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1.
PLoS One ; 19(2): e0292190, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38359044

RESUMO

Amyotrophic lateral sclerosis (ALS) has been linked to overactivity of the protein kinase RNA-like ER kinase (PERK) branch of the unfolded protein response (UPR) pathway, both in ALS patients and mouse models. However, attempts to pharmacologically modulate PERK for therapeutic benefit have yielded inconsistent and often conflicting results. This study sought to address these discrepancies by comprehensively evaluating three commonly used, CNS-penetrant, PERK modulators (GSK2606414, salubrinal, and Sephin1) in the same experimental models, with the goal of assessing the viability of targeting the PERK pathway as a therapeutic strategy for ALS. To achieve this goal, a tunicamycin-challenge assay was developed using wild-type mice to monitor changes in liver UPR gene expression in response to PERK pathway modulation. Subsequently, multiple dosing regimens of each PERK modulator were tested in standardized, well-powered, gender-matched, and litter-matched survival efficacy studies using the SOD1G93A mouse model of ALS. The alpha-2-adrenergic receptor agonist clonidine was also tested to elucidate the results obtained from the Sephin1, and of the previously reported guanabenz studies, by comparing the effects of presence or absence of α-2 agonism. The results revealed that targeting PERK may not be an ideal approach for ALS treatment. Inhibiting PERK with GSK2606414 or activating it with salubrinal did not confer therapeutic benefits. While Sephin1 showed some promising therapeutic effects, it appears that these outcomes were mediated through PERK-independent mechanisms. Clonidine also produced some favorable therapeutic effects, which were unexpected and not linked to the UPR. In conclusion, this study highlights the challenges of pharmacologically targeting PERK for therapeutic purposes in the SOD1G93A mouse model and suggests that exploring other targets within, and outside, the UPR may be more promising avenues for ALS treatment.


Assuntos
Adenina/análogos & derivados , Esclerose Lateral Amiotrófica , Cinamatos , Guanabenzo , Guanabenzo/análogos & derivados , Indóis , Tioureia/análogos & derivados , Camundongos , Humanos , Animais , Guanabenzo/farmacologia , Guanabenzo/uso terapêutico , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/genética , Clonidina , Resposta a Proteínas não Dobradas , Agonistas de Receptores Adrenérgicos alfa 2
2.
Acta Pharmacol Sin ; 45(4): 790-802, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38191913

RESUMO

Osteoporosis results from overactivation of osteoclasts. There are currently few drug options for treatment of this disease. Since the successful development of allosteric inhibitors, phosphatases have become attractive therapeutic targets. Protein phosphatase 1, regulatory subunit 15 A (PPP1R15A), is a stress-responsive protein, which promotes the UPR (unfolded protein response) and restores protein homeostasis. In this study we investigated the role of PPP1R15A in osteoporosis and osteoclastogenesis. Ovariectomy (OVX)-induced osteoporosis mouse model was established, osteoporosis was evaluated in the left femurs using micro-CT. RANKL-stimulated osteoclastogenesis was used as in vitro models. We showed that PPP1R15A expression was markedly increased in BMMs derived from OVX mice and during RANKL-induced osteoclastogenesis in vitro. Knockdown of PPP1R15A or application of Sephin1 (a PPP1R15A allosteric inhibitor in a phase II clinical trial) significantly inhibited osteoclastogenesis in vitro. Sephin1 (0.78, 3.125 and 12.5 µM) dose-dependently mitigated the changes in NF-κB, MAPK, and c-FOS and the subsequent nuclear factor of activated T cells 1 (NFATc1) translocation in RANKL-stimulated BMMs. Both Sephin1 and PPP1R15A knockdown increased the phosphorylated form of eukaryotic initiation factor 2α (eIF2α); knockdown of eIF2α reduced the inhibitory effects of Sephin1 on NFATc1-luc transcription and osteoclast formation. Furthermore, Sephin1 or PPP1R15A knockdown suppressed osteoclastogenesis in CD14+ monocytes from osteoporosis patients. In OVX mice, injection of Sephin1 (4, 8 mg/kg, i.p.) every two days for 6 weeks significantly inhibited bone loss, and restored bone destruction and decreased TRAP-positive cells. This study has identified PPP1R15A as a novel target for osteoclast differentiation, and genetic inhibition or allosteric inhibitors of PPP1R15A, such as Sephin1, can be used to treat osteoporosis. This study revealed that PPP1R15A expression was increased in osteoporosis in both human and mice. Inhibition of PPP1R15A by specific knockdown or an allosteric inhibitor Sephin1 mitigated murine osteoclast formation in vitro and attenuated ovariectomy-induced osteoporosis in vivo. PPP1R15A inhibition also suppressed pathogenic osteoclastogenesis in CD14+ monocytes from osteoporosis patients. These results identify PPP1R15A as a novel regulator of osteoclastogenesis and a valuable therapeutic target for osteoporosis.


