RESUMO
Temperature profoundly affects macromolecular function, particularly in proteins with temperature sensitivity1,2. However, its impact is often overlooked in biophysical studies that are typically performed at non-physiological temperatures, potentially leading to inaccurate mechanistic and pharmacological insights. Here we demonstrate temperature-dependent changes in the structure and function of TRPM4, a temperature-sensitive Ca2+-activated ion channel3-7. By studying TRPM4 prepared at physiological temperature using single-particle cryo-electron microscopy, we identified a 'warm' conformation that is distinct from those observed at lower temperatures. This conformation is driven by a temperature-dependent Ca2+-binding site in the intracellular domain, and is essential for TRPM4 function in physiological contexts. We demonstrated that ligands, exemplified by decavanadate (a positive modulator)8 and ATP (an inhibitor)9, bind to different locations of TRPM4 at physiological temperatures than at lower temperatures10,11, and that these sites have bona fide functional relevance. We elucidated the TRPM4 gating mechanism by capturing structural snapshots of its different functional states at physiological temperatures, revealing the channel opening that is not observed at lower temperatures. Our study provides an example of temperature-dependent ligand recognition and modulation of an ion channel, underscoring the importance of studying macromolecules at physiological temperatures. It also provides a potential molecular framework for deciphering how thermosensitive TRPM channels perceive temperature changes.
Assuntos
Ativação do Canal Iônico , Canais de Cátion TRPM , Temperatura , Humanos , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Sítios de Ligação , Cálcio/metabolismo , Microscopia Crioeletrônica , Células HEK293 , Ativação do Canal Iônico/efeitos dos fármacos , Ligantes , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Especificidade por Substrato , Canais de Cátion TRPM/agonistas , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/química , Canais de Cátion TRPM/metabolismo , Vanadatos/química , Vanadatos/farmacologia , Vanadatos/metabolismoRESUMO
Hypothalamic neurons expressing Agouti-related peptide (AgRP) are critical for initiating food intake, but druggable biochemical pathways that control this response remain elusive. Thus, genetic ablation of insulin or leptin signaling in AgRP neurons is predicted to reduce satiety but fails to do so. FoxO1 is a shared mediator of both pathways, and its inhibition is required to induce satiety. Accordingly, FoxO1 ablation in AgRP neurons of mice results in reduced food intake, leanness, improved glucose homeostasis, and increased sensitivity to insulin and leptin. Expression profiling of flow-sorted FoxO1-deficient AgRP neurons identifies G-protein-coupled receptor Gpr17 as a FoxO1 target whose expression is regulated by nutritional status. Intracerebroventricular injection of Gpr17 agonists induces food intake, whereas Gpr17 antagonist cangrelor curtails it. These effects are absent in Agrp-Foxo1 knockouts, suggesting that pharmacological modulation of this pathway has therapeutic potential to treat obesity.
Assuntos
Proteína Relacionada com Agouti/metabolismo , Ingestão de Alimentos , Fatores de Transcrição Forkhead/metabolismo , Hipotálamo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteína Relacionada com Agouti/genética , Animais , Metabolismo Energético , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Glucose/metabolismo , Leptina/metabolismo , CamundongosRESUMO
Pannexin 1 (PANX1) is an ATP-permeable channel with critical roles in a variety of physiological functions such as blood pressure regulation1, apoptotic cell clearance2 and human oocyte development3. Here we present several structures of human PANX1 in a heptameric assembly at resolutions of up to 2.8 angström, including an apo state, a caspase-7-cleaved state and a carbenoxolone-bound state. We reveal a gating mechanism that involves two ion-conducting pathways. Under normal cellular conditions, the intracellular entry of the wide main pore is physically plugged by the C-terminal tail. Small anions are conducted through narrow tunnels in the intracellular domain. These tunnels connect to the main pore and are gated by a long linker between the N-terminal helix and the first transmembrane helix. During apoptosis, the C-terminal tail is cleaved by caspase, allowing the release of ATP through the main pore. We identified a carbenoxolone-binding site embraced by W74 in the extracellular entrance and a role for carbenoxolone as a channel blocker. We identified a gap-junction-like structure using a glycosylation-deficient mutant, N255A. Our studies provide a solid foundation for understanding the molecular mechanisms underlying the channel gating and inhibition of PANX1 and related large-pore channels.
