Reduced expression of BTBD10 in anterior horn cells with Golgi fragmentation and pTDP‐43‐positive inclusions in patients with sporadic amyotrophic lateral sclerosis

Overexpression of BTBD10 (BTB/POZ domain‐containing protein 10) suppresses G93A‐superoxide dismutase 1 (SOD1)‐induced motor neuron death in a cell‐based amyotrophic lateral sclerosis (ALS) model. In the present study, paraffin sections of spinal cords from 13 patients with sporadic ALS and 10 with non‐ALS disorders were immunostained using a polyclonal anti‐BTBD10 antibody. Reduced BTBD10 expression in the anterior horn cells was more frequent in spinal cords from ALS patients than in cords from patients with non‐ALS disorders. We further investigated the relationship between the level of BTBD10 immunoreactivity and the morphology of the Golgi apparatus (GA) and the presence of phosphorylated TAR‐DNA‐binding protein 43 (pTDP‐43). Mirror sections of spinal cords from five sporadic ALS cases were immunostained with antibodies against BTBD10 and trans‐Golgi‐network (TGN)‐46 or pTDP‐43. Whereas 89.7–96.5% of the neurons with normal BTBD10 immunoreactivity showed normal GA morphology and no pTDP‐43 cytoplasmic aggregates, 86.2–94.3% of the neurons with reduced BTBD10 expression showed GA fragmentation and abnormal pTDP‐43 aggregates. These findings suggest that reduced BTBD10 expression is closely linked to the pathogenesis of sporadic ALS.     

New routes in frontotemporal dementia drug discovery

Introduction: Research into the pathogenic mechanisms behind frontotemporal dementia (FTD) has yielded several new targets for therapeutic intervention; such targets include specific new pathways uncovered by mutations as well as targets involving the modulation, formation and degradation of protein aggregates.

Areas covered: Herein, the authors outline the principal molecular causes underlying FTD to date and the research that has been performed in these areas with respect to an eventual corrective strategy.

Expert opinion: While it is worthwhile targeting pathways affected by specific mutations with a causative loss of function linked to FTD, research still has to contend with issues including the remaining presence of protein aggregates or that treatments are rarely universally applicable. Aiming to recover function in a downstream target caused by the protein aggregates will likely be insufficient due to the large cascade of events affected. It is our belief that the clearance of these aggregates and the inhibition of protein misfolding are more appropriate and direct routes to an eventual therapy.     

间隙连接蛋白43突变与皮肤疾病相关性研究

间隙连接蛋白(Cx)在保证细胞间物质信息传递及维持皮肤屏障稳态方面发挥着重要作用,其基因突变,甚至表达水平异常均会引起多种疾病,严重影响患者生活质量。在编码人类Cx家族的21个基因中,与Cx基因突变相关的临床伴随疾病至少有14种。其中,Cx43分布最广,不仅在大多数器官组织中均有报道,也是伤口愈合、皮肤角质化,以及皮肤肿瘤发展等重要生理病理过程中的关键调控节点。本文总结了近年来Cx43基因(GJA1)在皮肤屏障中的作用、GJA1基因突变相关皮肤疾病及其潜在致病机制这几个快速发展领域中的研究成果,以期为Cx43临床伴发疾病防治及相关研究提供参考。     

连接蛋白43在骨关节炎发病机制中的研究进展

骨关节炎(osteoarthritis,OA)是常见的关节疾病,也是导致残疾的主要原因之一,给患者及家庭带来巨大的经济生活负担。虽然OA的发病机制至今尚未完全明确,但关节软骨代谢平衡的破坏被认为与骨关节炎的发生息息相关。研究发现连接蛋白43(connexin43)在维持关节软骨代谢平衡的稳态中发挥着至关重要的作用,它所参与形成的半通道(hemichannels)和缝隙连接(gap junctions)在细胞与细胞外基质,以及细胞之间建立起直接的物质信号交换通道。同时,连接蛋白43的高表达和(或)分布的变化都将影响软骨细胞结构和功能的完整性。因此,连接蛋白43被认为与骨关节炎的发生有着密切关系。本文就连接蛋白43与骨关节炎发病机制的研究进展做一综述。     

Mechanism of Regulatory Effect of MicroRNA-206 on Connexin 43 in Distant Metastasis of Breast Cancer

Background:

MicroRNA-206 (miR-206) and connexin 43 (Cx43) are related with the distant metastasis of breast cancer. It remains unclear whether the regulatory effect of miR-206 on Cx43 is involved in metastasis of breast cancer.