Assuntos
Guanabenzo , Osteoporose , Animais , Feminino , Humanos , Camundongos , Diferenciação Celular , Guanabenzo/análogos & derivados , Guanabenzo/uso terapêutico , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Osteoclastos , Osteogênese , Osteoporose/tratamento farmacológico , Ovariectomia , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 1/farmacologia , Ligante RANK/metabolismo
3.
Exp Parasitol ; 246: 108460, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36642299

RESUMO

Chronic toxoplasmosis which is positively correlated with many neuropsychiatric problems has no curative treatment till now; due to the resistant tissue cysts especially in the brain. In search of an effective treatment, guanabenz-loaded polyethylene glycol poly lactic-co-glycolic acid (PEG-PLGA) nanoparticles was evaluated against chronic experimental toxoplasmosis. For this purpose, each mouse was infected with 10 cysts of Toxoplasma gondii (ME 49 strain). Treated mice received either guanabenz alone (5 mg/kg/day) in subgroup IIa or guanabenz-loaded nanoparticles by full dose in subgroup IIb or guanabenz-loaded nanoparticles by the half dose (2.5 mg/kg/day) in subgroup IIc. Subgroup Ie was treated by pyrimethamine and sulfadiazine. The treatment started on day 25 post-infection for 19 successive days. Then Parasitological, histopathological, immunohistochemical, immunological and ultrastructural morphological studies were performed. The results showed that: subgroup IIb showed the highest statistically significant reduction in the neuroinflammation and brain tissue cysts (77%) with a significant higher efficacy in comparison with pyrimethamine and sulfadiazine and showed the highest level of IFN-γ, while the lowest level was in subgroup IIa. All group II mice showed similar changes of depression and compression of the wall of the cyst. This is marked in subgroup IIb with release of crescent shaped bradyzoite outside the cyst. PEG-PLGA nanoparticles had no toxic effect on the liver or the kidney of the mice. It could be concluded that guanabenz-loaded PEG-PLGA nanoparticles could be promising and safe for treatment of chronic toxoplasmosis.


Assuntos
Guanabenzo , Nanopartículas , Toxoplasma , Toxoplasmose , Animais , Camundongos , Guanabenzo/farmacologia , Guanabenzo/uso terapêutico , Nanopartículas/uso terapêutico , Pirimetamina/uso terapêutico , Pirimetamina/farmacologia , Sulfadiazina/uso terapêutico , Sulfadiazina/farmacologia , Toxoplasmose/tratamento farmacológico
4.
Cell Tissue Res ; 388(2): 239-258, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35195784

RESUMO

Alzheimer's disease (AD) pathology is characterized by cognitive impairment, increased acetylcholinesterase (AChE) activity, and impaired neuronal communication. Clinically, AChE inhibitors are being used to treat AD patients; however, these remain unable to prevent the disease progression. Therefore, further development of new therapeutic molecules is required having broad spectrum effects on AD-related various neurodegenerative events. Since repurposing is a quick mode to search the therapeutic molecules; henceforth, this study was conducted to evaluate the anti-Alzheimer activity of drug guanabenz which is already in use for the management of high blood pressure in clinics. The study was performed employing both cellular and rat models of AD along with donepezil as reference drug. Guanabenz treatment in both the experimental models showed significant protection against AD-specific behavioral and pathological indicators like AChE activity, tau phosphorylation, amyloid precursor protein, and memory retention. In conjunction, guanabenz also attenuated the AD-related oxidative stress, impaired mitochondrial functionality (MMP, cytochrome-c translocation, ATP level, and mitochondrial complex I activity), endoplasmic reticulum stress (GRP78, GADD153, cleaved caspase-12), neuronal apoptosis (Bcl-2, Bax, cleaved caspase-3), and DNA fragmentation. In conclusion, findings suggested the panoptic protective effect of guanabenz on disease-related multiple degenerative markers and signaling. Furthermore, clinical trial may shed light and expedite the availability of new therapeutic anti-Alzheimer's molecule for the wellbeing of AD patients.


Assuntos
Doença de Alzheimer , Acetilcolinesterase/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Biomarcadores/metabolismo , Morte Celular , Guanabenzo/metabolismo , Guanabenzo/uso terapêutico , Humanos , Neurônios/metabolismo , Ratos
5.
Mol Biochem Parasitol ; 245: 111411, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34492239

RESUMO

Toxoplasma gondii is a protozoan parasite that causes opportunistic infection in immunocompromised individuals. The parasite forms latent tissue cysts that are refractory to current treatments and give rise to life-threatening reactivated infection following immune suppression. Previously, we showed that guanabenz sharply reduces brain cyst count in BALB/c mice harboring latent toxoplasmosis; however, whether cyst count would change once drug treatment stopped was not addressed. In the present study, we observed a rebound in brain cysts following the discontinuation of guanabenz or a guanabenz-pyrimethamine combination therapy. The re-expansion of brain cysts was not accompanied by symptoms of acute toxoplasmosis. We also tested whether the rebound in cyst counts could be ameliorated by administering pyrimethamine during or after guanabenz treatment.


