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1.
Cell Mol Neurobiol ; 41(8): 1817-1828, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32856232

RESUMO

Prothymosin alpha (ProTα) is involved in multiple cellular processes. Upon serum-free stress, ProTα lacking a signal peptide sequence is non-classically released from C6 glioma cells as a complex with Ca2+-binding cargo protein S100A13. Thus, ProTα and S100A13 are conceived to be members of damage-associated molecular patterns (DAMPs)/alarmins. However, it remains to be determined whether stress-induced release of ProTα and S100A13 involves SNARE proteins in the mechanisms underlying membrane tethering of the multiprotein complex. In the present study, we used C6 glioma cells as a model of ProTα release. In pull-down assay, p40 synaptotagmin-1 (Syt-1), a vesicular SNARE, formed a hetero-oligomeric complex with homodimeric S100A13 in a Ca2+-dependent manner. The interaction between p40 Syt-1 and S100A13 was also Ca2+-dependent in surface plasmon resonance (SPR). Immunoprecipitation using conditioned medium (CM) revealed that p40 Syt-1 was co-released with ProTα and S100A13 upon serum-free stress. In in situ proximity ligation assay (PLA), Syt-1 interacted with S100A13 upon serum-free stress in C6 glioma cells. The intracellular delivery of anti-Syt-1 IgG blocked serum free-induced release of ProTα and S100A13. Serum free-induced ProTα-EGFP release was significantly blocked by botulinum neurotoxin/C1 (BoNT/C1), which cleaves target SNARE syntaxin-1 (Stx-1). In immunocytochemistry, the cellular loss of ProTα-EGFP, S100A13, and Syt-1 was also blocked by BoNT/C1. Furthermore, the intracellular delivery of anti-Stx-1 IgG or Stx-1 siRNA treatment blocked Syt-1, S100A13 and ProTα release from C6 glioma cells. All these findings suggest that SNARE proteins play roles in stress-induced non-classical release of DAMPs/alarmins proteins, ProTα and S100A13 from C6 glioma cells.


Assuntos
Alarminas/metabolismo , Precursores de Proteínas/metabolismo , Proteínas S100/metabolismo , Proteínas SNARE/metabolismo , Timosina/análogos & derivados , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Ligação Proteica/fisiologia , Ratos , Timosina/metabolismo
2.
J Neurochem ; 153(6): 772-789, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31454420

RESUMO

Tissue plasminogen activator (tPA) administration beyond 4.5 h of stroke symptoms is beneficial for patients but has an increased risk of cerebral hemorrhage. Thus, increasing the therapeutic window of tPA is important for stroke recovery. We previously showed that prothymosin alpha (ProTα) or its mimetic hexapeptide (P6Q) has anti-ischemic activity. Here, we examined the beneficial effects of ProTα or P6Q against delayed tPA-induced brain damage following middle cerebral artery occlusion (MCAO) or photochemically induced thrombosis in mice. Brain hemorrhage was observed by tPA administration during reperfusion at 4.5 and 6 h after MCAO. Co-administration of ProTα with tPA at 4.5 h inhibited hemorrhage and motor dysfunction 2-4 days, but not 7 days after MCAO. ProTα administration at 2 and 4.5 h after MCAO significantly inhibited tPA (4.5 h)-induced motor dysfunction and death more than 7 days. Administration of tPA caused the loss of tight junction proteins, zona occulden-1 and occludin, and up-regulation of matrix metalloproteinase-2/9, in a ProTα-reversible manner. P6Q administration abolished tPA (4.5 h)-induced hemorrhage and reversed tPA (6 h)-induced vascular damage and matrix metalloproteinase-2 and 9 up-regulation. Twice administrations of P6Q at 2 h alone and 6 h with tPA significantly improved motor dysfunction more than 7 days. In photochemically induced thrombosis ischemia, similar vascular leakage and neuronal damage (infarction and motor dysfunction) by late tPA (4.5 or 6 h) were also inhibited by P6Q. Thus, these studies suggest that co-administration with ProTα or P6Q would be beneficial to inhibit delayed tPA-induced hemorrhagic mechanisms in acute ischemic stroke.