Methods:

Using quantitative real-time polymerase chain reaction and Western blot, the expressions of miR-206 and Cx43 were determined in breast cancer tissues, hepatic and pulmonary metastasis (PM), and cell lines (MCF-10A, MCF-7, and MDA-MB-231). MCF-7/MDA-M-231 cells were transfected with lentivirus-shRNA vectors to enhance/inhibit miR-206, and then Cx43 expression was observed. Cell counting kit-8 assay and Transwell method were used to detect their changes in proliferation, migration, and invasion activity. The mutant plasmids of Cx43-3’ untranslated region (3’UTR) at position 478–484 and position 1609–1615 were constructed. Luciferase reporter assay was performed to observe the effects of miR-206 on luciferase expression of different mutant plasmids and to confirm the potential binding sites of Cx43.

Results:

Cx43 protein expression in hepatic and PM was significantly higher than that in the primary tumor, while no significant difference was showed in messenger RNA (mRNA) expression. MiR-206 mRNA expression in hepatic and PM was significantly lower than that in the primary tumor. Cx43 mRNA and protein levels, as well as cell proliferation, migration, and invasion capabilities, were all significantly improved in MDA-MB-231 cells after reducing miR-206 expression but decreased in MCF-7 cells after elevating miR-206 expression, which demonstrated a significantly negative correlation between miR-206 and Cx43 expression (P = 0.03). MiR-206 can drastically decrease Cx43 expression of MCF-7 cells but exerts no effects on Cx43 expression in 293 cells transfected with the Cx43 coding region but the lack of Cx43-3’UTR, suggesting that Cx43-3’UTR may be the key in Cx43 regulated by miR-206. Luciferase expression showed that the inhibition efficiency was reduced by 46.80% in position 478–484 mutant, 16.72% in position 1609–1615 mutant; the inhibition was totally disappeared in double mutant (P = 0.02).

Conclusions:

MiR-206 can regulate the expression of Cx43, the cytobiological activity, and the metastasis of breast cancer through binding to the two binding sites in Cx43-3’UTR: position 478–484 and position 1609–1615.     

Chronic antidepressant treatment selectively increases expression of plasticity-related proteins in the hippocampus and medial prefrontal cortex of the rat

Antidepressants protect against hippocampal volume loss in humans and reverse stress-induced atrophic changes in animals thus supporting the hypothesis that the pathophysiology of stress-related disorders such as depression involves reductions in neuronal connectivity and this effect is reversible by antidepressant treatment. However, it is unclear which brain areas demonstrate such alterations in plasticity in response to antidepressant treatment. The aim of the present study was to examine the effect of antidepressant treatment on the expression of three plasticity-associated marker proteins, the polysialylated form of nerve cell adhesion molecule (PSA-NCAM), phosphorylated cyclic-AMP response element binding protein (pCREB) and growth-associated protein 43 (GAP-43), in the rat brain. To this end, rats were treated either acutely (60 min) or chronically (21 days) with imipramine (30 and 15 mg/kg, respectively) and the expression of PSA-NCAM, pCREB, and GAP-43 was assessed using immunohistochemistry. Initial mapping revealed that chronic imipramine treatment increased expression of these plasticity-associated proteins in the hippocampus, medial prefrontal cortex and piriform cortex but not in the other brain regions examined. Since PSA-NCAM and pCREB are expressed in recently-generated neurons in the dentate gyrus, it is likely that chronic imipramine treatment increased their expression in the hippocampus at least partially by increasing neurogenesis. In contrast, since chronic imipramine treatment is not associated with neurogenesis in the medial prefrontal cortex, increased expression of PSA-NCAM and pCREB in the prelimbic cortex implicates changes in synaptic connectivity in this brain region. Acute treatment with imipramine increased the number of pCREB positive nuclei in the hippocampus and the prefrontal cortex but did not alter expression of GAP-43 or PSA-NCAM in any of the brain regions examined. Taken together, the results of the present study suggest that antidepressant treatment increases synaptic plasticity and connectivity in brain regions associated with mood disorders.     