Assuntos
Guanabenzo , Toxoplasma , Toxoplasmose , Animais , Guanabenzo/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Recidiva , Toxoplasmose/tratamento farmacológico
6.
Curr Neurovasc Res ; 18(1): 85-92, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34011258

RESUMO

BACKGROUND: Neovascular age-related macular degeneration (AMD) with choroidal neovascularization (CNV) is a leading cause of blindness in elderly people. Anti-vascular endothelial growth factor (anti-VEGF)-drugs are used to treat AMD patients; however, some patients are resistant to these therapies. OBJECTIVE: The purpose of this study was to investigate the anti-angiogenic effects of α2-adrenergic agonists, including guanabenz and clonidine. METHODS: We evaluated the anti-angiogenic effects of α2-adrenergic agonists in human retinal microvascular endothelial cells (HRMECs). A proliferation assay was conducted, and the migration ratio was evaluated. In a laser-induced CNV model, guanabenz and clonidine were delivered via intraperitoneal injection or implantation of an osmotic pump device. Fourteen days following CNV induction, CNV lesion size and fundus fluorescein angiography (FFA) were evaluated. RESULTS: Guanabenz and clonidine inhibited VEGF-induced retinal endothelial cell growth and migration. In the CNV model mice, CNV lesion sizes were reduced by intraperitoneal administration of guanabenz or clonidine. Data, including body weight, systolic blood pressure, and heart rate showed that guanabenz (0.5 and 2.0 mg/kg/day) had little effect on these parameters; conversely, a high dose of clonidine (1.0 mg/kg/day) did affect these parameters. Additionally, clonidine did not affect CNV size, but continuous administration of guanabenz attenuated both CNV size and leakage from neovessels. CONCLUSION: Our study suggests a key role for α2-adrenergic receptors during CNV formation. Therefore, we suggest that α2-adrenergic receptor agonists may represent novel therapeutic drugs for patients with neovascular AMD.


Assuntos
Agonistas de Receptores Adrenérgicos alfa 2/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Neovascularização de Coroide/tratamento farmacológico , Clonidina/uso terapêutico , Guanabenzo/uso terapêutico , Agonistas de Receptores Adrenérgicos alfa 2/administração & dosagem , Animais , Movimento Celular/efeitos dos fármacos , Neovascularização de Coroide/patologia , Clonidina/administração & dosagem , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Guanabenzo/administração & dosagem , Humanos , Masculino , Camundongos , Retina/efeitos dos fármacos , Retina/patologia , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/farmacologia
7.
Brain ; 144(9): 2635-2647, 2021 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-33905493

RESUMO

Strong evidence suggests that endoplasmic reticulum stress plays a critical role in the pathogenesis of amyotrophic lateral sclerosis (ALS) through altered regulation of proteostasis. Robust preclinical findings demonstrated that guanabenz selectively inhibits endoplasmic reticulum stress-induced eIF2α-phosphatase, allowing misfolded protein clearance, reduces neuronal death and prolongs survival in in vitro and in vivo models. However, its safety and efficacy in patients with ALS are unknown. To address these issues, we conducted a multicentre, randomized, double-blind trial with a futility design. Patients with ALS who had displayed an onset of symptoms within the previous 18 months were randomly assigned in a 1:1:1:1 ratio to receive 64 mg, 32 mg or 16 mg of guanabenz or placebo daily for 6 months as an add-on therapy to riluzole. The purpose of the placebo group blinding was to determine safety but not efficacy. The primary outcome was the proportion of patients progressing to higher stages of disease within 6 months as measured using the ALS Milano-Torino staging system, compared with a historical cohort of 200 patients with ALS. The secondary outcomes were the rate of decline in the total revised ALS functional rating scale score, slow vital capacity change, time to death, tracheotomy or permanent ventilation and serum light neurofilament level at 6 months. The primary assessment of efficacy was performed using intention-to-treat analysis. The treatment arms using 64 mg and 32 mg guanabenz, both alone and combined, reached the primary hypothesis of non-futility, with the proportions of patients who progressed to higher stages of disease at 6 months being significantly lower than that expected under the hypothesis of non-futility and a significantly lower difference in the median rate of change in the total revised ALS functional rating scale score. This effect was driven by patients with bulbar onset, none of whom (0/18) progressed to a higher stage of disease at 6 months compared with those on 16 mg guanabenz (4/8; 50%), the historical cohort alone (21/49; 43%; P = 0.001) or plus placebo (25/60; 42%; P = 0.001). The proportion of patients who experienced at least one adverse event was higher in any guanabenz arm than in the placebo arm, with higher dosing arms having a significantly higher proportion of drug-related side effects and the 64 mg arm a significantly higher drop-out rate. The number of serious adverse events did not significantly differ between the guanabenz arms and the placebo. Our findings indicate that a larger trial with a molecule targeting the unfolded protein response pathway without the alpha-2 adrenergic related side-effect profile of guanabenz is warranted.