Assuntos
Materiais Biomiméticos/uso terapêutico , Lesões Encefálicas/tratamento farmacológico , Isquemia Encefálica/tratamento farmacológico , Precursores de Proteínas/uso terapêutico , Timosina/análogos & derivados , Ativador de Plasminogênio Tecidual/toxicidade , Animais , Lesões Encefálicas/induzido quimicamente , Lesões Encefálicas/patologia , Isquemia Encefálica/induzido quimicamente , Isquemia Encefálica/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Timosina/uso terapêutico
3.
J Pharmacol Sci ; 143(2): 127-131, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32156464

RESUMO

The inhibition of retinal ischemia-induced damage by post-ischemic prothymosin alpha (ProTα) was not affected in toll-like receptor 4 knockout (TLR4-/-) mice but blocked by the pretreatment with antibody against F0/F1 ATPase α- or ß-subunit, novel candidate for ProTα-receptor. In addition to the previous observation of ProTα-induced ATP release from cells, the present study showed a ProTα-induced enhancement of ATP hydrolysis activity of recombinant ATP5A1/5B complex. As the protection of retinal function by post-ischemic ProTα was abolished by anti-P2Y12 antibody, the activation of F0/F1 ATPase and subsequent P2Y12 receptor system may play roles in beneficial actions by post-ischemic ProTα.


Assuntos
Isquemia/metabolismo , Isquemia/prevenção & controle , Precursores de Proteínas/administração & dosagem , Precursores de Proteínas/farmacologia , ATPases Translocadoras de Prótons/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Retina , Timosina/análogos & derivados , Animais , Hidrólise/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Proteínas Recombinantes/metabolismo , Timosina/administração & dosagem , Timosina/farmacologia
4.
BMC Physiol ; 16: 2, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26932824

RESUMO

BACKGROUND: The cell type, cell status and specific localization of Prothymosin α (PTMA) within cells seemingly determine its function. PTMA undergoes 2 types of protease proteolytic modifications that are useful in elucidating its interactions with other molecules; a factor that typifies its roles. Preferably a nuclear protein, PTMA has been shown to function in the cytoplasm and extracellularly with much evidence leaning on pathognomonic status. As such, determination of its cellular distribution under normal physiological context while utilizing varied techniques is key to illuminating prospective validation of its distinct functions in different tissues. Differential distribution insights at normal physiology would also portent better basis for further clarification of its interactions and proteolytic modifications under pathological conditions like numerous cancer, ischemic stroke and immunomodulation. We therefore raised an antibody against the C terminal of PTMA to use in tandem with available antibody against the N terminal in a murine model to explicate the differences in its distribution in brain cell types and major peripheral organs through western blotting and immunohistochemical approaches. RESULTS: The newly generated antibody was applied against the N-terminal antibody to distinguish truncated versions of PTMA or deduce possible masking of the protein by other interacting molecules. Western blot analysis indicated presence of a truncated form of the protein only in the thymus, while immunohistochemical analysis showed that in brain hippocampus the full-length PTMA was stained prominently in the nucleus whereas in the stomach full-length PTMA staining was not observed in the nucleus but in the cytoplasm. CONCLUSION: Truncated PTMA could not be detected by western blotting when both antibodies were applied in all tissues examined except the thymus. However, immunohistochemistry revealed differential staining by these antibodies suggesting possible masking of epitopes by interacting molecules. The differential localization patterns observed in the context of nucleic versus cytoplasmic presence as well as punctate versus diffuse pattern in tissues and cell types, warrant further investigations as to the forms of PTMA interacting partners.


Assuntos
Precursores de Proteínas/metabolismo , Timosina/análogos & derivados , Animais , Anticorpos/imunologia , Western Blotting/métodos , Núcleo Celular/metabolismo , Feminino , Imuno-Histoquímica/métodos , Masculino , Camundongos , Precursores de Proteínas/imunologia , Ratos , Timosina/imunologia , Timosina/metabolismo
5.
J Neurochem ; 135(6): 1161-77, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26364961

