Differential effect of calmodulin antagonists on MG132-induced mitochondrial dysfunction and cell death in PC12 cells

Defects in proteasome function have been suggested to be involved in the pathogenesis of neurodegenerative diseases. We examined the effect of calmodulin antagonists on proteasome inhibitor-induced mitochondrial dysfunction and cell viability loss in undifferentiated PC12 cells. Caspase inhibitors (z-IETD.fmk, z-LEHD.fmk and z-DQMD.fmk) and antioxidants attenuated cell death and decrease in GSH contents in PC12 cells treated with 20 microM MG132, a proteasome inhibitor. Calmodulin antagonists (trifluoperazine, W-7 and calmidazolium) had a differential inhibitory effect on the MG132-induced cell death and GSH depletion depending on concentration with a maximal inhibitory effect at 0.5-1 microM. Addition of trifluoperazine and W-7 reduced the MG132-induced nuclear damage, loss of the mitochondrial transmembrane potential followed by cytochrome c release, formation of reactive oxygen species and elevation of intracellular Ca(2+) levels in PC12 cells. Calmodulin antagonists at 5 microM exhibited a cytotoxic effect on PC12 cells but attenuated the cytotoxicity of MG132. The results suggest that the toxicity of MG132 on PC12 cells is mediated by activation of caspase-8, -9 and -3. Trifluoperazine and W-7 at the concentrations of 0.5-1 microM may attenuate the MG132-induced viability loss in PC12 cells by suppressing change in the mitochondrial membrane permeability and by lowering of the intracellular Ca(2+) levels as well as calmodulin inhibition.     

Calcium-dependent proteasome activation is required for axonal neurofilament degradation

Even though many studies have identified roles of proteasomes in axonal degeneration, the mo- lecular mechanisms by which axonal injury regulates proteasome activity are still unclear. In the present study, we found evidence indicating that extracellular calcium influx is an upstream regulator of proteasome activity during axonal degeneration in injured peripheral nerves. In degenerating axons, the increase in proteasome activity and the degradation of ubiquitinated proteins were sig- nificantly suppressed by extracellular calcium chelation. In addition, electron microscopic findings revealed selective inhibition of neurofilament degradation, but not microtubule depolymerization or mitochondrial swelling, by the inhibition of calpain and proteasomes. Taken together, our findings suggest that calcium increase and subsequent proteasome activation are an essential initiator of neurofilament degradation in Wallerian degeneration.     

Proteasome inhibition arrests neurite outgrowth and causes "dying-back" degeneration in primary culture

Proteasome inhibitors such as lactacystin were first isolated when assaying their ability to stimulate neurite outgrowth in neuronal-like cell lines; however, their effect on neurites in primary culture has been largely neglected. We report here that lactacystin causes immediate arrest of nerve growth factor (NGF)-stimulated neurite outgrowth in sympathetic and sensory explant cultures. This is followed by neurite degeneration that in sympathetic cultures has a distinctive "dying-back" morphology. Remarkably, this occurs even at concentrations below that required to induce neurite outgrowth in PC12 cells. Thus, lactacystin opposes rather than potentiates the effect of NGF on sympathetic neurite outgrowth and the role of the ubiquitin proteasome pathway in growth and long-term maintenance of axons and dendrites differs from that in neuritogenesis in neuronal-like cell lines. Retrograde degeneration caused by blocking of the ubiquitin proteasome pathway may mimic some aspects of gracile axonal dystrophy, a dying-back axonopathy in mice caused by ubiquitin hydrolase (Uch-l1) deficiency, and may be relevant to human neurodegenerative diseases involving ubiquitination or proteasome abnormalities.     