Assuntos
Conexinas/química , Conexinas/metabolismo , Microscopia Crioeletrônica , Ativação do Canal Iônico , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Técnicas de Patch-Clamp , Trifosfato de Adenosina/metabolismo , Animais , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestrutura , Apoptose , Sítios de Ligação/efeitos dos fármacos , Carbenoxolona/química , Carbenoxolona/metabolismo , Carbenoxolona/farmacologia , Caspase 7/metabolismo , Linhagem Celular , Conexinas/ultraestrutura , Junções Comunicantes , Glicosilação , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Modelos Moleculares , Mutação , Proteínas do Tecido Nervoso/ultraestrutura , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Células Sf9RESUMO
Importance: Continuous glucose monitoring (CGM) has been shown to be beneficial for adults with type 2 diabetes using intensive insulin therapy, but its use in type 2 diabetes treated with basal insulin without prandial insulin has not been well studied. Objective: To determine the effectiveness of CGM in adults with type 2 diabetes treated with basal insulin without prandial insulin in primary care practices. Design, Setting, and Participants: This randomized clinical trial was conducted at 15 centers in the US (enrollment from July 30, 2018, to October 30, 2019; follow-up completed July 7, 2020) and included adults with type 2 diabetes receiving their diabetes care from a primary care clinician and treated with 1 or 2 daily injections of long- or intermediate-acting basal insulin without prandial insulin, with or without noninsulin glucose-lowering medications. Interventions: Random assignment 2:1 to CGM (n = 116) or traditional blood glucose meter (BGM) monitoring (n = 59). Main Outcomes and Measures: The primary outcome was hemoglobin A1c (HbA1c) level at 8 months. Key secondary outcomes were CGM-measured time in target glucose range of 70 to 180 mg/dL, time with glucose level at greater than 250 mg/dL, and mean glucose level at 8 months. Results: Among 175 randomized participants (mean [SD] age, 57 [9] years; 88 women [50%]; 92 racial/ethnic minority individuals [53%]; mean [SD] baseline HbA1c level, 9.1% [0.9%]), 165 (94%) completed the trial. Mean HbA1c level decreased from 9.1% at baseline to 8.0% at 8 months in the CGM group and from 9.0% to 8.4% in the BGM group (adjusted difference, -0.4% [95% CI, -0.8% to -0.1%]; P = .02). In the CGM group, compared with the BGM group, the mean percentage of CGM-measured time in the target glucose range of 70 to 180 mg/dL was 59% vs 43% (adjusted difference, 15% [95% CI, 8% to 23%]; P < .001), the mean percentage of time at greater than 250 mg/dL was 11% vs 27% (adjusted difference, -16% [95% CI, -21% to -11%]; P < .001), and the means of the mean glucose values were 179 mg/dL vs 206 mg/dL (adjusted difference, -26 mg/dL [95% CI, -41 to -12]; P < .001). Severe hypoglycemic events occurred in 1 participant (1%) in the CGM group and in 1 (2%) in the BGM group. Conclusions and Relevance: Among adults with poorly controlled type 2 diabetes treated with basal insulin without prandial insulin, continuous glucose monitoring, as compared with blood glucose meter monitoring, resulted in significantly lower HbA1c levels at 8 months. Trial Registration: ClinicalTrials.gov Identifier: NCT03566693.
