Neurotrophic factors stabilize microtubules and protect against rotenone toxicity on dopaminergic neurons

Parkinson disease is characterized by the selective degeneration of dopaminergic (DA) neurons in substantia nigra. Long term epidemiological studies have implicated exposure to agricultural pesticides as a significant risk factor. Systemic administration of rotenone, a widely used pesticide, causes selective degeneration of nigral DA neurons and Parkinson disease-like symptoms in rats. Our previous study has shown that the microtubule depolymerizing activity of rotenone plays a critical role in its selective toxicity on DA neurons. Rotenone toxicity is mimicked by the microtubule-depolymerizing drug colchicine and attenuated by the microtubule-stabilizing agent taxol. Here we show that nerve growth factor (NGF) significantly reduced rotenone toxicity on TH(+) neurons in midbrain neuronal cultures. The protective effect of NGF was completely abolished by inhibiting the microtubule-associated protein kinase kinase (MEK) and partially reversed by blocking phosphatidylinositol 3-kinase. In addition, NGF decreased colchicine toxicity on TH(+) neurons in a manner dependent on MEK but not phosphatidylinositol 3-kinase. The protective effect of NGF against rotenone toxicity was occluded by the microtubule-stabilizing drug taxol. In a MEK-dependent manner, NGF significantly attenuated rotenone- or colchicine-induced microtubule depolymerization and ensuing accumulation of vesicles in the soma and elevation in protein carbonyls. Moreover, other neurotrophic factors such as brain-derived neurotrophic factor and glia cell line-derived neurotrophic factor also reduced rotenone- or colchicine-induced microtubule depolymerization and death of TH(+) through a MEK-dependent mechanism. Thus, our results suggest that neurotrophic factors activate the microtubule-associated protein kinase pathway to stabilize microtubules, and this action significantly attenuates rotenone toxicity on dopaminergic neurons.     

NSAIDs protect dopaminergic neurons against 6‐OHDA and MPP+ toxicity

Endogenous and environmental neurotoxins are among the suspected causes of the loss of dopaminergic (DA) neurons in Parkinson's disease (PD). Non‐steroidal anti‐inflammatory drugs (NSAIDs) reduce inflammation by inhibiting cyclooxygenase (COX)‐dependent synthesis of prostaglandins (PG) from arachidonic acid. NSAIDs decrease the incidence of Alzheimer's disease, but little is known about their potential benefit for PD. Therefore, we examined whether NSAIDs could protect DA neurons from neurotoxic insults. NSAIDs can protect DA neurons against excitotoxicity (Casper et al. 2000), and against 6‐hydroxydopamine (6‐OHDA) toxicity (Carrasco et al. 2001). Here, we compared in primary mesencephalic/DA neuron cultures the effect of NSAIDs on the toxicity of 1‐methyl‐phenylpyridinium (MPP⁺) or 6‐OHDA. 6‐OHDA significantly (*p < 0.0001) increased PG production, whereas MPP⁺ did not (p < 0.05). We then compared the competitive/unspecific COX inhibitors ibuprofen and naproxen and the noncompetitive/unspecific inhibitor acetylsalicylic acid (ASA, aspirin) for their ability to protect DA neurons against either 6‐OHDA or MPP⁺ toxicity. Interestingly, all three nonselective COX inhibitors protected DA neurons in cultures against both 6‐OHDA and MPP⁺ (p < 0.05), despite the difference in PG induction by 6‐OHDA vs. MPP⁺. The selective COX‐2 inhibitor NS398 did protect DA neurons against 5 μm MPP⁺ (*p < 0.05), but failed to protect DA neurons against 5 μm 6‐OHDA (p < 0.05). Our results suggest that COX‐inhibitors may have neuroprotective benefits unrelated to inhibition of PG synthesis, and that 6‐OHDA and MPP⁺ have partially overlapping mechanisms of neurodegeneration possibly involving COX activity. Acknowledgement: Supported, in part, by the International Federation for Parkinson's disease, NY, NY.     