Differentiation-dependent sensitivity to cell death induced in the developing retina by inhibitors of the ubiquitin-proteasome proteolytic pathway

The effects of inhibitors of proteasome function were studied in the retina of developing rats. Explants from the retina of neonatal rats at postnatal day (P) 3 or P6 were incubated with various combinations of the proteasome inhibitor carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132), the protein synthesis inhibitor anisomycin, or the adenylyl cyclase activator forskolin. MG132 induced cell death in a subset of cells within the neuroblastic (proliferative) layer of the retinal tissue. The cells sensitive to degeneration induced by either MG132 or anisomycin, were birthdated by bromodeoxyuridine injections. This showed that the MG132-sensitive population includes both proliferating cells most likely in their last round of cell division, and postmitotic undifferentiated cells, at a slightly earlier stage than the population, sensitive to anisomycin-induced cell death. The results show that sensitivity to cell death induced by proteasome inhibitors defines a window of development in the transition from the cell cycle to the differentiated state in retinal cells.     

The neuroprotective effects of the WldS gene are correlated with proteasome expression rather than apoptosis

The Wld(s) gene (slow Wallerian degeneration) specifically delays axonal degeneration following injury and in several models of neurodegenerative diseases. It thus provides an interesting tool to study mechanisms of neurodegeneration. We previously crossed the Wld(s) mice with a mouse mutant that has a motoneuron disease (pmn for progressive motor neuronopathy) and showed that the Wld(s) gene prevented axonal loss, increased the life-span and prolonged the survival of the motoneuron cell bodies. In this study we show that spinal motoneurons of pmn/pmn mice, as opposed to axons, die by apoptosis that cannot be prevented by the Wld(s) gene. However, this same gene could partially rescue the proteasome impairment observed in motoneuron cell bodies and axons of pmn/pmn mice. We conclude that the neuroprotective effect of the Wld(s) gene is not related to an inhibition of apoptosis but could possibly be linked to a regulation in proteasome expression.     

Proteasome inhibitor MG-132 induces dopaminergic degeneration in cell culture and animal models

Impairment in ubiquitin-proteasome system (UPS) has recently been implicated in Parkinson's disease, as demonstrated by reduced proteasomal activities, protein aggregation and mutation of several genes associated with UPS. However, experimental studies with proteasome inhibitors failed to yield consensus regarding the effect of proteasome inhibition on dopaminergic degeneration. In this study, we systematically examined the effect of the proteasome inhibitor MG-132 on dopaminergic degeneration in cell culture and animal models of Parkinson's disease. Exposure of immortalized dopaminergic neuronal cells (N27) to low doses of MG-132 (2-10 microM) resulted in dose- and time-dependent cytotoxicity. Further, exposure to MG-132 (5 microM) for 10 min led to dramatic reduction of proteasomal activity (>70%) accompanied by a rapid accumulation of ubiquitinated proteins in these cells. MG-132 treatment also induced increases in caspase-3 activity in a time-dependent manner, with significant activation occurring between 90 and 150 min. We also noted a 12-fold increase in DNA fragmentation in MG-132 treated N27 cells. Similarly, primary mesencephalic neurons exposed to 5 microM MG-132 also induced >60% loss of TH positive neurons but only a minimal loss of non-dopaminergic cells. Stereotaxic injection of MG-132 (0.4 microg in 4 microl) into the substantia nigra compacta (SNc) in C57 black mice resulted in significant depletion of ipisilateral striatal dopamine and DOPAC content as compared to the vehicle-injected contralateral control sides. Also, we observed a significant decrease in the number of TH positive neurons in the substantia nigra of MG-132-injected compared to the vehicle-injected sites. Collectively, these results demonstrate that the proteasomal inhibitor MG-132 induces dopamine depletion and nigral dopaminergic degeneration in both cell culture and animal models, and suggest that proteasomal dysfunction may promote nigral dopaminergic degeneration in Parkinson's disease.     

蛋白酶体抑制剂MG132对动脉粥样硬化的影响

目的观察蛋白酶体抑制剂MG132对动脉粥样硬化的影响。方法将新西兰白兔30只随机分成高脂组、MG132(1)组和MG132(2)组。3组兔普通饲料喂养1w后行颈动脉球囊损伤术,术后高脂饲料(含1%胆固醇、3%猪油和15%蛋黄)喂养;MG132(1)组在术后高脂饲料喂养的同时血管局部应用蛋白酶体抑制剂MG132;MG132(2)组在术后高脂饲料喂养4w后血管局部应用蛋白酶体抑制剂MG132;3组均喂养8w后取颈总动脉血管制成病理切片行HE染色。结果高脂组兔的右颈总动脉管壁呈动脉粥样硬化改变。MG132(1)组兔右颈动脉血管组织结构基本正常。MG132(2)组兔的右颈总动脉管腔介于二者之间。结论局部应用蛋白酶体抑制剂MG132能够抑制血管内膜增生及动脉粥样形成,同时可能具有稳定斑块的作用。     