Assuntos
Glicemia/análise , Diabetes Mellitus Tipo 2/tratamento farmacológico , Controle Glicêmico/métodos , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Idoso , Intervalos de Confiança , Diabetes Mellitus Tipo 2/sangue , Feminino , Hemoglobinas Glicadas/análise , Humanos , Masculino , Pessoa de Meia-Idade , Satisfação do Paciente , Período Pós-Prandial , Tamanho da Amostra , Fatores de Tempo , Resultado do TratamentoRESUMO
M-current-mediating KCNQ (Kv7) channels play an important role in regulating the excitability of neuronal cells, as highlighted by mutations in Kcnq2 and Kcnq3 that underlie certain forms of epilepsy. In addition to their expression in brain, KCNQ2 and -3 are also found in the somatosensory system. We have now detected both KCNQ2 and KCNQ3 in a subset of dorsal root ganglia neurons that correspond to D-hair Aδ-fibers and demonstrate KCNQ3 expression in peripheral nerve endings of cutaneous D-hair follicles. Electrophysiological recordings from single D-hair afferents from Kcnq3(-/-) mice showed increased firing frequencies in response to mechanical ramp-and-hold stimuli. This effect was particularly pronounced at slow indentation velocities. Additional reduction of KCNQ2 expression further increased D-hair sensitivity. Together with previous work on the specific role of KCNQ4 in rapidly adapting skin mechanoreceptors, our results show that different KCNQ isoforms are specifically expressed in particular subsets of mechanosensory neurons and modulate their sensitivity directly in sensory nerve endings.
Assuntos
Folículo Piloso/inervação , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/metabolismo , Mecanorreceptores/metabolismo , Pele/inervação , Animais , Células Cultivadas , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Deleção de Genes , Expressão Gênica , Canal de Potássio KCNQ2/genética , Canal de Potássio KCNQ3/genética , Mecanorreceptores/citologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The dystrobrevin binding protein (DTNBP) 1 gene has emerged over the last decade as a potential susceptibility locus for schizophrenia. While no causative mutations have been found, reduced expression of the encoded protein, dysbindin, was reported in patients. Dysbindin likely plays a role in the neuronal trafficking of proteins including receptors. One important pathway suspected to be affected in schizophrenia is the fast excitatory glutamatergic transmission mediated by AMPA receptors. Here, we investigated excitatory synaptic transmission and plasticity in hippocampal neurons from dysbindin-deficient sandy mice bred on the DBA/2J strain. In cultured neurons an enhancement of AMPAR responses was observed. The enhancement of AMPAR-mediated transmission was confirmed in hippocampal CA3-CA1 synapses, and was not associated with changes in the expression of GluA1-4 subunits or an increase in GluR2-lacking receptor complexes. Lastly, an enhancement in LTP was also found in these mice. These data provide compelling evidence that dysbindin, a widely suspected susceptibility protein in schizophrenia, is important for AMPAR-mediated synaptic transmission and plasticity in the developing hippocampus.
Assuntos
Proteínas de Transporte/metabolismo , Potenciação de Longa Duração , Neurônios/metabolismo , Receptores de AMPA/metabolismo , Transmissão Sináptica , Animais , Proteínas de Transporte/genética , Células Cultivadas , Disbindina , Proteínas Associadas à Distrofina , Ácido Glutâmico/metabolismo , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Hipocampo/fisiologia , Camundongos , Camundongos Endogâmicos DBA , Neurônios/fisiologia , Receptores de AMPA/genética , Esquizofrenia/genéticaRESUMO
Context: Radiofrequency ablation (RFA) is used in the United States to treat benign thyroid nodules; however, experience with treating cervical recurrence/persistence of papillary thyroid cancer (PTC) is limited. Objective: To evaluate the efficacy RFA for the treatment of cervical recurrence/persistence of PTC in the United States. Methods: This is a retrospective, multicenter study of 8 patients who underwent RFA of 11 cervical metastatic PTC lesions between July 2020 and December 2021. The volume reduction (VR) of the lesions, thyroglobulin (Tg) levels and complications following RFA were assessed. Energy applied per unit volume (E/V) during RFA was also determined. Results: Nine out of 11 (81.8%) lesions had initial volume under 0.5â mL and showed a complete (n = 8) or near-complete (n = 1) response. The 2 lesions with initial volume over 1.1â mL had a partial response, 1 of which had regrowth. There was a median VR of 100% (range 56.3-100%) after a median follow-up period of 453 days (range 162-570 days), with corresponding decline in Tg levels from a median of 0.7â ng/mL (range 0-15.2â ng/mL) to a median of 0.3â ng/mL (range 0-1.3â ng/mL). All patients with an E/V of at least 4483 J/mL or higher had a complete or near-complete response. There were no complications. Conclusion: RFA performed in an endocrinology practice is an efficacious treatment option for selected patients with cervical metastases of PTC, particularly those who cannot or do not want to undergo further surgery.