RESUMO

Prothymosin-alpha protects the brain and retina from ischemic damage. Although prothymosin-alpha contributes to toll-like receptor (TLR4)-mediated immnunopotentiation against viral infection, the beneficial effects of prothymosin-alpha-TLR4 signaling in protecting against ischemia remain to be elucidated. In this study, intravitreal administration of prothymosin-alpha 48 h before induction of retinal ischemia prevented retinal cellular damage as evaluated by histology, and retinal functional deficits as evaluated by electroretinography. Prothymosin-alpha preconditioning completely prevented the ischemia-induced loss of ganglion cells with partial survival of bipolar and photoreceptor cells, but not amacrine cells, in immunohistochemistry experiments. Prothymosin-alpha treatment in the absence of ischemia caused mild activation, proliferation, and migration of retinal microglia, whereas the ischemia-induced microglial activation was inhibited by prothymosin-alpha preconditioning. All these preventive effects of prothymosin-alpha preconditioning were abolished in TLR4 knock-out mice and by pre-treatments with anti-TLR4 antibodies or minocycline, a microglial inhibitor. Prothymosin-alpha preconditioning inhibited the retinal ischemia-induced up-regulation of TLR4-related injury genes, and increased expression of TLR4-related protective genes. Furthermore, the prothymosin-alpha preconditioning-induced prevention of retinal ischemic damage was abolished in TIR-domain-containing adapter-inducing interferon-ß knock-out mice, but not in myeloid differentiation primary response gene 88 knock-out mice. Taken together, the results of this study suggest that prothymosin-alpha preconditioning selectively drives TLR4-TIR-domain-containing adapter-inducing interferon-ß signaling and microglia in the prevention of retinal ischemic damage. We propose the following mechanism for prothymosin-alpha (ProTα) preconditioning-induced retinal prevention against ischemia: ProTα preconditioning-induced prevention of retinal ischemic damage is mediated by selective activation of the TIR-domain-containing adapter-inducing interferon-ß (TRIF)- interferon regulatory factor 3 (IRF3) pathway downstream of toll-like receptor 4 (TLR4) in microglia, resulting in up-regulation of TRIF-IRF3-dependent protective genes and down-regulation of myeloid differentiation primary response gene 88 (MyD88)-Nuclear factor (NF)κB-dependent injury genes. Detailed investigations would be helpful to test the efficacy of ProTα as a therapeutic agent for the prevention of ischemic disorders.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Isquemia/metabolismo , Precursores de Proteínas/farmacologia , Doenças Retinianas/metabolismo , Transdução de Sinais/fisiologia , Timosina/análogos & derivados , Receptor 4 Toll-Like/metabolismo , Animais , Lipopolissacarídeos/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/citologia , Timosina/farmacologia , Regulação para Cima/efeitos dos fármacos
6.
Biochim Biophys Acta ; 1831(1): 61-73, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22960381

RESUMO

Acute inflammatory pain signal originates from transient hypersensitivity in afferent fibers when depolarized via injured tissues or proinflammatory cells-derived pronociceptive ligand binding. This pain is sensitive to opioids and NSAIDs. In neuropathic pain, however, damage to the nerve along the pain pathway results in spontaneous generation of action potential and lowered nociceptive threshold, as seen in allodynia and hyperalgesia. This abnormal pain transmission had been linked to LPA production in the spinal cord, through activation of NMDA and NK1 activation by glutamate and SP in iPLA(2)/cPLA(2)/ATX-dependent pathway. In a bifurcated response involving G(q/11) and G(12/13) coupling, Schwann cell LPA(1) mediates degradation and transcriptional suppression of myelin proteins, respectively. The loss of contact inhibition on axonal growth creates cytoskeletal framework for axonal sprouting. LPA causes an amplification of LPA production through activation of LPA(3) signaling in microglia immediately after nerve injury. LPA(1) deficient mice (LPA(1)(-/-)) show no neuropathic-pain behavior or demyelination in response to intrathecal LPA injection or nerve injury. Given these bodies of research evidence, LPA therefore presents as the chemical signature for the initiation of neuropathic pain. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.


Assuntos
Lisofosfolipídeos/metabolismo , Neuralgia/metabolismo , Transdução de Sinais , Animais , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/patologia , Humanos , Modelos Biológicos , Neuralgia/patologia , Neurônios/metabolismo , Neurônios/patologia
7.
Front Mol Neurosci ; 17: 1222935, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38495551