Chlortetracycline and demeclocycline inhibit calpains and protect mouse neurons against glutamate toxicity and cerebral ischemia

Minocycline is a potent neuroprotective tetracycline in animal models of cerebral ischemia. We examined the protective properties of chlortetracycline (CTC) and demeclocycline (DMC) and showed that these two tetracyclines were also potent neuroprotective against glutamate-induced neuronal death in vitro and cerebral ischemia in vivo. However, CTC and DMC appeared to confer neuroprotection through a unique mechanism compared with minocycline. Rather than inhibiting microglial activation and caspase, CTC and DMC suppressed calpain activities. In addition, CTC and DMC only weakly antagonized N-methyl-D-aspartate (NMDA) receptor activities causing 16 and 14%, respectively, inhibition of NMDA-induced whole cell currents and partially blocked NMDA-induced Ca2+ influx, commonly regarded as the major trigger of neuronal death. In vitro and in vivo experiments demonstrated that the two compounds selectively inhibited the activities of calpain I and II activated following glutamate treatment and cerebral ischemia. In contrast, minocycline did not significantly inhibit calpain activity. Taken together, these results suggested that CTC and DMC provide neuroprotection through suppression of a rise in intracellular Ca2+ and inhibition of calpains.     

Small heat shock proteins protect against alpha-synuclein-induced toxicity and aggregation

Protein misfolding and inclusion formation are common events in neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD) or Huntington's disease (HD). Alpha-synuclein (aSyn) is the main protein component of inclusions called Lewy bodies (LB) which are pathognomic of PD, Dementia with Lewy bodies (DLB), and other diseases collectively known as LB diseases. Heat shock proteins (HSPs) are one class of the cellular quality control system that mediate protein folding, remodeling, and even disaggregation. Here, we investigated the role of the small heat shock proteins Hsp27 and alphaB-crystallin, in LB diseases. We demonstrate, via quantitative PCR, that Hsp27 messenger RNA levels are approximately 2-3-fold higher in DLB cases compared to control. We also show a corresponding increase in Hsp27 protein levels. Furthermore, we found that Hsp27 reduces aSyn-induced toxicity by approximately 80% in a culture model while alphaB-crystallin reduces toxicity by approximately 20%. In addition, intracellular inclusions were immunopositive for endogenous Hsp27, and overexpression of this protein reduced aSyn aggregation in a cell culture model.     

Cell divisions and mammalian aging: integrative biology insights from genes that regulate longevity

Despite recent progress in the identification of genes that regulate longevity, aging remains a mysterious process. One influential hypothesis is the idea that the potential for cell division and replacement are important factors in aging. In this work, we review and discuss this perspective in the context of interventions in mammals that appear to accelerate or retard aging. Rather than focus on molecular mechanisms, we interpret results from an integrative biology perspective of how gene products affect cellular functions, which in turn impact on tissues and organisms. We review evidence suggesting that mutations that give rise to features resembling premature aging tend to be associated with cellular phenotypes such as increased apoptosis or premature replicative senescence. In contrast, many interventions in mice that extend lifespan and might delay aging, including caloric restriction, tend to either hinder apoptosis or result in smaller animals and thus may be the product of fewer cell divisions. Therefore, it appears plausible that changes in the number of times that cells, and particularly stem cells, divide during an organism's lifespan influence longevity and aging. We discuss possible mechanisms related to this hypothesis and propose experimental paradigms.     

Insight into drug resistance mechanisms and discovery of potential inhibitors against wild-type and L1196M mutant ALK from FDA-approved drugs