RESUMO
The Ca2+-activated TRPM5 channel plays essential roles in taste perception and insulin secretion. However, the mechanism by which Ca2+ regulates TRPM5 activity remains elusive. We report cryo-EM structures of the zebrafish TRPM5 in an apo closed state, a Ca2+-bound open state, and an antagonist-bound inhibited state. We define two novel ligand binding sites: a Ca2+ site (CaICD) in the intracellular domain and an antagonist site in the transmembrane domain (TMD). The CaICD site is unique to TRPM5 and has two roles: modulating the voltage dependence and promoting Ca2+ binding to the CaTMD site, which is conserved throughout TRPM channels. Conformational changes initialized from both Ca2+ sites cooperatively open the ion-conducting pore. The antagonist NDNA wedges into the space between the S1-S4 domain and pore domain, stabilizing the transmembrane domain in an apo-like closed state. Our results lay the foundation for understanding the voltage-dependent TRPM channels and developing new therapeutic agents.
Assuntos
Membrana Celular/fisiologia , Canais de Cátion TRPM/metabolismo , Peixe-Zebra/metabolismo , Animais , Sítios de Ligação/fisiologia , Cálcio/metabolismo , Microscopia Crioeletrônica , Ativação Enzimática , Humanos , Técnicas de Patch-Clamp , Conformação Proteica , Canais de Cátion TRPM/antagonistas & inibidoresRESUMO
OBJECTIVE: To explore the effect of discontinuing continuous glucose monitoring (CGM) after 8 months of CGM use in adults with type 2 diabetes treated with basal without bolus insulin. RESEARCH DESIGN AND METHODS: This multicenter trial had an initial randomization to either real-time CGM or blood glucose monitoring (BGM) for 8 months followed by 6 months in which the BGM group continued to use BGM (n = 57) and the CGM group was randomly reassigned either to continue CGM (n = 53) or discontinue CGM with resumption of BGM for glucose monitoring (n = 53). RESULTS: In the group that discontinued CGM, mean time in range (TIR) 70-180 mg/dL, which improved from 38% before initiating CGM to 62% after 8 months of CGM, decreased after discontinuing CGM to 50% at 14 months (mean change from 8 to 14 months -12% [95% CI -21% to -3%], P = 0.01). In the group that continued CGM use, little change was found in TIR from 8 to 14 months (baseline 44%, 8 months 56%, 14 months 57%, mean change from 8 to 14 months 1% [95% CI -11% to 12%], P = 0.89). Comparing the two groups at 14 months, the adjusted treatment group difference in mean TIR was -6% (95% CI -16% to 4%, P = 0.20). CONCLUSIONS: In adults with type 2 diabetes treated with basal insulin who had been using real-time CGM for 8 months, discontinuing CGM resulted in a loss of about one-half of the initial gain in TIR that had been achieved during CGM use.