Assuntos
Agonistas de Receptores Adrenérgicos alfa 2/uso terapêutico , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/metabolismo , Guanabenzo/uso terapêutico , Resposta a Proteínas não Dobradas/fisiologia , Agonistas de Receptores Adrenérgicos alfa 2/farmacologia , Idoso , Esclerose Lateral Amiotrófica/diagnóstico , Método Duplo-Cego , Feminino , Guanabenzo/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Resposta a Proteínas não Dobradas/efeitos dos fármacos
8.
Mol Neurobiol ; 57(5): 2206-2219, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31981074

RESUMO

Prion diseases are fatal infectious neurodegenerative disorders in human and animals caused by misfolding of the cellular prion protein (PrPC) into the infectious isoform PrPSc. These diseases have the potential to transmit within or between species, and no cure is available to date. Targeting the unfolded protein response (UPR) as an anti-prion therapeutic approach has been widely reported for prion diseases. Here, we describe the anti-prion effect of the chemical compound Sephin1 which has been shown to protect in mouse models of protein misfolding diseases including amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS) by selectively inhibiting the stress-induced regulatory subunit of protein phosphatase 1, thus prolonging eIF2α phosphorylation. We show here that Sephin1 dose and time dependently reduced PrPSc in different neuronal cell lines which were persistently infected with various prion strains. In addition, prion seeding activity was reduced in Sephin1-treated cells. Importantly, we found that Sephin1 significantly overcame the endoplasmic reticulum (ER) stress induced in treated cells, as measured by lower expression of stress-induced aberrant prion protein. In a mouse model of prion infection, intraperitoneal treatment with Sephin1 significantly prolonged survival of prion-infected mice. When combining Sephin1 with the neuroprotective drug metformin, the survival of prion-infected mice was also prolonged. These results suggest that Sephin1 could be a potential anti-prion drug selectively targeting one component of the UPR pathway.


Assuntos
Guanabenzo/análogos & derivados , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Príons/efeitos dos fármacos , Scrapie/tratamento farmacológico , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/metabolismo , Guanabenzo/administração & dosagem , Guanabenzo/farmacologia , Guanabenzo/uso terapêutico , Metformina/administração & dosagem , Metformina/farmacologia , Metformina/uso terapêutico , Camundongos , Neuroblastoma/patologia , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 1/antagonistas & inibidores , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Scrapie/patologia
9.
Chem Res Toxicol ; 33(1): 162-171, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31524377

RESUMO

Endoplasmic reticulum (ER) stress has been shown to be involved in the hepatotoxicity of acetaminophen (APAP). Guanabenz (GA), a widely known antihypertensive drug, is reported to exhibit an anti-ER stress effect. In this study, we investigated the potential of GA as an antidote against APAP-induced hepatotoxicity. The underlying biochemical mechanisms for the hepatoprotective effect of GA were explored. Here we found that treatment of mice with GA (10 mg/kg) before APAP overdose dramatically prevented APAP-induced liver enzyme elevation and resultant toxicity in mice, as indicated by suppression of elevated serum alanine aminotransferase (ALT) levels and liver histological analysis. Importantly, delayed administration of GA within 6 h after APAP overdose also showed an almost equivalent protective effect against APAP liver toxicity. Mechanistically, several pathways are involved in the protective effect of GA against APAP-induced live toxicity, including attenuation of ER stress and oxidative stress, increased levels of nontoxic phase I and II metabolites of APAP, decrease in the formation of toxic N-acetyl-p-benzoquinone imine (NAPQI), and its subsequent protein binding. Importantly, combination of GA with APAP exhibited synergistic interaction in the latter's analgesic activity, while sparing its antipyretic action. These findings provide the preclinical evidence of GA as a promising antidote for treatment of APAP-induced liver toxicity and raise a possibility of its combination with APAP in clinical settings.


Assuntos
Acetaminofen , Analgésicos não Narcóticos/uso terapêutico , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Guanabenzo/uso terapêutico , Substâncias Protetoras/uso terapêutico , Alanina Transaminase/sangue , Analgesia , Analgésicos não Narcóticos/farmacologia , Animais , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Sinergismo Farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Glutationa/metabolismo , Guanabenzo/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia
10.
Front Immunol ; 10: 134, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30809223

RESUMO

The guanabenz derivative Sephin1 has recently been proposed to increase the levels of translation initiation factor 2 (eIF2α) phosphorylation by inhibiting dephosphorylation by the protein phosphatase 1-GADD34 (PPP1R15A) complex. As phosphorylation of eIF2α by protein kinase R (PKR) is a prominent cellular antiviral pathway, we evaluated the consequences of Sephin1 treatment on virus replication. Our results provide evidence that Sephin1 downregulates replication of human respiratory syncytial virus, measles virus, human adenovirus 5 virus, human enterovirus D68, human cytomegalovirus, and rabbit myxoma virus. However, Sephin1 proved to be inactive against influenza virus, as well as against Japanese encephalitis virus. Sephin1 increased the levels of phosphorylated eIF2α in cells exposed to a PKR agonist. By contrast, in virus-infected cells, the levels of phosphorylated eIF2α did not always correlate with the inhibition of virus replication by Sephin1. This work identifies Sephin1 as an antiviral molecule in cell culture against RNA, as well as DNA viruses belonging to phylogenetically distant families.