Anaplastic lymphoma kinase (ALK) plays a crucial role in multiple malignant cancers. It is known as a well-established target for the treatment of ALK-dependent cancers. Even though substantial efforts have been made to develop ALK inhibitors, only crizotinib, ceritinib, and alectinib had been approved by the U.S. Food and Drug Administration for patients with ALK-positive non-small cell lung cancer (NSCLC). The secondary mutations with drug-resistance bring up difficulties to develop effective drugs for ALK-positive cancers. To give a comprehensive understanding of molecular mechanism underlying inhibitor response to ALK tyrosine kinase mutations, we established an accurate assessment for the extensive profile of drug against ALK mutations by means of computational approaches. The molecular mechanics-generalized Born surface area (MM-GBSA) method based on molecular dynamics (MD) simulation was carried out to calculate relative binding free energies for receptor-drug systems. In addition, the structure-based virtual screening was utilized to screen effective inhibitors targeting wild-type ALK and the gatekeeper mutation L1196M from 3180 approved drugs. Finally, the mechanism of drug resistance was discussed, several novel potential wild-type and L1196M mutant ALK inhibitors were successfully identified.     

Antioxidants and antioxidant enzymes protect against pulmonary oxygen toxicity in the rabbit

Two major lines of defense exist against oxidant lung injury: tissue antioxidants and antioxidant enzymes. We studied pretreatment with the antioxidants, vitamin E and butylated hydroxyanisole (BHA), and the antioxidant enzymes, superoxide dismutase (SOD) and catalase, in rabbits exposed to 100% O2 for 48 h. BHA (200 mg/kg ip) or vitamin E (50-100 mg/kg po) were given for 2 or 3 days, respectively, before O2 exposure. Combined therapy with polyethylene glycol- (PEG) conjugated SOD (12 mg/kg) and catalase (200,000 U/kg) was given intraperitoneally 1 h before and 24 h after beginning 100% O2. Hyperoxia significantly increased the pulmonary content of malondialdehyde, indicating enhanced lipid peroxidation. One hundred percent O2 also increased lung weight gain and alveolar-capillary permeability to aerosolized 99mTc-labeled diethylenetriaminepentaacetate (99mTc-DTPA, 500 mol wt) and fluorescein isothiocyanate-labeled dextran (7,000 mol wt). Pretreatment with vitamin E, BHA, or the combination of PEG-SOD and PEG-catalase prevented the increase in malondialdehyde, lung weight gain, and alveolar-capillary permeability caused by hyperoxia. These results indicate that augmenting either tissue antioxidants or antioxidant enzymes can prevent the pulmonary injury caused by 48 h of 100% O2 in rabbits.     

Cholesterol-depleting statin drugs protect postmitotically differentiated human neurons against ethanol- and human immunodeficiency virus type 1-induced oxidative stress in vitro

The majority of human immunodeficiency virus type 1 (HIV-1)-infected individuals are either alcoholics or prone to alcoholism. Upon ingestion, alcohol is easily distributed into the various compartments of the body, particularly the brain, by crossing through the blood-brain barrier. Both HIV-1 and alcohol induce oxidative stress, which is considered a precursor for cytotoxic responses. Several reports have suggested that statins exert antioxidant as well as anti-inflammatory pleiotropic effects, besides their inherent cholesterol-depleting potentials. In our studies, postmitotically differentiated neurons were cocultured with HIV-1-infected monocytes, T cells, or their cellular supernatants in the presence of physiological concentrations of alcohol for 72 h. Parallel cultures were pretreated with statins (atorvastatin and simvastatin) with the appropriate controls, i.e., postmitotically differentiated neurons cocultured with uninfected cells and similar cultures treated with alcohol. The oxidative stress responses in the presence/absence of alcohol in these cultures were determined by the production of the well-characterized oxidative stress markers, 8-isoprostane-F2-alpha, total nitrates as an indicator for various isoforms of nitric oxide synthase activity, and heat shock protein 70 (Hsp70). An in vitro culture of postmitotically differentiated neurons with HIV-1-infected monocytes or T cells as well as supernatants from these cells enhanced the release of 8-isoprostane-F2-alpha in the conditioned medium six- to sevenfold (monocytes) and four- to fivefold (T cells). It was also observed that coculturing of HIV-1-infected primary monocytes over a time period of 72 h significantly elevated the release of Hsp70 compared with that of uninfected controls. Cellular supernatants of HIV-1-infected monocytes or T cells slightly increased Hsp70 levels compared to neurons cultured with uninfected monocytes or T-cell supernatants (controls). Ethanol (EtOH) presence further elevated Hsp70 in both infected and uninfected cultures. The amount of total nitrates was significantly elevated in the coculture system when both infected cells and EtOH were present. Surprisingly, pretreatment of postmitotic neurons with clinically available inhibitors of HMG-coenzyme A reductase (statins) inhibited HIV-1-induced release of stress/toxicity-associated parameters, i.e., Hsp70, isoprostanes, and total nitrates from HIV-1-infected cells. The results of this study provide new insights into HIV-1 neuropathogenesis aimed at the development of future HIV-1 therapeutics to eradicate viral reservoirs from the brain.     