Assuntos
Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Adulto , Glicemia , Automonitorização da Glicemia/métodos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hemoglobinas Glicadas/análise , Humanos , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêuticoRESUMO
Human primary aldosteronism (PA) can be caused by mutations in several ion channel genes but mouse models replicating this condition are lacking. We now show that almost all known PA-associated CLCN2 mutations markedly increase ClC-2 chloride currents and generate knock-in mice expressing a constitutively open ClC-2 Cl- channel as mouse model for PA. The Clcn2op allele strongly increases the chloride conductance of zona glomerulosa cells, provoking a strong depolarization and increasing cytoplasmic Ca2+ concentration. Clcn2op mice display typical features of human PA, including high serum aldosterone in the presence of low renin activity, marked hypertension and hypokalemia. These symptoms are more pronounced in homozygous Clcn2op/op than in heterozygous Clcn2+/op mice. This difference is attributed to the unexpected finding that only ~50 % of Clcn2+/op zona glomerulosa cells are depolarized. By reproducing essential features of human PA, Clcn2op mice are a valuable model to study the pathological mechanisms underlying this disease.
Assuntos
Canais de Cloreto/genética , Modelos Animais de Doenças , Hiperaldosteronismo/genética , Hipertensão/genética , Camundongos , Zona Glomerulosa/metabolismo , Animais , Canais de Cloro CLC-2 , Técnicas de Introdução de Genes , Heterozigoto , Homozigoto , Hiperaldosteronismo/complicações , Hipertensão/etiologia , Hipopotassemia/etiologia , Hipopotassemia/genética , MutaçãoRESUMO
In the last decade, multiple lines of transgenic APP overexpressing mice have been created that recapitulate certain aspects of Alzheimer's disease (AD). However, none of the previously reported transgenic APP overexpressing rat models developed AD-like beta-amyloid (Abeta) deposits, or age-related learning and memory deficits. In the present study, we have characterized a transgenic rat model overexpressing transgenes with three, familial AD mutations (two in APP and one in PS1) that were developed by Flood et al. [Flood, D.G., et al., Abeta deposition in a transgenic rat model of Alzheimer's disease. Society for Neuroscience 2003, Washington, DC, 2003]. From the age of 9 months, these rats develop Abeta deposits in both diffuse and compact forms, with the latter being closely associated with activated microglia and reactive astrocytes. Impaired long-term potentiation (LTP) was revealed by electrophysiological recordings performed on hippocampal slices from rats at 7 months of age, which is 2 months before the appearance of amyloid plaques. The deficit in LTP was accompanied by impaired spatial learning and memory in the Morris water maze, which became more pronounced in transgenic rats of 13 months of age. For Tg rats of both ages, there was a trend for cognitive impairment to correlate with total Abeta42 levels in the hippocampus. The rat model therefore recapitulates AD-like amyloid pathology and cognitive impairment. The advantage of the rat model over the available mouse models is that rats provide better opportunities for advanced studies, such as serial CSF sampling, electrophysiology, neuroimaging, cell-based transplant manipulations, and complex behavioral testing.
Assuntos
Doença de Alzheimer/patologia , Encéfalo/patologia , Transtornos Cognitivos/etiologia , Plasticidade Neuronal/fisiologia , Placa Amiloide/patologia , Doença de Alzheimer/complicações , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/metabolismo , Transtornos Cognitivos/fisiopatologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Potenciais Pós-Sinápticos Excitadores , Immunoblotting , Imuno-Histoquímica , Masculino , Aprendizagem em Labirinto , Mutação , Técnicas de Cultura de Órgãos , Placa Amiloide/metabolismo , Presenilinas/genética , Presenilinas/metabolismo , Ratos , Ratos Sprague-Dawley , Transmissão Sináptica/fisiologiaRESUMO
Primary aldosteronism is the most common and curable form of secondary arterial hypertension. We performed whole-exome sequencing in patients with early-onset primary aldosteronism and identified a de novo heterozygous c.71G>A/p.Gly24Asp mutation in the CLCN2 gene, encoding the voltage-gated ClC-2 chloride channel 1 , in a patient diagnosed at 9 years of age. Patch-clamp analysis of glomerulosa cells of mouse adrenal gland slices showed hyperpolarization-activated Cl- currents that were abolished in Clcn2-/- mice. The p.Gly24Asp variant, located in a well-conserved 'inactivation domain'2,3, abolished the voltage- and time-dependent gating of ClC-2 and strongly increased Cl- conductance at resting potentials. Expression of ClC-2Asp24 in adrenocortical cells increased expression of aldosterone synthase and aldosterone production. Our data indicate that CLCN2 mutations cause primary aldosteronism. They highlight the important role of chloride in aldosterone biosynthesis and identify ClC-2 as the foremost chloride conductor of resting glomerulosa cells.