RESUMO

This study reports on biallelic homozygous and monoallelic de novo variants in SLITRK3 in three unrelated families presenting with epileptic encephalopathy associated with a broad neurological involvement characterized by microcephaly, intellectual disability, seizures, and global developmental delay. SLITRK3 encodes for a transmembrane protein that is involved in controlling neurite outgrowth and inhibitory synapse development and that has an important role in brain function and neurological diseases. Using primary cultures of hippocampal neurons carrying patients' SLITRK3 variants and in combination with electrophysiology, we demonstrate that recessive variants are loss-of-function alleles. Immunostaining experiments in HEK-293 cells showed that human variants C566R and E606X change SLITRK3 protein expression patterns on the cell surface, resulting in highly accumulating defective proteins in the Golgi apparatus. By analyzing the development and phenotype of SLITRK3 KO (SLITRK3-/-) mice, the study shows evidence of enhanced susceptibility to pentylenetetrazole-induced seizure with the appearance of spontaneous epileptiform EEG as well as developmental deficits such as higher motor activities and reduced parvalbumin interneurons. Taken together, the results exhibit impaired development of the peripheral and central nervous system and support a conserved role of this transmembrane protein in neurological function. The study delineates an emerging spectrum of human core synaptopathies caused by variants in genes that encode SLITRK proteins and essential regulatory components of the synaptic machinery. The hallmark of these disorders is impaired postsynaptic neurotransmission at nerve terminals; an impaired neurotransmission resulting in a wide array of (often overlapping) clinical features, including neurodevelopmental impairment, weakness, seizures, and abnormal movements. The genetic synaptopathy caused by SLITRK3 mutations highlights the key roles of this gene in human brain development and function.

8.
J Neurochem ; 125(5): 713-23, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23278181

RESUMO

Prothymosin alpha (ProTα), a nuclear protein, is implicated in the inhibition of ischemia-induced necrosis as well as apoptosis in the brain and retina. Although ProTα has multiple biological functions through distinct regions in its sequence, it has remained which region is involved in this neuroprotection. This study reported that the active core peptide sequence P30 (amino acids 49-78) of ProTα exerts its full survival effect in cultured cortical neurons against ischemic stress. Our in vivo study revealed that intravitreous administration of P30 at 24 h after retinal ischemia significantly blocks the ischemia-induced functional damages of retina at day 7. In addition, P30 completely rescued the retinal ischemia-induced ganglion cell damages at day 7 after the ischemic stress, along with partial blockade of the loss of bipolar, amacrine, and photoreceptor cells. On the other hand, intracerebroventricular (3 nmol) or systemic (1 mg/kg; i.v.) injection of P30 at 1 h after cerebral ischemia (1 h tMCAO) significantly blocked the ischemia-induced brain damages and disruption of blood vessels. Systemic P30 delivery (1 mg/kg; i.v.) also significantly ameliorated the ischemic brain caused by photochemically induced thrombosis. Taken together, this study confers a precise demonstration about the novel protective activity of ProTα-derived small peptide P30 against the ischemic damages in vitro and in vivo.


Assuntos
Isquemia Encefálica/prevenção & controle , Fármacos Neuroprotetores/uso terapêutico , Precursores de Proteínas/uso terapêutico , Doenças Retinianas/prevenção & controle , Timosina/análogos & derivados , Sequência de Aminoácidos , Animais , Isquemia Encefálica/patologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fármacos Neuroprotetores/farmacologia , Precursores de Proteínas/genética , Precursores de Proteínas/farmacologia , Ratos , Doenças Retinianas/patologia , Timosina/genética , Timosina/farmacologia , Timosina/uso terapêutico
9.
J Neurochem ; 126(2): 243-60, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23574143

RESUMO

Reprogramming of toll-like receptor 4 (TLR4) by brief ischemia or lipopolysacharide (LPS) contributes to superintending tolerance against destructive ischemia in brain. However, beneficial roles of TLR4 signaling in ischemic retina are not well known. This study demonstrated that preconditioning with LPS 48 h prior to the retinal ischemia prevents the cellular damage in morphology with hematoxylin and eosin (H&E) staining and functions of retina with electroretinogram (ERG), while post-ischemia treatment deteriorated it. The preventive effects of LPS preconditioning showed the cell type-specificity of retinal cells. There was complete rescue of ganglion cells, partial rescue of bipolar and photoreceptor cells or no rescue of amacrine cells, respectively. LPS treatment caused the proliferation and migration of retinal microglia and its preconditioning prevented the ischemia-induced microglial activation. Preventive actions from cell damages following LPS preconditioning prior to retinal ischemia were abolished in TLR4 knock-out mice, and by pre-treatments with anti-TLR4 antibody or minocycline, a microglia inhibitor, which themselves had no effects on the retinal ischemia-induced damages or microglia activation. Thus, this study revealed that TLR4 mediates the LPS preconditioning-induced preventive effects through microglial activation in the retinal ischemia model.


