Systematic spatiotemporal mapping reveals divergent cell death pathways in three mouse models of hereditary retinal degeneration

Calcium (Ca²⁺) dysregulation has been linked to neuronal cell death, including in hereditary retinal degeneration. Ca²⁺ dysregulation is thought to cause rod and cone photoreceptor cell death. Spatial and temporal heterogeneities in retinal disease models have hampered validation of this hypothesis. We examined the role of Ca²⁺ in photoreceptor degeneration, assessing the activation pattern of Ca²⁺-dependent calpain proteases, generating spatiotemporal maps of the entire retina in the cpfl1 mouse model for primary cone degeneration, and in the rd1 and rd10 models for primary rod degeneration. We used Gaussian process models to distinguish the temporal sequences of degenerative molecular processes from other variability sources.In the rd1 and rd10 models, spatiotemporal pattern of increased calpain activity matched the progression of primary rod degeneration. High calpain activity coincided with activation of the calpain-2 isoform but not with calpain-1, suggesting differential roles for both calpain isoforms. Primary rod loss was linked to upregulation of apoptosis-inducing factor, although only a minute fraction of cells showed activity of the apoptotic marker caspase-3. After primary rod degeneration concluded, caspase-3 activation appeared in cones, suggesting apoptosis as the dominant mechanism for secondary cone loss. Gaussian process models highlighted calpain activity as a key event during primary rod photoreceptor cell death. Our data suggest a causal link between Ca²⁺ dysregulation and primary, nonapoptotic degeneration of photoreceptors and a role for apoptosis in secondary degeneration of cones, highlighting the importance of the spatial and temporal location of key molecular events, which may guide the evaluation of new therapies.     

L‐type calcium channel blocker ameliorates diabetic encephalopathy by modulating dysregulated calcium homeostasis

Diabetic encephalopathy is a complication of diabetes characterized by impaired cognitive functions. The objective of the present study was to examine the beneficial effect of the calcium channel blocker, nimodipine, on diabetes‐induced cognitive deficits and altered calcium homeostasis in the cerebral cortex. Diabetes was induced in mice by intraperitoneal injection of streptozotocin (40 mg/kg body wt) for 5 days. Nimodipine (10 mg/kg body weight) was administered intraperitoneally to the animals every 48 hr for 8 weeks. A significant impairment in spatial learning and memory was observed in diabetic animals, which was reversed by nimodipine treatment. Diabetic animals showed increased CaV1.2 mRNA and protein expression, which might be responsible for enhanced synaptosomal calcium uptake. Nimodipine treatment was found to lower CaV1.2 mRNA, protein expression, and calcium uptake. Mitochondrial Ca²⁺ uptake was reduced in diabetic brains, which was reversed with nimodipine treatment. Plasma membrane and sarcoplasmic reticulum Ca²⁺‐ATPase activity was found to be significantly decreased in diabetic animals, whereas nimodipine supplementation restored the activity of both Ca²⁺‐ATPases nearly to control values. Nimodipine treatment was shown to normalize intracellular free Ca²⁺ levels in diabetic animals. Nimodipine was shown to attenuate increased calpain activity measured in terms of hydrolysis of fluorogenic substrate and αII‐spectrin degradation. Nimodipine supplementation also reduced reactive oxygen species production and lipid peroxidation in diabetic animals. The data suggests that L‐type calcium channel blocker is beneficial in preventing cognitive deficits associated with diabetic encephalopathy through modulation of dysregulated calcium homeostasis. © 2014 Wiley Periodicals, Inc.     

Brain-derived neurotrophic factor mediates neuroprotection against Aβ-induced toxicity through a mechanism independent on adenosine 2A receptor activation

Abstract

Brain-derived neurotrophic factor (BDNF) promotes neuronal survival through TrkB-FL activation. The activation of adenosine A_2A receptors (A_2AR) is essential for most of BDNF-mediated synaptic actions, such as synaptic plasticity, transmission and neurotransmitter release. We now aimed at evaluating the A_2AR influence upon BDNF-mediated neuroprotection against Aβ_25–35 toxicity in cultured neurons. Results showed that BDNF increases cell survival and reduces the caspase-3 and calpain activation induced by amyloid-β (Aβ) peptide, in a mechanism probably dependent on PLCγ pathway. This BDNF-mediated neuroprotection is not affected by A_2AR activation or inhibition. Moreover neither activation nor inhibition of A_2AR, per se, significantly influenced Aβ-induced neuronal death on calpain-mediated cleavage of TrkB induced by Aβ. In conclusion, these results suggest that, in opposition to the fast synaptic actions of BDNF, the neuroprotective actions of this neurotrophin against a strong Aβ insult do not require the activation of A_2AR.     