Autophagy inhibition in endogenous and nutrient‐deprived conditions reduces dorsal root ganglia neuron survival and neurite growth in vitro

Peripheral neuropathies can result in cytoskeletal changes in axons, ultimately leading to Wallerian degeneration and cell death. Recently, autophagy has been studied as a potential target for improving axonal survival and growth during peripheral nerve damage. This study investigates the influence of autophagy on adult dorsal root ganglia (DRG) neuron survival and axonal growth under control and nutrient deprivation conditions. Constitutive autophagy was modulated with pharmacological activators (rapamycin; Rapa) and inhibitors (3‐methyladenine, bafilomycin A1) in conjunction with either a nutrient‐stable environment (standard culture medium) or a nutrient‐deprived environment (Hank's balanced salt solution + Ca²⁺/Mg²⁺). The results demonstrated that autophagy inhibition decreased cell viability and reduced neurite growth and branching complexity. Although autophagy was upregulated with nutrient deprivation compared with the control, it was not further activated by rapamycin, suggesting a threshold level of autophagy. Overall, both cellular and biochemical approaches combined to show the influence of autophagy on adult DRG neuron survival and growth. © 2016 Wiley Periodicals, Inc.     

Long-term administration of mouse nerve growth factor to adult rats with partial lesions of the cholinergic septohippocampal pathway

Nerve growth factor (NGF), a neurotrophic factor acting on cholinergic neurons of the basal forebrain, has been proposed as a treatment for Alzheimer's disease. Experimental support for its pharmacological use is derived from short-term studies showing that intraventricular administration of NGF during 2-4 weeks protects cholinergic cell bodies from lesion-induced degeneration, stimulates synthesis of choline acetyltransferase, and improves various behavioral impairments. To investigate the consequences of long-term NGF administration, we tested whether cholinergic cell bodies are protected from lesion-induced degeneration and whether cholinergic axons are stimulated to regrow into the denervated hippocampus following fimbrial transections. We found that intraventricular injections of NGF twice a week for 5 months to adult rats resulted in extended protection of cholinergic cell bodies from lesion-induced degeneration and did not produce obvious detrimental effects on the animals. NGF treatment mildly stimulated growth of cholinergic neurites within the 2-mm area directly adjacent to the fimbrial lesion but it failed to induce significant homotypic growth of cholinergic neurites into the deafferented hippocampus.     

Elevation of heme oxygenase‐1 by proteasome inhibition affords dopaminergic neuroprotection

Postmortem studies have shown that heme oxygenase‐1 (HO‐1) immunoreactivity is increased in patients with Parkinson disease. HO‐1 expression is highly upregulated by a variety of stress. Since the proteasome activity is decreased in patients with Parkinson disease, we investigated whether proteasome activity regulates HO‐1 content. MG‐132, a proteasome inhibitor, increased the amount of HO‐1 protein mainly in astrocytes of primary mesencephalic cultures. Quantitative RT‐PCR analysis revealed that lactacystin upregulated HO‐1 mRNA expression. Proteasome inhibition with MG132 also increased the cytomegalovirus promoter‐driven expression of Flag‐HO‐1 protein and resulted in an accumulation of ubiquitinated Flag‐HO‐1 in Flag‐HO‐1‐overexpressing PC12 cells. In addition, a cycloheximide chase assay demonstrated that the degradation of Flag‐HO‐1 protein was slowed by MG‐132. Next, the function of HO‐1 which was upregulated by proteasome inhibitors was examined. Proteasome inhibitors protected dopaminergic neurons from 6‐hydroxydopamine (6‐OHDA)‐induced toxicity and this neuroprotection was abrogated by co‐treatment with zinc protoporphyrin IX, a HO‐1 inhibitor. Furthermore, 6‐OHDA‐induced toxicity was blocked by bilirubin and carbon monoxide, products of the HO‐1‐catalyzed degradation of heme. These results suggest that mesencephalic HO‐1 protein level is regulated by proteasome activity and the elevation by proteasome inhibition affords neuroprotection. © 2010 Wiley‐Liss, Inc.     

