HO-1, MOR, DOR, and CB2R was also assessed. The results show that treatment with CoPP increased the local antinociceptive effects produced by morphine, DPDPE, or JWH-015 during chronic inflammatory pain, and these effects were blocked by the subplantar administration of SnPP, indicating the participation of HO-1 in the antinociceptive actions. CoPP treatment, apart from inducing the expression of HO-1, also enhanced the expression of MOR, did not alter CB2R, and avoided the decreased expression of DOR induced by inflammatory pain. This study shows that the HO-1 inducer (CoPP) increased the local antinociceptive effects of MOR, DOR, and CB2R agonists during inflammatory pain by altering the peripheral expression of MOR and DOR. Therefore, the coadministration of CoPP with local morphine, DPDPE, or JWH-015 may be a good strategy for the management of chronic inflammatory pain.