The physiological response of protease inhibition in dystrophic muscle

Duchenne muscular dystrophy (DMD) is caused by the production of a non‐functional dystrophin gene product and a failure to accumulate functional dystrophin protein in muscle cells. This leads to membrane instability, loss of Ca²⁺ homoeostasis and widespread cellular injury. Associated with these changes are increased protease activities in a variety of proteolytic systems. As such, there have been numerous investigations directed towards determining the therapeutic potential of protease inhibition. In this review, evidence from genetic and/or pharmacological inhibition of proteases as a treatment strategy for DMD is systematically evaluated. Specifically, we review the potential roles of calpain, proteasome, caspase, matrix metalloproteinase and serine protease inhibition as therapeutic approaches for DMD. We conclude that despite early results to the contrary, inhibition of calpain proteases is unlikely to be successful. Conversely, evidence suggests that inhibition of proteasome, matrix metalloproteinases and serine proteases does appear to decrease disease severity. An important caveat to these conclusions, however, is that the fundamental cause of DMD, dystrophin deficiency, is not corrected by this strategy. Hence, this should not be viewed as a cure, but rather, protease inhibitors should be considered for inclusion in a therapeutic cocktail. Physiological Relevance. Selective modulation of protease activity has the potential to profoundly change intracellular physiology resulting in a possible treatment for DMD. However, alteration of protease activities could also lead to worsening of disease progression by promoting the accumulation of substrates in the cell. The balance of benefit and potential damage caused by protease inhibition in human DMD patients is largely unexplored.     

Altered ubiquitin causes perturbed calcium homeostasis,hyperactivation of calpain,dysregulated differentiation,and cataract

Although the ocular lens shares many features with other tissues, it is unique in that it retains its cells throughout life, making it ideal for studies of differentiation/development. Precipitation of proteins results in lens opacification, or cataract, the major blinding disease. Lysines on ubiquitin (Ub) determine fates of Ub-protein substrates. Information regarding ubiquitin proteasome systems (UPSs), specifically of K6 in ubiquitin, is undeveloped. We expressed in the lens a mutant Ub containing a K6W substitution (K6W-Ub). Protein profiles of lenses that express wild-type ubiquitin (WT-Ub) or K6W-Ub differ by only ∼2%. Despite these quantitatively minor differences, in K6W-Ub lenses and multiple model systems we observed a fourfold Ca²⁺ elevation and hyperactivation of calpain in the core of the lens, as well as calpain-associated fragmentation of critical lens proteins including Filensin, Fodrin, Vimentin, β-Crystallin, Caprin family member 2, and tudor domain containing 7. Truncations can be cataractogenic. Additionally, we observed accumulation of gap junction Connexin43, and diminished Connexin46 levels in vivo and in vitro. These findings suggest that mutation of Ub K6 alters UPS function, perturbs gap junction function, resulting in Ca²⁺ elevation, hyperactivation of calpain, and associated cleavage of substrates, culminating in developmental defects and a cataractous lens. The data show previously unidentified connections between UPS and calpain-based degradative systems and advance our understanding of roles for Ub K6 in eye development. They also inform about new approaches to delay cataract and other protein precipitation diseases.Many age-related diseases such as cataracts, macular degeneration, Alzheimer’s, Parkinson’s, Huntington’s, and several premature aging syndromes, appear to be causally associated with accumulation of abnormal proteins (1, 2). The accumulation of damaged proteins in many age-related diseases involves a vicious cycle of stress-induced postsynthetic modifications to bulk and catalytically critical molecules and limited capacity to remove the damaged proteins, thus accelerating accumulation of damaged proteins and protein precipitation (1–3). Clarity is essential for lens function. Age-related cataract is due to the aggregation and precipitation of proteins from the normally clear milieu and is the leading cause of adult blindness worldwide, affecting more than 18 million people (4). Congenital cataracts also involve protein precipitation (5).The lens is an excellent system to study specific relationships between proteolytic pathways, stress, and maintenance of protein quality because all of the cells are retained throughout life. The oldest lens tissue is found at the center or core of the lens. Crystallins, the major gene products of the lens, are very long-lived proteins, with half-lives of decades, and their aberrant synthesis or modification results in aggregation, insolubilization, and cataract (6). Common age-related stresses that confront proteins in the lens and other tissues during aging include oxidation, glycation, and methylation, as well as their sequels (3, 6–9). Effective stress-reducing systems including antioxidants, antioxidant and repair enzymes, and chaperone and proteolytic capacities help limit damage and maintain solubility and function in younger tissues (3).There are three general systems for intracellular proteolysis: lysosomal/autophagic mechanisms, calcium-activated proteases, or calpains, and the ubiquitin proteasome system (UPS). Because nuclei and lysosomes are removed from most of the lens cells in regions where most cataracts form, this leaves only cytoplasmic proteases, including the UPSs and calpains, to remove damaged proteins to retain lens function (3). There are two basic steps to the UPS: conjugation of substrates to multiple ubiquitins (Ubs) followed by degradation of the protein substrate. Ubiquitin is a highly conserved protein with seven lysines (10). The lysines are used, in the first step of the UPS, to form inter-Ub linkages that lead to Ub polymers that are conjugated to protein substrates. Commonly, proteins with K48-linked Ub oligomers attached are scheduled for degradation by the 26S proteasome. The UPS is also involved at multiple critical stages of proliferation, differentiation, and development in most tissues.It was recently noted that only a minute proportion of Ub conjugates use K6 on Ub (11). Thus, we were surprised to find that expression of higher levels of K6W-Ub in the lens produced cataracts (12). Other work showed that Ub mutations in which K6 is replaced by various amino acids, i.e., K6W-Ub, are conjugation competent but proteolytically incompetent. Thus, cells and tissues in which K6W-Ub is expressed accumulate Ub conjugates (13). Exchanging K for A or R has the same effects. Although structural information about Ub conjugates built using K6 is becoming available (14, 15), a complete understanding of features that render such conjugates biologically stable remain to be elucidated.Calpain is up-regulated by increased Ca²⁺. Gap junction proteins, or Connexins (Cxs), are required for maintaining Ca²⁺ homeostasis (16). UPS-dependent degradation of Cx has been observed in CHO and BWEM cells (17). The formation of cataracts in lenses that express K6W-Ub is compatible with novel functional connections between UPS activity, regulation of Cx and Ca²⁺, calpain activity, and lens clarity. To test the hypothesis, we targeted expression of K6W-Ub to the lens, and we monitored stability of multiple proteins, Cx function and ubiquitination, Ca²⁺, calpain activity, protein integrity, solubility, and localization, as well as lens clarity.     