The duration of sprouted cerebrovascular axons following intracranial infusion of nerve growth factor

We reported previously that a 2-week infusion of the trophic protein nerve growth factor (NGF) into the lateral ventricle of the adult rat brain elicits a sprouting response by perivascular axons associated with the intradural segment of the internal carotid artery. In the present study, we used electron microscopy to determine whether the sprouted axons persist following cessation of NGF delivery and, if not, to determine the time course of their disappearance. Our results demonstrate that NGF-induced sprouted axons do not persist following cessation of NGF delivery. The total number of axons at 1 week following the end of the NGF infusion was elevated compared to control values, but significantly reduced compared with NGF cases sacrificed immediately following the infusion period. Three weeks following the end of the NGF infusion, the total number of axons was similar to controls although there were no signs of axonal degeneration. These results suggest that continued elevation of NGF levels is necessary to maintain the sprouted axons and that endogenous levels of NGF, or other factors produced by the vascular target tissue, are not sufficient to maintain the newly formed axons. The demonstration that mature perivascular axons proliferate and disappear as a function of exogenous NGF exposure supports the accumulating evidence for continued plasticity in the mature nervous system.     

Collapsin response mediator protein-2 plays a major protective role in acute axonal degeneration

Axonal degeneration is a key pathological feature in many neurological diseases. It often leads to persistent deficits due to the inability of axons to regenerate in the central nervous system.Therefore therapeutic ap-proaches should optimally both attenuate axonal degeneration and foster axonal regeneration. Compelling evidence suggests that collapsin response mediator protein-2 (CRMP2) might be a molecular target fulfill-ing these requirements. In this mini-review, we give a compact overview of the known functions of CRMP2 and its molecular interactors in neurite outgrowth and in neurodegenerative conditions. Moreover, we dis-cuss in detail our recent findings on the role of CRMP2 in acute axonal degeneration in the optic nerve. We found that the calcium influx induced by the lesion activates the protease calpain which cleaves CRMP2, leading to impairment of axonal transport. Both calpain inhibition and CRMP2 overexpression effec-tively protected the proximal axons against acute axonal degeneration. Taken together, CRMP2 is further characterized as a central molecular player in acute axonal degeneration and thus evolves as a promising therapeutic target to both counteract axonal degeneration and foster axonal regeneration in neurodegener-ative and neurotraumatic diseases.     

Nerve Growth Factor-Induced Protection of Brain Capillary Endothelial Cells Exposed to Oxygen–Glucose Deprivation Involves Attenuation of Erk Phosphorylation

Nerve growth factor (NGF) was recently characterized as an angiogenic factor inducing proliferation, migration, and capillary sprouting in endothelial cells (ECs) of different vascular beds. While NGF neuroprotective effects on neurons were described, its survival-inducing effects on brain capillary ECs were not yet addressed. Using a model of oxygen–glucose deprivation (OGD) followed by reoxygenation, we demonstrated that NGF conferred protection in brain capillary ECs. These cells express TrkA and p75^NTR receptors and respond to NGF by stimulation of Erk1/2 phosphorylation and stimulation of proliferation and migration. The NGF protective effect was dose-dependent, inhibited by NGF/TrkA antagonist, K252a, and required presence of NGF during both OGD and reoxygenation phases while the major protective effect was related to decreased cell death during the reoxygenation phase. A causal relationship was found between NGF-induced protection and attenuation of OGD-induced Erk1/2 phosphorylation, supporting the death-promoting role of insult-induced Erk1/2 phosphorylation in the brain capillary ECs. These results emphasize the importance of NGF in the process of EC survival in response to ischemic injury and suggest fine-tuning regulation of Erk1/2 phosphorylation, extending the neuroprotective impact of NGF from sympathetic neuroendocrine cells to brain capillary ECs as the other element in the neurovascular tandem.     