Assuntos
Antivirais/farmacologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Guanabenzo/análogos & derivados , Animais , Antivirais/uso terapêutico , Linhagem Celular , Vírus de DNA/efeitos dos fármacos , Vírus de DNA/fisiologia , Guanabenzo/farmacologia , Guanabenzo/uso terapêutico , Humanos , Camundongos , Fosforilação/efeitos dos fármacos , Infecções por Poxviridae/tratamento farmacológico , Vírus de RNA/efeitos dos fármacos , Vírus de RNA/fisiologia , Coelhos , Infecções Tumorais por Vírus/tratamento farmacológico , Replicação Viral/efeitos dos fármacos
11.
Brain ; 142(2): 344-361, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30657878

RESUMO

Multiple sclerosis is a chronic autoimmune demyelinating disorder of the CNS. Immune-mediated oligodendrocyte cell loss contributes to multiple sclerosis pathogenesis, such that oligodendrocyte-protective strategies represent a promising therapeutic approach. The integrated stress response, which is an innate cellular protective signalling pathway, reduces the cytotoxic impact of inflammation on oligodendrocytes. This response is initiated by phosphorylation of eIF2α to diminish global protein translation and selectively allow for the synthesis of protective proteins. The integrated stress response is terminated by dephosphorylation of eIF2α. The small molecule Sephin1 inhibits eIF2α dephosphorylation, thereby prolonging the protective response. Herein, we tested the effectiveness of Sephin1 in shielding oligodendrocytes against inflammatory stress. We confirmed that Sephin1 prolonged eIF2α phosphorylation in stressed primary oligodendrocyte cultures. Moreover, by using a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis, we demonstrated that Sephin1 delayed the onset of clinical symptoms, which correlated with a prolonged integrated stress response, reduced oligodendrocyte and axon loss, as well as diminished T cell presence in the CNS. Sephin1 is reportedly a selective inhibitor of GADD34 (PPP1R15A), which is a stress-induced regulatory subunit of protein phosphatase 1 complex that dephosphorylates eIF2α. Consistent with this possibility, GADD34 mutant mice presented with a similar ameliorated experimental autoimmune encephalomyelitis phenotype as Sephin1-treated mice, and Sephin1 did not provide additional therapeutic benefit to the GADD34 mutant animals. Results presented from the adoptive transfer of encephalitogenic T cells between wild-type and GADD34 mutant mice further indicate that the beneficial effects of Sephin1 are mediated through a direct protective effect on the CNS. Of particular therapeutic relevance, Sephin1 provided additive therapeutic benefit when combined with the first line multiple sclerosis drug, interferon ß. Together, our results suggest that a neuroprotective treatment based on the enhancement of the integrated stress response would likely have significant therapeutic value for multiple sclerosis patients.


Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/imunologia , Guanabenzo/análogos & derivados , Imunidade Inata/fisiologia , Oligodendroglia/imunologia , Animais , Células Cultivadas , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Guanabenzo/farmacologia , Guanabenzo/uso terapêutico , Humanos , Imunidade Inata/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Ratos
12.
Neuropathol Appl Neurobiol ; 44(4): 391-403, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-28953319

RESUMO

AIM: Vanishing White Matter (VWM) is a devastating leucoencephalopathy without effective treatment options. Patients have mutations in the EIF2B1-5 genes, encoding the five subunits of eIF2B, a guanine exchange factor that is an important regulator of protein translation. We recently developed mouse models for VWM that replicate the human disease. To study disease improvement after treatment in these mice, it is essential to have sensitive biomarkers related to disease stage. The Bergmann glia of the cerebellum, an astrocytic subpopulation, translocate into the molecular layer in symptomatic VWM mice and patients. This study looked at the prospects of using Bergmann glia pathology as an objective disease marker for VWM. METHODS: We defined a new quantitative measurement of Bergmann glia pathology in the cerebellum of VWM mice and patients. To test the sensitivity of this new marker for improvement, VWM mutant mice received long-term treatment with Guanabenz, an FDA-approved anti-hypertensive agent affecting eIF2B activity. RESULTS: Bergmann glia translocation was significantly higher in symptomatic VWM mice and VWM patients than in controls and worsened over the disease course. Both Bergmann glia pathology and cerebellar myelin pathology improved with Guanabenz treatment in mice, showing that Bergmann glia translocation is a sensitive measurement for improvement. CONCLUSIONS: Bergmann glia translocation can be used to objectively assess effects of treatment in VWM mice. Future treatment strategies involving compounds regulating eIF2 phosphorylation might benefit VWM patients.


Assuntos
Agonistas de Receptores Adrenérgicos alfa 2/uso terapêutico , Astrócitos/patologia , Guanabenzo/uso terapêutico , Leucoencefalopatias/patologia , Animais , Biomarcadores , Modelos Animais de Doenças , Progressão da Doença , Leucoencefalopatias/tratamento farmacológico , Camundongos , Fosforilação , Resultado do Tratamento
13.
Dis Model Mech ; 10(12): 1465-1480, 2017 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-29061563

RESUMO

Polyglutamine expansion diseases are a group of hereditary neurodegenerative disorders that develop when a CAG repeat in the causative genes is unstably expanded above a certain threshold. The expansion of trinucleotide CAG repeats causes hereditary adult-onset neurodegenerative disorders, such as Huntington's disease, dentatorubral-pallidoluysian atrophy, spinobulbar muscular atrophy and multiple forms of spinocerebellar ataxia (SCA). The most common dominantly inherited SCA is the type 3 (SCA3), also known as Machado-Joseph disease (MJD), which is an autosomal dominant, progressive neurological disorder. The gene causatively associated with MJD is ATXN3 Recent studies have shown that this gene modulates endoplasmic reticulum (ER) stress. We generated transgenic Caenorhabditiselegans strains expressing human ATXN3 genes in motoneurons, and animals expressing mutant ATXN3-CAG89 alleles showed decreased lifespan, impaired movement, and rates of neurodegeneration greater than wild-type ATXN3-CAG10 controls. We tested three neuroprotective compounds (Methylene Blue, guanabenz and salubrinal) believed to modulate ER stress and observed that these molecules rescued ATXN3-CAG89 phenotypes. Furthermore, these compounds required specific branches of the ER unfolded protein response (UPRER), reduced global ER and oxidative stress, and polyglutamine aggregation. We introduce new C. elegans models for MJD based on the expression of full-length ATXN3 in a limited number of neurons. Using these models, we discovered that chemical modulation of the UPRER reduced neurodegeneration and warrants investigation in mammalian models of MJD.