Assuntos
Canais de Cloreto/genética , Mutação com Ganho de Função , Hiperaldosteronismo/genética , Adulto , Animais , Canais de Cloro CLC-2 , Criança , Canais de Cloreto/metabolismo , Estudos de Coortes , Análise Mutacional de DNA , Feminino , Mutação em Linhagem Germinativa , Humanos , Hiperaldosteronismo/patologia , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Linhagem , Sequenciamento do Exoma , Adulto Jovem , Zona Glomerulosa/metabolismo , Zona Glomerulosa/patologiaRESUMO
The oligomeric amyloid-ß (Aß) peptide is thought to contribute to the subtle amnesic changes in Alzheimer's disease (AD) by causing synaptic dysfunction. Here, we examined the time course of synaptic changes in mouse hippocampal neurons following exposure to Aß42 at picomolar concentrations, mimicking its physiological levels in the brain. We found opposite effects of the peptide with short exposures in the range of minutes enhancing synaptic plasticity, and longer exposures lasting several hours reducing it. The plasticity reduction was concomitant with an increase in the basal frequency of spontaneous neurotransmitter release, a higher basal number of functional presynaptic release sites, and a redistribution of synaptic proteins including the vesicle-associated proteins synapsin I, synaptophysin, and the post-synaptic glutamate receptor I. These synaptic alterations were mediated by cytoskeletal changes involving actin polymerization and p38 mitogen-activated protein kinase. These in vitro findings were confirmed in vivo with short hippocampal infusions of picomolar Aß enhancing contextual memory and prolonged infusions impairing it. Our findings provide a model for initiation of synaptic dysfunction whereby exposure to physiologic levels of Aß for a prolonged period of time causes microstructural changes at the synapse which result in increased transmitter release, failure of synaptic plasticity, and memory loss.
Assuntos
Doença de Alzheimer/diagnóstico , Peptídeos beta-Amiloides/farmacologia , Transtornos da Memória/diagnóstico , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Regulação da Expressão Gênica , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Transtornos da Memória/genética , Transtornos da Memória/metabolismo , Transtornos da Memória/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Neurônios/patologia , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/efeitos dos fármacos , Cultura Primária de Células , Multimerização Proteica , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Sinapses/efeitos dos fármacos , Sinapsinas/genética , Sinapsinas/metabolismo , Sinaptofisina/genética , Sinaptofisina/metabolismo , Fatores de Tempo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Learning and memory and the underlying cellular correlate, long-term synaptic plasticity, involve regulation by posttranslational modifications (PTMs). Here we demonstrate that conjugation with the small ubiquitin-like modifier (SUMO) is a novel PTM required for normal synaptic and cognitive functioning. Acute inhibition of SUMOylation impairs long-term potentiation (LTP) and hippocampal-dependent learning. Since Alzheimer's disease (AD) prominently features both synaptic and PTM dysregulation, we investigated SUMOylation under pathology induced by amyloid-ß (Aß), a primary neurotoxic molecule implicated in AD. We observed that SUMOylation is dysregulated in both human AD brain tissue and the Tg2576 transgenic AD mouse model. While neuronal activation normally induced upregulation of SUMOylation, this effect was impaired by Aß42 oligomers. However, supplementing SUMOylation via transduction of its conjugating enzyme, Ubc9, rescued Aß-induced deficits in LTP and hippocampal-dependent learning and memory. Our data establish SUMO as a novel regulator of LTP and hippocampal-dependent cognition and additionally implicate SUMOylation impairments in AD pathogenesis.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Cognição/fisiologia , Plasticidade Neuronal/fisiologia , Proteína SUMO-1/metabolismo , Idoso , Idoso de 80 Anos ou mais , Peptídeos beta-Amiloides/metabolismo , Animais , Modelos Animais de Doenças , Hipocampo/metabolismo , Hipocampo/fisiologia , Hipocampo/fisiopatologia , Humanos , Potenciação de Longa Duração/fisiologia , Masculino , Memória/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Sumoilação/fisiologiaRESUMO