丹曲林对大鼠缺血再灌注心肌嘌呤核苷酸代谢的影响

目的 探讨丹曲林对缺血再灌注心肌嘌呤核苷酸代谢的影响.方法 24只健康雄性Wistar大鼠(n=24)建立Langendorff离体心脏灌注模型,分为两组,对照组即缺血再灌注组和实验组即丹曲林处理组,应用高效液相色谱仪(HPLC)测定嘌呤核苷酸代谢产物腺苷,AMP,尿酸的含量变化,以揭示5'-核苷酸酶(5'-nu-cleotidase,5'-NT),腺苷脱氛酶(adenosine deaminase,ADA),黄嘌呤氧化酶(xanthine oxidase,XO)的活性.结果 丹曲林处理组与缺血再灌注组相比,5'-核苷酸酶,腺苷脱氛酶,黄嘌呤氧化酶活性均明显增加(P<0.05).结论 丹曲林通过抑制Ca2+超载,降低Ca2+活化的钙蛋白酶对细胞膜骨架蛋白和结构蛋白的降解,以及增强膜磷脂的稳定性,维持细胞完整性,保存细胞内的各种代谢酶,对缺血再灌注心肌起到保护作用.     

钙激活中性蛋白酶-2对肝癌细胞阿霉素耐药性的影响

目的观察阿霉素作用下,钙激活中性蛋白酶-2(cal-pain-2)在肝癌细胞(HepG2)中的表达及其对细胞存活率的影响,评价其作为抗癌药靶的可能性。方法以倍比稀释的ADM作用于HepG2细胞,用四甲基偶氮唑盐比色(MTT)实验检测阿霉素(Adriamycin,ADM)72 h的半数致死量(LD50);用蛋白质印迹(Western blot)检测的ADM(LD50)作用下,calpain-2和cyclin E的表达和酶解情况;给予calpain-2抑制剂(calpastatin peptide)干预后,用MTT法观察ADM对HepG2细胞存活率的影响。结果 MTT实验结果显示,ADM对HepG2的LD50为2.5μmol.L-1;Western blot发现,随ADM作用时间的延长,calpain-2的表达水平增高(P<0.01),而且被剪切的程度增加(P<0.05);MTT检测ADM对HepG2的作用发现,calpastatin peptide干预的细胞存活率明显低于对照组(P<0.01),Cyclin E被剪切的程度减轻(P<0.01)。结论肝癌细胞HepG2在ADM作用下发生凋亡的同时,也上调calpain-2的表达水平和活性,并通过其信号通路降低对ADM的敏感性,而针对calpain-2的活性抑制可以提高HepG2对ADM敏感性,提示calpain-2可以作为降低肿瘤化疗耐药性的潜在靶点。     