Assuntos
Ataxina-3/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Estresse do Retículo Endoplasmático , Neurônios Motores/patologia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/efeitos dos fármacos , Cinamatos/farmacologia , Cinamatos/uso terapêutico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Guanabenzo/farmacologia , Guanabenzo/uso terapêutico , Humanos , Longevidade , Azul de Metileno/farmacologia , Azul de Metileno/uso terapêutico , Mutação/genética , Degeneração Neural/complicações , Degeneração Neural/patologia , Estresse Oxidativo/efeitos dos fármacos , Paralisia/complicações , Paralisia/tratamento farmacológico , Fenótipo , Agregados Proteicos/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Tioureia/análogos & derivados , Tioureia/farmacologia , Tioureia/uso terapêutico , Transgenes , Resposta a Proteínas não Dobradas/efeitos dos fármacos
14.
Brain Res ; 1648(Pt B): 594-602, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27055915

RESUMO

Myelin is vital to the proper function of the nervous system. Oligodendrocytes in the CNS and Schwann cells in the PNS are the glial cells responsible for generating the myelin sheath. Myelination requires the production of a vast amount of proteins and lipid-rich membrane, which puts a heavy load on the secretory pathway of myelinating glia and leaves them susceptible to endoplasmic reticulum (ER) stress. Cells respond to ER stress by activating the unfolded protein response (UPR). The UPR is initially protective but in situations of prolonged unresolved stress the UPR can lead to the apoptotic death of the stressed cell. There is strong evidence that ER stress and the UPR play a role in a number of disorders of myelin and myelinating glia, including multiple sclerosis, Pelizaeus-Merzbacher disease, Vanishing White Matter Disease, and Charcot-Marie-Tooth disease. In this review we discuss the role that ER stress and the UPR play in these disorders of myelin. In addition, we discuss the progress that has been made in our understanding of the effect genetic and pharmacological manipulation of the UPR has in mouse models of these disorders and the novel therapeutic potential of targeting the UPR that these studies support. This article is part of a Special Issue entitled SI:ER stress.


Assuntos
Doenças Desmielinizantes/patologia , Estresse do Retículo Endoplasmático/fisiologia , Neuroglia/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Agonistas de Receptores Adrenérgicos alfa 2/uso terapêutico , Animais , Doenças Desmielinizantes/tratamento farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Guanabenzo/análogos & derivados , Guanabenzo/uso terapêutico , Humanos , Neuroglia/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos
15.
Antimicrob Agents Chemother ; 59(11): 6939-45, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26303803

RESUMO

Toxoplasma gondii is a protozoan parasite that persists as a chronic infection. Toxoplasma evades immunity by forming tissue cysts, which reactivate to cause life-threatening disease during immune suppression. There is an urgent need to identify drugs capable of targeting these latent tissue cysts, which tend to form in the brain. We previously showed that translational control is critical during infections with both replicative and latent forms of Toxoplasma. Here we report that guanabenz, an FDA-approved drug that interferes with translational control, has antiparasitic activity against replicative stages of Toxoplasma and the related apicomplexan parasite Plasmodium falciparum (a malaria agent). We also found that inhibition of translational control interfered with tissue cyst biology in vitro. Toxoplasma bradyzoites present in these abnormal cysts were diminished and misconfigured, surrounded by empty space not seen in normal cysts. These findings prompted analysis of the efficacy of guanabenz in vivo by using established mouse models of acute and chronic toxoplasmosis. In addition to protecting mice from lethal doses of Toxoplasma, guanabenz has a remarkable ability to reduce the number of brain cysts in chronically infected mice. Our findings suggest that guanabenz can be repurposed into an effective antiparasitic with a unique ability to reduce tissue cysts in the brain.


Assuntos
Antiparasitários/uso terapêutico , Guanabenzo/uso terapêutico , Plasmodium falciparum/efeitos dos fármacos , Toxoplasmose Animal/tratamento farmacológico , Animais , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium falciparum/patogenicidade , Toxoplasma/efeitos dos fármacos , Toxoplasma/patogenicidade , Toxoplasmose Animal/parasitologia
16.
PLoS One ; 9(11): e109703, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25386686