Assuntos
Microglia/metabolismo , Retina/patologia , Doenças Retinianas/prevenção & controle , Transdução de Sinais/fisiologia , Receptor 4 Toll-Like/metabolismo , Animais , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Interações Medicamentosas , Eletrorretinografia , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Isquemia/complicações , Isquemia/patologia , Lipopolissacarídeos/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/efeitos dos fármacos , Minociclina/farmacologia , Retina/efeitos dos fármacos , Retina/fisiopatologia , Doenças Retinianas/etiologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/imunologia
10.
J Neurochem ; 123(2): 262-75, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22853710

RESUMO

Prothymosin alpha (ProTα), a nuclear protein devoid of signal sequence, has been shown to possess a number of cellular functions including cell survival. Most recently, we demonstrated that ProTα is localized in the nuclei of neurons, while it is found in both nuclei and cytoplasm in the astrocytes and microglia of adult brain. However, the cell type-specific non-classical release of ProTα under cerebral ischemia is yet unknown. In this study, we report that ProTα is non-classically released along with S100A13 from neurons in the hippocampus, striatum and somatosensory cortex at 3 h after cerebral ischemia, but amlexanox (an anti-allergic compound) reversibly blocks this neuronal ProTα release. We found that none of ProTα is released from astrocytes and microglia under ischemic stress. Indeed, ProTα intensity is increased gradually in astrocytes and microglia through 24 h after the cerebral ischemia. Interestingly, Z-Val-Ala-Asp fluoromethyl ketone, a caspase 3 inhibitor, pre-treatment induces ProTα release from astrocytes in the ischemic brain, but this release is reversibly blocked by amlexanox. However, Z-Val-Ala-Asp fluoromethyl ketone as well as amlexanox has no effect on ProTα distribution in microglia upon cerebral ischemia. Taken together, these results suggest that only neurons have machineries to release ProTα upon cerebral ischemic stress in vivo.


Assuntos
Isquemia Encefálica/prevenção & controle , Encéfalo/metabolismo , Neurônios/metabolismo , Fármacos Neuroprotetores , Precursores de Proteínas/metabolismo , Timosina/análogos & derivados , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Encéfalo/patologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Camundongos , Microglia/metabolismo , Microglia/patologia , Neurônios/patologia , Fármacos Neuroprotetores/metabolismo , Timosina/metabolismo
11.
J Cell Biol ; 176(6): 853-62, 2007 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-17353361

RESUMO

We initially identified a nuclear protein, prothymosin-alpha1 (ProTalpha), as a key protein inhibiting necrosis by subjecting conditioned media from serum-free cultures of cortical neurons to a few chromatography steps. ProTalpha inhibited necrosis of cultured neurons by preventing rapid loss of cellular adenosine triphosphate levels by reversing the decreased membrane localization of glucose transporters but caused apoptosis through up-regulation of proapoptotic Bcl(2)-family proteins. The apoptosis caused by ProTalpha was further inhibited by growth factors, including brain-derived neurotrophic factor. The ProTalpha-induced cell death mode switch from necrosis to apoptosis was also reproduced in experimental ischemia-reperfusion culture experiments, although the apoptosis level was markedly reduced, possibly because of the presence of growth factors in the reperfused serum. Knock down of PKCbeta(II) expression prevented this cell death mode switch. Collectively, these results suggest that ProTalpha is an extracellular signal protein that acts as a cell death mode switch and could be a promising candidate for preventing brain strokes with the help of known apoptosis inhibitors.


Assuntos
Apoptose , Córtex Cerebral/citologia , Necrose , Neurônios/citologia , Precursores de Proteínas/fisiologia , Timosina/análogos & derivados , Sequência de Aminoácidos , Animais , Células Cultivadas , Meios de Cultivo Condicionados , Dados de Sequência Molecular , Neurônios/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/isolamento & purificação , Ratos , Timosina/genética , Timosina/isolamento & purificação , Timosina/fisiologia
12.
Commun Biol ; 5(1): 935, 2022 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-36085162

RESUMO

SLITRK1 is an obsessive-compulsive disorder spectrum-disorders-associated gene that encodes a neuronal transmembrane protein. Here we show that SLITRK1 suppresses noradrenergic projections in the neonatal prefrontal cortex, and SLITRK1 functions are impaired by SLITRK1 mutations in patients with schizophrenia (S330A, a revertant of Homo sapiens-specific residue) and bipolar disorder (A444S). Slitrk1-KO newborns exhibit abnormal vocalizations, and their prefrontal cortices show excessive noradrenergic neurites and reduced Semaphorin3A expression, which suppresses noradrenergic neurite outgrowth in vitro. Slitrk1 can bind Dynamin1 and L1 family proteins (Neurofascin and L1CAM), as well as suppress Semaphorin3A-induced endocytosis. Neurofascin-binding kinetics is altered in S330A and A444S mutations. Consistent with the increased obsessive-compulsive disorder prevalence in males in childhood, the prefrontal cortex of male Slitrk1-KO newborns show increased noradrenaline levels, and serotonergic varicosity size. This study further elucidates the role of noradrenaline in controlling the development of the obsessive-compulsive disorder-related neural circuit.