钙蛋白酶参与tau蛋白过度磷酸化和截断的机制研究进展

阿尔茨海默病(AD)是最常见的神经退行性疾病之一,临床表现为认知和记忆功能障碍。钙蛋白酶(calpain)在细胞中广泛地被激活,钙蛋白酶的紊乱会引起AD病理学中tau蛋白过度磷酸化和tau蛋白的异常剪切。钙蛋白酶通过影响糖原合成酶激酶3和蛋白磷酸酶2A的活性,从而使tau蛋白多个位点发生异常过度磷酸化,并且介导tau蛋白单体的截断,诱导神经变性。钙蛋白酶有望成为AD药物治疗的潜在靶点。     

急性离心运动后骨骼肌超微结构、钙依赖性蛋白酶和泛素的动态变化

目的:探讨1次离心运动后大鼠骨骼肌超微结构、Calpains和Ubiquitin的动态变化。方法:30只雄性SD大鼠随机分为对照组和1次离心运动后即刻组、24 h组和7天组。各运动组大鼠进行1次下坡跑运动,速度16 m/min,坡度?16°,运动100 min后休息10 min,再运动100 min,总时间200 min。分别在急性运动后即刻、24 h和第7天取材,测定血清LDH、CK活性和股四头肌超微结构及Calpain-1、Calpain-2和Ubiquitin含量。结果:①1次离心运动后即刻组大鼠股四头肌肌丝排列混杂、卷曲;运动后24 h出现轻度溶解、断裂,Z线不规则,局部Z线消失;运动后7天超微结构与对照组相比无显著变化。血清CK和LDH活性变化与股四头肌超微结构变化一致。②与对照组相比,运动后即刻组大鼠股四头肌Calpain-1、Calpain-2和Ubiquitin含量均有所降低,但均无显著性差异;运动后24 h组股四头肌Calpain-1、Calpain-2和Ubiquitin含量均显著高于对照组和运动后即刻组;运动后7天组与运动后24 h组相比均显著下降,而与对照组相比均无显著性差异。结论:①1次离心运动所致的骨骼肌损伤有一定的延迟性,骨骼肌损伤在运动后即刻从整体看并不严重,但在运动后24 h出现明显损伤,运动后7天基本恢复。②急性离心运动后骨骼肌Calpain和Ubiquitin含量变化与骨骼肌损伤的动态变化基本一致。     

实验性自身免疫性脑脊髓炎小鼠脑组织和脊髓中脑型肌酸激酶、钙泵和钙中性蛋白酶的变化

目的：观察实验性自身免疫性脑脊髓炎（EAE）小鼠模型脑组织和脊髓中脑型肌酸激酶（CK-BB）、钙泵（CaATPase）和钙中性蛋白酶（calpain）的变化。方法：C57BL/6小鼠随机分为：EAE组（n=9）,用髓鞘少突胶质细胞糖蛋白35-55多肽（MOG35-55）及完全弗氏佐剂混合抗原乳剂免疫小鼠;对照组（n=5）,注射生理盐水。用MOG35-55诱导C57BL/6小鼠,建立（EAE）动物模型（即多发性硬化模型）。观察记录EAE小鼠行为学变化;采用苏木精-伊红染色、LFB髓鞘染色,酶标仪和分光光度计法检测发病高峰期（免疫后第19天）的中枢神经组织病理学变化、CK-BB,CaATPase和calpain酶活性的改变。结果：EAE组与对照组相比：①日均神经功能评分和日累积评分增加（P〈0.01）;②苏木精-伊红染色：中枢炎症细胞浸润明显（P〈0.05）;③LFB染色：髓鞘脱失较多;④CK-BB和CaATPase活性降低,calpain活性增加（P〈0.05）;⑤EAE组小鼠（只）日均累积神经功能评分与CK-BB和CaATPase活性呈负相关（P〈0.05）;⑥EAE组小鼠（只）日均累积神经功能评分与calpain酶活性呈正相关（P〈0.05）。结论：EAE高峰期中枢CK-BB和CaATPase活性降低、calpain活性增高是EAE发生发展的后果,提示EAE时中枢能量代谢和CaATPase、calpain的病理性损害。