RESUMO

Activation of the endoplasmic reticulum stress response (ERSR) is a hallmark of various pathological diseases and/or traumatic injuries. Restoration of ER homeostasis can contribute to improvement in the functional outcome of these diseases. Using genetic and pharmacological inhibition of the PERK-CHOP arm of the ERSR, we recently demonstrated improvements in hindlimb locomotion after spinal cord injury (SCI) and implicated oligodendrocyte survival as a potential mechanism. Here, we investigated the contribution of stress-inducible PPP1R15A/GADD34, an ERSR signaling effector downstream of CHOP that dephosphorylates eIF2α, in the pathogenesis of SCI. We show that although genetic ablation of GADD34 protects oligodendrocyte precursor cells (OPCs) against ER stress-mediated cell death in vitro and results in differential ERSR attenuation in vivo after SCI, there is no improvement in hindlimb locomotor function. Guanabenz, a FDA approved antihypertensive drug, was recently shown to reduce the burden of misfolded proteins in the ER by directly targeting GADD34. Guanabenz protected OPCs from ER stress-mediated cell death in vitro and attenuated the ERSR in vivo after SCI. However, guanabenz administration failed to rescue the locomotor deficits after SCI. These data suggest that deletion of GADD34 alone is not sufficient to improve functional recovery after SCI.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Guanabenzo/uso terapêutico , Proteína Fosfatase 1/antagonistas & inibidores , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Agonistas de Receptores Adrenérgicos alfa 2/uso terapêutico , Animais , Morte Celular/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/genética , Feminino , Locomoção/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligodendroglia/citologia , Fosforilação/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Proteína Fosfatase 1/genética , Proteínas Serina-Treonina Quinases/metabolismo , Recuperação de Função Fisiológica/fisiologia , Células-Tronco/citologia , Fator de Transcrição CHOP/antagonistas & inibidores , Fator de Transcrição CHOP/genética , Tunicamicina/farmacologia , eIF-2 Quinase/antagonistas & inibidores
17.
Neurobiol Dis ; 71: 317-24, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25134731

RESUMO

Approximately 20% of familial amyotrophic lateral sclerosis (FALS) cases are caused by mutant superoxide dismutase type 1 (mtSOD1). Although the mechanisms of mtSOD1-induced toxicity remain poorly understood, evidence suggests that accumulation of misfolded SOD1 is fundamental to its toxicity and the death of motor neurons. Misfolded mtSOD1 can accumulate inside the endoplasmic reticulum (ER), leading to ER stress, with activation of the unfolded protein response (UPR). We have previously carried out genetic studies focused on PERK (which is an eIF2α kinase that is rapidly activated in response to ER stress and leads to a repression in translation) and GADD34 (which participates in the dephosphorylation of eIF2α). We reported that mtSOD1 transgenic mice that are haploinsufficient for PERK have a significantly accelerated ALS disease, while mtSOD1 mice that are mutated for GADD34 have a remarkably ameliorated disease. Guanabenz, a centrally acting oral drug approved for the treatment of hypertension, enhances the PERK pathway by selectively inhibiting GADD34-mediated dephosphorylation of eIF2α. We have now treated G93A mtSOD1 transgenic mice with guanabenz and found a significant amelioration of disease with a delay in the onset and prolongation of the early phase of disease and survival. Guanabenz-treated G93A mice have less accumulation of mtSOD1 and an enhanced phosphorylation of eIF2α at endstage. This study further emphasizes the importance of the PERK pathway in the pathogenesis of FALS and as a therapeutic target in ALS, and identifies guanabenz as a candidate drug for the treatment of ALS patients.


Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/genética , Guanabenzo/uso terapêutico , Desdobramento de Proteína/efeitos dos fármacos , Superóxido Dismutase/genética , Fatores Etários , Esclerose Lateral Amiotrófica/mortalidade , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/metabolismo , Guanabenzo/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Análise de Sobrevida
18.
J Clin Invest ; 123(12): 5119-34, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24231350

RESUMO

A systems pharmacological approach that capitalizes on the characterization of intracellular signaling networks can transform our understanding of human diseases and lead to therapy development. Here, we applied this strategy to identify pharmacological targets for the treatment of Stargardt disease, a severe juvenile form of macular degeneration. Diverse GPCRs have previously been implicated in neuronal cell survival, and crosstalk between GPCR signaling pathways represents an unexplored avenue for pharmacological intervention. We focused on this receptor family for potential therapeutic interventions in macular disease. Complete transcriptomes of mouse and human samples were analyzed to assess the expression of GPCRs in the retina. Focusing on adrenergic (AR) and serotonin (5-HT) receptors, we found that adrenoceptor α 2C (Adra2c) and serotonin receptor 2a (Htr2a) were the most highly expressed. Using a mouse model of Stargardt disease, we found that pharmacological interventions that targeted both GPCR signaling pathways and adenylate cyclases (ACs) improved photoreceptor cell survival, preserved photoreceptor function, and attenuated the accumulation of pathological fluorescent deposits in the retina. These findings demonstrate a strategy for the identification of new drug candidates and FDA-approved drugs for the treatment of monogenic and complex diseases.