Assuntos
Norepinefrina , Córtex Pré-Frontal , Axônios , Humanos , Recém-Nascido , Masculino , Proteínas de Membrana , Proteínas do Tecido Nervoso , Neuritos , Crescimento Neuronal
13.
Front Mol Neurosci ; 15: 856315, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35615067

RESUMO

The striatum is involved in action selection, and its disturbance can cause movement disorders. Here, we show that leucine-rich repeats and transmembrane domain 2 (Lrtm2) controls protein sorting in striatal projection systems, and its deficiency causes disturbances in monoamine dynamics and behavior. The Lrtm2 protein was broadly detected in the brain, but it was enhanced in the olfactory bulb and dorsal striatum. Immunostaining revealed a strong signal in striatal projection output, including GABAergic presynaptic boutons of the SNr. In subcellular fractionation, Lrtm2 was abundantly recovered in the synaptic plasma membrane fraction, synaptic vesicle fraction, and microsome fraction. Lrtm2 KO mice exhibited altered motor responses in both voluntary explorations and forced exercise. Dopamine metabolite content was decreased in the dorsal striatum and hypothalamus, and serotonin turnover increased in the dorsal striatum. The prefrontal cortex showed age-dependent changes in dopamine metabolites. The distribution of glutamate decarboxylase 67 (GAD67) protein and gamma-aminobutyric acid receptor type B receptor 1 (GABA B R1) protein was altered in the dorsal striatum. In cultured neurons, wild-type Lrtm2 protein enhanced axon trafficking of GAD67-GFP and GABA B R1-GFP whereas such activity was defective in sorting signal-abolished Lrtm2 mutant proteins. The topical expression of hemagglutinin-epitope-tag (HA)-Lrtm2 and a protein sorting signal abolished HA-Lrtm2 mutant differentially affected GABA B R1 protein distribution in the dorsal striatum. These results suggest that Lrtm2 is an essential component of striatal projection neurons, contributing to a better understanding of striatal pathophysiology.

14.
iScience ; 25(7): 104604, 2022 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35789858

RESUMO

SLITRK2 encodes a transmembrane protein that modulates neurite outgrowth and synaptic activities and is implicated in bipolar disorder. Here, we addressed its physiological roles in mice. In the brain, the Slitrk2 protein was strongly detected in the hippocampus, vestibulocerebellum, and precerebellar nuclei-the vestibular-cerebellar-brainstem neural network including pontine gray and tegmental reticular nucleus. Slitrk2 knockout (KO) mice exhibited increased locomotor activity in novel environments, antidepressant-like behaviors, enhanced vestibular function, and increased plasticity at mossy fiber-CA3 synapses with reduced sensitivity to serotonin. A serotonin metabolite was increased in the hippocampus and amygdala, and serotonergic neurons in the raphe nuclei were decreased in Slitrk2 KO mice. When KO mice were treated with methylphenidate, lithium, or fluoxetine, the mood stabilizer lithium showed a genotype-dependent effect. Taken together, Slitrk2 deficiency causes aberrant neural network activity, synaptic integrity, vestibular function, and serotonergic function, providing molecular-neurophysiological insight into the brain dysregulation in bipolar disorders.