Defects in the astrocytic membrane protein MLC1, the adhesion molecule GlialCAM or the chloride channel ClC-2 underlie human leukoencephalopathies. Whereas GlialCAM binds ClC-2 and MLC1, and modifies ClC-2 currents in vitro, no functional connections between MLC1 and ClC-2 are known. Here we investigate this by generating loss-of-function Glialcam and Mlc1 mouse models manifesting myelin vacuolization. We find that ClC-2 is unnecessary for MLC1 and GlialCAM localization in brain, whereas GlialCAM is important for targeting MLC1 and ClC-2 to specialized glial domains in vivo and for modifying ClC-2's biophysical properties specifically in oligodendrocytes (OLs), the cells chiefly affected by vacuolization. Unexpectedly, MLC1 is crucial for proper localization of GlialCAM and ClC-2, and for changing ClC-2 currents. Our data unmask an unforeseen functional relationship between MLC1 and ClC-2 in vivo, which is probably mediated by GlialCAM, and suggest that ClC-2 participates in the pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts.
Assuntos
Moléculas de Adesão Celular Neurônio-Glia/metabolismo , Moléculas de Adesão Celular/metabolismo , Canais de Cloreto/metabolismo , Leucoencefalopatias/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Astrócitos/ultraestrutura , Western Blotting , Encéfalo/metabolismo , Encéfalo/patologia , Canais de Cloro CLC-2 , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular Neurônio-Glia/genética , Cerebelo/metabolismo , Cerebelo/patologia , Canais de Cloreto/genética , Modelos Animais de Doenças , Feminino , Células HEK293 , Células HeLa , Humanos , Leucoencefalopatias/genética , Leucoencefalopatias/patologia , Potenciais da Membrana , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microscopia Eletrônica , Proteínas do Tecido Nervoso/genética , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Oligodendroglia/ultraestrutura , Técnicas de Patch-ClampRESUMO
Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). beta-amyloid (A beta), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A beta is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A beta produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A beta1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A beta(1-42;). Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A beta(1-42;) compared to slices in a control experiment where no A beta(1-42;) exposure had occurred.
Assuntos
Peptídeos beta-Amiloides/síntese química , Peptídeos beta-Amiloides/farmacologia , Hipocampo/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Oligopeptídeos/síntese química , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/farmacologia , Animais , Hipocampo/fisiologia , Potenciação de Longa Duração/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Sinapses/efeitos dos fármacos , Sinapses/fisiologiaRESUMO
Genes encoding polyadenylated mRNAs depend on their poly(A) signals for termination of transcription. Typically, transcription downstream of the poly(A) signal gradually declines to zero, but often there is a transient increase in polymerase density immediately preceding the decline. Special elements called pause sites are traditionally invoked to account for this increase. Using run-on transcription from the nuclei of transfected cells, we show that both the pause and the gradual decline that follow a poly(A) site are generated entirely by the poly(A) signal itself in a series of model constructs. We found no other elements to be involved and argue that the elements called pause sites do not function through pausing. Both the poly(A)-dependent pause and the subsequent decline occurred earlier for a stronger poly(A) signal than for a weaker one. Because the gradual decline resembles the abortive elongation that occurs downstream of many promoters, one model has proposed that the poly(A) signal flips the polymerase from the elongation mode to the abortive mode like a binary switch. We compared abortive elongators with poly(A) terminators and found a 4-fold difference in processivity. We conclude that poly(A) terminating polymerases do not merely revert to their prior state of low processivity but rather convert to a new termination-prone condition.