Assuntos
Inibidores de Adenilil Ciclases , Agonistas alfa-Adrenérgicos/uso terapêutico , Antagonistas Adrenérgicos alfa/uso terapêutico , Degeneração Macular/congênito , Terapia de Alvo Molecular , Proteínas do Tecido Nervoso/biossíntese , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Receptor 5-HT2A de Serotonina/biossíntese , Receptores Adrenérgicos alfa 2/biossíntese , Antagonistas da Serotonina/uso terapêutico , Transportadores de Cassetes de Ligação de ATP/deficiência , Transportadores de Cassetes de Ligação de ATP/genética , Adenina/análogos & derivados , Adenina/farmacologia , Adenina/uso terapêutico , Agonistas alfa-Adrenérgicos/farmacologia , Antagonistas Adrenérgicos alfa/farmacologia , Oxirredutases do Álcool/deficiência , Oxirredutases do Álcool/genética , Animais , Sobrevivência Celular , Modelos Animais de Doenças , Doxazossina/farmacologia , Doxazossina/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Guanabenzo/farmacologia , Guanabenzo/uso terapêutico , Humanos , Luz/efeitos adversos , Macaca fascicularis , Degeneração Macular/tratamento farmacológico , Degeneração Macular/genética , Degeneração Macular/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Células Fotorreceptoras de Vertebrados/patologia , Células Fotorreceptoras de Vertebrados/fisiologia , Células Fotorreceptoras de Vertebrados/efeitos da radiação , Espécies Reativas de Oxigênio , Receptor 5-HT2A de Serotonina/genética , Receptores Adrenérgicos alfa 2/genética , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/genética , Antagonistas da Serotonina/farmacologia , Transdução de Sinais , Doença de Stargardt
19.
Neurobiol Dis ; 55: 64-75, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23567652

RESUMO

C. elegans and D. rerio expressing mutant TAR DNA Binding Protein 43 (TDP-43) are powerful in vivo animal models for the genetics and pharmacology of amyotrophic lateral sclerosis (ALS). Using these small-animal models of ALS, we previously identified methylene blue (MB) as a potent suppressor of TDP-43 toxicity. Consequently here we investigated how MB might exert its neuroprotective properties and found that it acts through reduction of the endoplasmic reticulum (ER) stress response. We tested other compounds known to be active in the ER unfolded protein response in worms and zebrafish expressing mutant human TDP-43 (mTDP-43). We identified three compounds: salubrinal, guanabenz and a new structurally related compound phenazine, which also reduced paralysis, neurodegeneration and oxidative stress in our mTDP-43 models. Using C. elegans genetics, we showed that all four compounds act as potent suppressors of mTDP-43 toxicity through reduction of the ER stress response. Interestingly, these compounds operate through different branches of the ER unfolded protein pathway to achieve a common neuroprotective action. Our results indicate that protein-folding homeostasis in the ER is an important target for therapeutic development in ALS and other TDP-43-related neurodegenerative diseases.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Estresse do Retículo Endoplasmático/genética , Síndromes Neurotóxicas/genética , Síndromes Neurotóxicas/fisiopatologia , Análise de Variância , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Cinamatos/farmacologia , Cinamatos/uso terapêutico , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Reação de Fuga/efeitos dos fármacos , Reação de Fuga/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Guanabenzo/farmacologia , Guanabenzo/uso terapêutico , Humanos , Microinjeções , Transtornos dos Movimentos/tratamento farmacológico , Transtornos dos Movimentos/etiologia , Mutação/genética , Neurônios/efeitos dos fármacos , Neurônios/patologia , Síndromes Neurotóxicas/tratamento farmacológico , Síndromes Neurotóxicas/patologia , Fenazinas , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tioureia/análogos & derivados , Tioureia/farmacologia , Tioureia/uso terapêutico , Fatores de Tempo , Tato/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
20.
Antimicrob Agents Chemother ; 57(4): 1815-22, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23380722

RESUMO

Toxoplasma gondii is an obligate intracellular parasite that permanently infects warm-blooded vertebrates through its ability to convert into a latent tissue cyst form. The latent form (bradyzoite) can reinitiate a life-threatening acute infection if host immunity wanes, most commonly in AIDS or organ transplant patients. We have previously shown that bradyzoite development is accompanied by phosphorylation of the parasite eukaryotic initiation factor 2 alpha subunit (eIF2α), which dampens global protein synthesis and reprograms gene expression. In this study, we analyzed the activities of two specific inhibitors of eIF2α dephosphorylation, salubrinal (SAL) and guanabenz (GA). We establish that these drugs are able to inhibit the dephosphorylation of Toxoplasma eIF2α. Our results show that SAL and GA reduce tachyzoite replication in vitro and in vivo. Furthermore, both drugs induce bradyzoite formation and inhibit the reactivation of latent bradyzoites in vitro. To address whether the antiparasitic activities of SAL and GA involve host eIF2α phosphorylation, we infected mutant mouse embryonic fibroblast (MEF) cells incapable of phosphorylating eIF2α, which had no impact on the efficacies of SAL and GA against Toxoplasma infection. Our findings suggest that SAL and GA may serve as potential new drugs for the treatment of acute and chronic toxoplasmosis.


Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Toxoplasma/efeitos dos fármacos , Toxoplasma/metabolismo , Animais , Cinamatos/farmacologia , Cinamatos/uso terapêutico , Guanabenzo/farmacologia , Guanabenzo/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação/efeitos dos fármacos , Tioureia/análogos & derivados , Tioureia/farmacologia , Tioureia/uso terapêutico , Toxoplasmose/tratamento farmacológico , Toxoplasmose/metabolismo
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