15.
Front Neural Circuits ; 15: 634875, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33790745

RESUMO

Trans-regulation of G protein-coupled receptors (GPCRs) by leucine-rich repeat (LRR) transmembrane proteins has emerged as a novel type of synaptic molecular interaction in the last decade. Several studies on LRR-GPCR interactions have revealed their critical role in synapse formation and in establishing synaptic properties. Among them, LRR-GPCR interactions between extracellular LRR fibronectin domain-containing family proteins (Elfn1 and Elfn2) and metabotropic glutamate receptors (mGluRs) are particularly interesting as they can affect a broad range of synapses through the modulation of signaling by glutamate, the principal excitatory transmitter in the mammalian central nervous system (CNS). Elfn-mGluR interactions have been investigated in hippocampal, cortical, and retinal synapses. Postsynaptic Elfn1 in the hippocampus and cerebral cortex mediates the tonic regulation of excitatory input onto somatostatin-positive interneurons (INs) through recruitment of presynaptic mGluR7. In the retina, presynaptic Elfn1 binds to mGluR6 and is necessary for synapse formation between rod photoreceptor cells and rod-bipolar cells. The repertoire of binding partners for Elfn1 and Elfn2 includes all group III mGluRs (mGluR4, mGluR6, mGluR7, and mGluR8), and both Elfn1 and Elfn2 can alter mGluR-mediated signaling through trans-interaction. Importantly, both preclinical and clinical studies have provided support for the involvement of the Elfn1-mGluR7 interaction in attention-deficit hyperactivity disorder (ADHD), post-traumatic stress disorder (PTSD), and epilepsy. In fact, Elfn1-mGluR7-associated disorders may reflect the altered function of somatostatin-positive interneuron inhibitory neural circuits, the mesolimbic and nigrostriatal dopaminergic pathway, and habenular circuits, highlighting the need for further investigation into this interaction.


Assuntos
Receptores de Glutamato Metabotrópico , Animais , Ácido Glutâmico , Hipocampo/metabolismo , Interneurônios/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Sinapses/metabolismo
16.
Cells ; 10(3)2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33807671

RESUMO

Prothymosin alpha (ProTα) and S100A13 are released from C6 glioma cells under serum-free conditions via membrane tethering mediated by Ca2+-dependent interactions between S100A13 and p40 synaptotagmin-1 (Syt-1), which is further associated with plasma membrane syntaxin-1 (Stx-1). The present study revealed that S100A13 interacted with annexin A2 (ANXA2) and this interaction was enhanced by Ca2+ and p40 Syt-1. Amlexanox (Amx) inhibited the association between S100A13 and ANXA2 in C6 glioma cells cultured under serum-free conditions in the in situ proximity ligation assay. In the absence of Amx, however, the serum-free stress results in a flop-out of ANXA2 through the membrane, without the extracellular release. The intracellular delivery of anti-ANXA2 antibody blocked the serum-free stress-induced cellular loss of ProTα, S100A13, and Syt-1. The stress-induced externalization of ANXA2 was inhibited by pretreatment with siRNA for P4-ATPase, ATP8A2, under serum-free conditions, which ablates membrane lipid asymmetry. The stress-induced ProTα release via Stx-1A, ANXA2 and ATP8A2 was also evidenced by the knock-down strategy in the experiments using oxygen glucose deprivation-treated cultured neurons. These findings suggest that starvation stress-induced release of ProTα, S100A13, and p40 Syt-1 from C6 glioma cells is mediated by the ANXA2-flop-out via energy crisis-dependent recovery of membrane lipid asymmetry.


Assuntos
Alarminas/metabolismo , Anexina A2/metabolismo , Glioma/genética , Humanos
17.
J Neurochem ; 114(5): 1333-43, 2010 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-20534002

RESUMO

The present study demonstrates a novel high-affinity neuronal target for endocrine disrupting chemicals (EDCs), which potentially cause psychological disorders. EDCs competitively inhibited the binding of bovine serum albumin-conjugated progesterone to recombinant human microtubule-associated protein 2C (rhMAP2C) with an inhibition constant at picomolar levels. In the rhMAP2C-stimulated tubulin assembly assay, agonistic enhancement was observed with dibutyl phthalate and pentachlorphenol and pregnenolone, while an inverse agonistic effect was observed with 4-nonylphenol. In contrast, progesterone and many of the EDCs, including bisphenol A, antagonized the pregnenolone-induced enhancement of rhMAP2C-stimulated tubulin assembly. These agonistic and inverse agonistic actions were not observed in tubulin assembly stimulated with Delta1-71 rhMAP2C, which lacks the steroid-binding site. Using a dark-field microscopy, pregnenolone and pentachlorphenol were observed to generate characteristic filamentous microtubules in a progesterone- or bisphenol A-reversible manner. In cultured hippocampal neurons, similar agonist-antagonist relationships were reproduced in terms of dendritic outgrowth. Fluorescent recovery after photobleaching of hippocampal neurons showed that pregnenolone and agonistic EDCs enhanced, but that 4-nonylphenol inhibited the MAP2-mediated neurite outgrowth in a progesterone- or antagonistic EDC-reversible manner. Furthermore, none of the examined effects were affected by mifepristone or ICI-182,786 i.e. the classical progesterone and estrogen receptor antagonists. Taken together, these results suggest that EDCs cause a wide variety of significant disturbances to dendritic outgrowth in hippocampal neurons, which may lead to psychological disorders following chronic exposure during early neuronal development.