Connexin 43 Is Necessary for Murine Tendon Enthesis Formation and Response to Loading

The enthesis is a mineralized fibrocartilage transition that attaches tendon to bone and is vital for musculoskeletal function. Despite recent studies demonstrating the necessity of muscle loading for enthesis formation, the mechanisms that regulate enthesis formation and mechanoresponsiveness remain unclear. Therefore, the current study investigated the role of the gap junction protein connexin 43 in these processes by deleting Gja1 (the Cx43 gene) in the tendon and enthesis. Compared with their wild-type (WT) counterparts, mice lacking Cx43 showed disrupted entheseal cell alignment, reduced mineralized fibrocartilage, and impaired biomechanical properties of the supraspinatus tendon entheses during postnatal development. Cx43-deficient mice also exhibited reduced ability to complete a treadmill running protocol but no apparent deficits in daily activity, metabolic indexes, shoulder muscle size, grip strength, and major trabecular bone properties of the adjacent humeral head. To examine enthesis mechanoresponsiveness, young adult mice were subjected to modest treadmill exercise. Gja1 deficiency in the tendon and enthesis reduced entheseal anabolic responses to treadmill exercise: WT mice had increased expression of Sox9, Ihh, and Gli1 and increased Brdu incorporation, whereas Cx43-deficient mice showed no changes or decreased levels with exercise. Collectively, the results demonstrated an essential role for Cx43 in postnatal tendon enthesis formation, function, and response to loading; results further provided evidence implicating a link between Cx43 function and the hedgehog signaling pathway. © 2020 American Society for Bone and Mineral Research.     

七氟醚对低温全心缺血-再灌注心脏电传导及Cx43 Ser368磷酸化的影响

目的 观察七氟醚对低温全心缺血-再灌注心室肌电传导及Cx43 Ser368磷酸化的影响。
方法 制备成功的离体灌注工作心脏24只,随机分为三组：对照组（C组）、低温全心缺血-再灌注组（IR组）和1.0 MAC七氟醚处理组（Sev组）,每组8只。C组：37 ℃ K-H液平衡灌注15 min后继续灌注37 ℃ K-H液105 min;IR组：37 ℃ K-H液平衡灌注15 min后继续灌注37 ℃ K-H液15 min,注射Thomas液（4 ℃,20 ml/kg）使心脏停搏60 min,4 ℃ K-H液保护心脏,停搏30 min时半量复灌Thomas液（4 ℃,10 ml/kg）,60 min时使用37 ℃ K-H液再灌注30 min;Sev组：37 ℃ K-H液平衡灌注15 min后继续灌注含饱和1.0 MAC七氟醚的37 ℃ K-H液15 min,注射Thomas液（4 ℃,20 ml/kg）使心脏停搏60 min,4 ℃ K-H液保护心脏,停搏30 min时半量复灌Thomas液（4 ℃,10 ml/kg）,60 min时使用含饱和1.0 MAC七氟醚的37 ℃ K-H液再灌注30 min。于再灌注即刻至灌注结束,记录离体心脏复跳时间（再灌注即刻至心脏首次跳动所需的时间）,室性心律失常（室性早搏、室性心动过速、室性颤动）发生情况和持续时间。采用心脏刺激仪行程控刺激,测定并记录有效不应期（ERP）、传导速度(CV）。采用免疫印迹法检测心室肌组织Cx43和Cx43 Ser368蛋白相对含量。
结果 与C组比较,IR组和Sev组ERP明显延长,CV明显减慢（P＜0.05）;IR组Cx43及Cx43 Ser368蛋白相对含量明显降低（P＜0.05）。与IR组比较,Sev组心脏复跳时间明显缩短,心律失常发生率明显降低,心律失常持续时间明显缩短,ERP明显缩短,CV明显增快,Cx43及Cx43 Ser368蛋白相对含量明显升高（P＜0.05）。
结论 七氟醚可以上调低温全心缺血-再灌注心室肌组织Cx43和Cx43 Ser368的表达,促使心室肌电传导增快、有效不应期缩短,降低再灌注心律失常的发生。     