Glutamate induces rapid loss of axonal neurofilament proteins from cortical neurons in vitro

One of the primary hallmarks of glutamate excitotoxicity is degradation of the neuronal cytoskeleton. Using a tissue culture approach, we have investigated the relationship between excitotoxicity and cytoskeletal degradation within axons, with particular reference to the axon specific neurofilament proteins. Neurofilaments were rapidly lost from axons over a 24-h period in response to excitotoxic insult (as observed by immunocytochemistry and western blotting), while other axonal cytoskeletal markers (such as betaIII-tubulin) remained intact. Treatment with kainic acid and NMDA, or complementary experiments using the pharmacological glutamate receptors blockers CNQX (kainate/AMPA receptor antagonist) and MK-801 (NMDA receptor antagonist), demonstrated that neurofilament degeneration was mediated primarily by NMDA receptor activity. This work suggests that excitotoxicity triggers a progressive pathway of cytoskeletal degeneration within axons, initially characterised by the loss of neurofilament proteins.     

Inhibition of MG132-induced mitochondrial dysfunction and cell death in PC12 cells by 3-morpholinosydnonimine

The effect of 3-morpholinosydnonimine (SIN-1) against the cytotoxicity of MG132, a proteasome inhibitor, in differentiated PC12 cells was assessed by measuring the effect on the mitochondrial membrane permeability. Treatment of PC12 cells with MG132 resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species (ROS), and depletion of GSH. Addition of SIN-1, a producer of nitric oxide (NO) and superoxide, differentially reduced the MG132-induced cell death and GSH depletion concentration dependently with a maximal inhibitory effect at 150 microM. Carboxy-PTIO, superoxide dismutase, Mn-TBAP, and ascorbate prevented the inhibitory effect of SIN-1 on the cytotoxicity of MG132. SIN-1 inhibited the MG132-induced change in the mitochondrial membrane permeability, ROS formation and decrease in GSH contents in PC12 cells. S-nitroso-N-acetyl-DL-penicillamine reduced the MG132-induced cell death in PC12 cells, whereas peroxynitrite and H2O2 did not affect the cytotoxicity of MG132. The results suggest that NO and superoxide liberated from SIN-1 exert an inhibitory effect against the cytotoxicity of MG132. SIN-1 may inhibit the MG132-induced viability loss in PC12 cells by suppressing change in the mitochondrial membrane permeability that is associated with oxidative damage.     

Nitric oxide stimulates cGMP formation in rat optic nerve axons, providing a specific marker of axon viability

A major transduction pathway for nitric oxide (NO) is stimulation of soluble guanylyl cyclase and the generation of cyclic GMP (cGMP). In the central nervous system, the NO-cGMP pathway has previously been associated primarily with synapses, particularly glutamatergic synapses. We report here that NO caused a large increase in the levels of cGMP in a central white matter tract devoid of synapses, namely in the rat isolated optic nerve. Cyclic GMP immunohistochemistry indicated that this response was confined to the axons. Accordingly, nerves previously subjected to 1 h of oxygen/glucose deprivation, which leads to irreversible axonal damage, displayed an 80% reduction in their subsequent capacity to generate cGMP in response to NO and a corresponding reduction in the numbers of cGMP-immunostained axons. Protection of the axon cGMP response against this insult was achieved by omission of Ca2 + or Na + from the incubation medium, and by the pharmacological agents tetrodotoxin, lamotrigine, BW619C89 and BW1003C87, all of which protect axonal structure from oxygen/glucose deprivation-induced damage. The results suggest that the NO-cGMP pathway has a hitherto unsuspected function in the optic nerve. Additionally, the expression of NO-stimulated guanylyl cyclase in optic nerve axons provides a simple, sensitive and specific marker of their functional integrity that is likely to be valuable in investigating the mechanisms responsible for axon degeneration in ischaemia and other conditions.     