Assuntos
Dendritos/metabolismo , Disruptores Endócrinos/metabolismo , Inibidores do Crescimento/metabolismo , Hipocampo/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Bovinos , Crescimento Celular/efeitos dos fármacos , Células Cultivadas , Dendritos/efeitos dos fármacos , Dendritos/fisiologia , Regulação para Baixo/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Humanos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Ratos , Regulação para Cima/efeitos dos fármacos
18.
Histochem Cell Biol ; 133(6): 677-82, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20424853

RESUMO

Although anthracycline antibiotics daunorubicin (DR), doxorubicin (DX), and epirubicin (ER) possess minor differences in their chemical structures, large differences are noted in their clinical use, as well as in cellular and plasma pharmacokinetic parameters in vivo. Immunocytochemistry for DR, DX, or ER was developed using an anti-DR monoclonal antibody (ADM-1-11), which has been demonstrated to react equally well with each of the three drugs, and therefore it was used for comparing their accumulation in several rat tissue cells after a single i.v. injection of each drug. In the kidney, immunoreactivity for each drug was distributed in essentially the same pattern and in the same strength 2 h after injection, but quite differently distributed in kidney cells thereafter, so that at 120-h post-injection significant amounts of DX and ER remained, but DR had almost completely vanished. Similar patterns of accumulation were observed in cells of other tissues including the pancreas, hair follicle, and stomach, with the exception of the intestine in which none of the three drugs remained after 120 h. These results appear to be supported by previous pharmacokinetic studies on the anthracyclines. The mechanism for such differences among the three drugs remains obscure, but the hydroxyl group at C-14 of DX and ER molecule might be related to the strong propensity of DX and ER to accumulate in tissue cells. The present results should contribute to the understanding of the mechanisms of the differences in the pharmacokinetics, as well as the possibly in anti-tumor activities of the anthracyclines.


Assuntos
Antraciclinas/farmacocinética , Antineoplásicos/farmacocinética , Túbulos Renais Proximais/metabolismo , Animais , Daunorrubicina/farmacocinética , Doxorrubicina/farmacocinética , Epirubicina/farmacocinética , Imuno-Histoquímica , Masculino , Ratos , Ratos Wistar
20.
Peptides ; 126: 170265, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31982448

RESUMO

Prothymosin alpha (ProTα)-mimetic hexapeptide (amino acid: NEVDQE, P6Q) inhibits cerebral or retinal ischemia-induced behavioral, electrophysiological and histological damage. P6Q also abolishes cerebral hemorrhage induced by ischemia with tissue plasminogen activator (tPA). In the present study we examined the beneficial effects of P6Q on other post-stroke prognostic psychology-related symptoms, which obstruct the motivation toward physical therapy. Intravenous (i.v.) administration with tPA (10 mg/kg) at 6 h after photochemically induced thrombosis (PIT) in mice resulted in bilateral central post-stroke pain in thermal and mechanical nociception tests and loss of social activity in the nest building test, both of which were significantly blocked by P6Q (30 mg/kg, i.v.) given at 5 h after PIT. P6Q (30 mg/kg, i.v.) also improved the memory-learning deficit in the step-through test and depression-like behavior in the tail suspension test when it was given 1 day after bilateral common carotid arteries occlusion (BCCAO) in mice. Thus, these studies suggest that P6Q could be a promising candidate to prevent negative prognostic psychological symptoms following focal and global ischemia.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Depressão/tratamento farmacológico , Transtornos da Memória/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Dor/tratamento farmacológico , Precursores de Proteínas/farmacologia , Acidente Vascular Cerebral/tratamento farmacológico , Timosina/análogos & derivados , Animais , Isquemia Encefálica/induzido quimicamente , Isquemia Encefálica/patologia , Isquemia Encefálica/psicologia , Depressão/etiologia , Depressão/patologia , Aprendizagem , Masculino , Transtornos da Memória/etiologia , Transtornos da Memória/patologia , Camundongos , Camundongos Endogâmicos C57BL , Dor/etiologia , Dor/patologia , Fragmentos de Peptídeos/farmacologia , Acidente Vascular Cerebral/induzido quimicamente , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/psicologia , Timosina/farmacologia , Ativador de Plasminogênio Tecidual/toxicidade
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