Acceleration of TDP43 and FUS/TLS protein expressions in the preconditioned hippocampus following repeated transient ischemia

The 43‐kDa transactivation response DNA binding protein (TDP43), fused in sarcoma/translocated in liposarcoma (FUS/TLS), heat shock protein 70 (HSP70), and β‐amyloid (Aβ) are induced and involved in cerebral ischemia, amyotrophic lateral sclerosis (ALS), and Alzheimer's disease (AD), but their relationships in ischemic tolerance have never been examined, although they could be involved in endogenous neuroprotection under ischemic preconditioning. In the present study, Mongolian gerbils were subjected to one or three incidents of basically nonlethal 2‐min transient common carotid arteries occlusion (tCCAO). Hippocampal CA1 neurons were lost only in the 2‐min three times group at 3 and 7 days, which then gradually recovered from 1 to 6 months. Inductions of TDP43 and FUS/TLS were accelerated from 3 months to 7 days or from 7 days to 1 day, respectively, after 2‐min three times ischemia compared with once. The cytoplasmic stainings of TDP43 and FUS/TLS showed a further acceleration of the peaks from 1 months to 3 days or from 1 months to 7 days, respectively, after 2‐min three times ischemia compared with once. In contrast, HSP70 was induced only at 7 days after 2‐min tCCAO for three times, with no expression for Aβ. These data show that ischemic preconditioning offers a way to induce endogenous neuroprotection and neurogenesis in gerbils, with TDP43, FUS/TLS, and HSP70 involved in this function. © 2013 Wiley Periodicals, Inc.     

Bunina bodies in motor and non‐motor neurons revisited: A pathological study of an ALS patient after long‐term survival on a respirator

Bunina bodies (BBs) are small eosinophilic neuronal cytoplasmic inclusions (NCIs) found in the remaining lower motor neurons (LMNs) of patients with sporadic amyotrophic lateral sclerosis (SALS), being a specific feature of the cellular pathology. We examined a case of SALS, unassociated with TDP‐43 or C9ORF72 mutation, of 12 years duration in a 75‐year‐old man, who had received artificial respiratory support for 9 years, and showed widespread multisystem degeneration with TDP‐43 pathology. Interestingly, in this patient, many NCIs reminiscent of BBs were observed in the oculomotor nucleus, medullary reticular formation and cerebellar dentate nucleus. As BBs in the cerebellar dentate nucleus have not been previously described, we performed ultrastructural and immunohistochemical studies of these NCIs to gain further insight into the nature of BBs. In each region, the ultrastructural features of these NCIs were shown to be identical to those of BBs previously described in LMNs. These three regions and the relatively well preserved sacral anterior horns (S1 and S2) and facial motor nucleus were immunostained with antibodies against cystatin C (CC) and TDP‐43. Importantly, it was revealed that BBs exhibiting immunoreactivity for CC were a feature of LMNs, but not of non‐motor neurons, and that in the cerebellar dentate nucleus, the ratio of neurons with BBs and TDP‐43 inclusions/neurons with BBs was significantly lower than in other regions. These findings suggest that the occurrence of BBs with CC immunoreactivity is intrinsically associated with the particular cellular properties of LMNs, and that the mechanism responsible for the formation of BBs is distinct from that for TDP‐43 inclusions.