人NGF促进感觉神经生长和诱导SP释放实验研究

目的采用体外模型对比研究观察,评估人痛性椎间盘细胞外基质对小鼠背根神经节神经轴突生长所产生的促进作用和神经肽（sP）的诱导作用。方法通过获取椎间盘源性下腰痛患者的椎间盘组织（退变组）,以及正常人腰椎间盘组织（正常组）提取细胞基质,对小鼠背根神经节神经元（DRG）进行培养,通过形态学观察神经元轴突生长和酶联免疫吸附法（ELISA）检测SP含量。结果实验组神经生长因子（NGF）浓度显著高于正常组;实验组神经元轴突平均长度为（134．17±2．21）μm,明显高于正常组的（121．28±4．10）μm,二者相比有明显差异性（P〈0．05）。在退变人椎间盘组织培养液中SP物质比率明显高于正常组（P〈0．001）,二者具有显著性差异。结论人退变椎间盘细胞外基质中高表达的NGF能促进感觉神经轴突的生长以及诱导和疼痛相关神经肽的释放。     

Overexpression of nerve growth factor by murine smooth muscle cells: Role of the p75 neurotrophin receptor on sympathetic and sensory sprouting

Elevating levels of nerve growth factor (NGF) can have pronounced effects on the survival and maintenance of distinct populations of neurons. We have generated a line of transgenic mice in which NGF is expressed under the control of the smooth muscle α‐actin promoter. These transgenic mice have augmented levels of NGF protein in the descending colon and urinary bladder, so these tissues display increased densities of NGF‐sensitive sympathetic efferents and sensory afferents. Here we provide a thorough examination of sympathetic and sensory axonal densities in the descending colon and urinary bladder of NGF transgenic mice with and without the expression of the p75 neurotrophin receptor (p75NTR). In response to elevated NGF levels, sympathetic axons (immunostained for tyrosine hydroxylase) undergo robust collateral sprouting in the descending colon and urinary bladder of adult transgenic mice (i.e., those tissues having smooth muscle cells); this sprouting is not augmented in the absence of p75NTR expression. As for sensory axons (immunostained for calcitonin gene‐related peptide) in the urinary bladders of transgenic mice, fibers undergo sprouting that is further increased in the absence of p75NTR expression. Sympathetic axons are also seen invading the sensory ganglia of transgenic mice; these fibers form perineuronal plexi around a subpopulation of sensory somata. Our results reveal that elevated levels of NGF in target tissues stimulate sympathetic and sensory axonal sprouting and that an absence of p75NTR by sensory afferents (but not by sympathetic efferents) leads to a further increase of terminal arborization in certain NGF‐rich peripheral tissues. J. Comp. Neurol. 521:2621–2643, 2013. © 2013 Wiley Periodicals, Inc.     

Overexpression of nerve growth factor in epidermis disrupts the distribution and properties of sympathetic innervation in footpads

Sympathetic and sensory neurons form distinct axonal arborizations in several peripheral targets. The developmental mechanisms responsible for partitioning sympathetic and sensory axons between potential target tissues are poorly understood. We have used rodent footpads to study this process because three populations of peripheral axons innervate topographically segregated targets in the footpad; cholinergic sympathetic axons innervate sweat glands, noradrenergic sympathetic axons innervate blood vessels, and sensory axons form a plexus at the epidermal/dermal junction. To examine how nerve growth factor (NGF), a trophic and survival factor for sympathetic and some sensory neurons, may contribute to the generation of the patterned distribution of axons among targets, we studied transgenic mice (K14-NGF mice) in which NGF expression was significantly increased in the epidermis. Whereas the temporal sequence in which sensory and sympathetic fibers arrived in the footpad was not affected, the normal partitioning of axons between target tissues was disrupted. The two sympathetic targets in footpads, sweat glands, and blood vessels lacked substantial innervation and instead a dense plexus of catecholaminergic sympathetic fibers was found commingled with sensory fibers in the dermis. Those sympathetic fibers present in sweat glands expressed an abnormal dual catecholaminergic/cholinergic phenotype. Our findings indicate that overexpression of NGF in skin interferes with the segregation of sensory and sympathetic axonal arbors and suggests a role for target-derived NGF in the establishment of distinct axonal territories. Our data also suggest that by determining where axon arbors form, NGF can indirectly influence the phenotypic properties of sympathetic neurons. J. Comp. Neurol. 393:231–243, 1998. © 1998 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.