Vulnerability to glutamate toxicity of dopaminergic neurons is dependent on endogenous dopamine and MAPK activation

Dopaminergic neurons are more vulnerable than other types of neurons in cases of Parkinson disease and ischemic brain disease. An increasing amount of evidence suggests that endogenous dopamine plays a role in the vulnerability of dopaminergic neurons. Although glutamate toxicity contributes to the pathogenesis of these disorders, the sensitivity of dopaminergic neurons to glutamate toxicity has not been clarified. In this study, we demonstrated that dopaminergic neurons were preferentially affected by glutamate toxicity in rat mesencephalic cultures. Glutamate toxicity in dopaminergic neurons was blocked by inhibiting extracellular signal-regulated kinase (ERK), c- jun N-terminal kinase, and p38 MAPK. Furthermore, depletion of dopamine by α-methyl- dl - p -tyrosine methyl ester (α-MT), an inhibitor of tyrosine hydroxylase (TH), protected dopaminergic neurons from the neurotoxicity. Exposure to glutamate facilitated phosphoryration of TH at Ser31 by ERK, which contributes to the increased TH activity. Inhibition of ERK had no additive effect on the protection offered by α-MT, whereas α-MT and c- jun N-terminal kinase or p38 MAPK inhibitors had additive effects and yielded full protection. These data suggest that endogenous dopamine is responsible for the vulnerability to glutamate toxicity of dopaminergic neurons and one of the mechanisms may be an enhancement of dopamine synthesis mediated by ERK.     

Activity of substituted thiophene sulfonamides against malarial and mammalian cyclin dependent protein kinases

Cyclin dependent protein kinases (CDKs) are pursued as drug targets for several eukaryotic pathogens. In this study, we identified thiophene and benzene sulfonamides as potent inhibitors of Pfmrk, a Plasmodium falciparum CDK with sequence homology to human CDK7. Several of the compounds demonstrated inhibitor selectivity for CDK7 over CDK1, CDK2, and CDK6. The compounds are moderate antimalarial agents against drug resistant parasites and possess encouraging in vitro therapeutic indices as determined against human cell lines. One particular sub-class of compounds, bromohydrosulfonylacetamides, was specific for Pfmrk with IC₅₀ values in the sub-micromolar range. These compounds represent the most potent Pfmrk inhibitors reported and provide support for further characterization and derivation as potential antimalarial agents.     

Choline derivatives and sodium fluoride protect acetylcholinesterase against irreversible inhibition and aging by DFP and paraoxon

A light addressable potentiometric sensor was used to measure acetylcholinesterase (AChE) activity in order to evaluate the protective effects of quaternary compounds and NaF against enzyme phosphorylation and aging by two organophosphates. The use of the immobilized AChE made possible the quick removal of reagents (i.e., organophosphate, 2-pralidoxime, and protectant), thereby permitting accurate determination of AChE activity before and after phosphorylation and aging. Paraoxon was 15-fold more potent in inhibiting AChE than DFP, while the percent aging following phosphorylation by diiso-propylfluorophosphate (DFP) was much higher. Sodium fluoride (NaF), the most effective protectant against phosphorylation and aging, and the quaternary ammonium compounds reduced significantly AChE inhibition by DFP and paraoxon, to similar degrees. Even though the percent AChE activity that was lost to aging was reduced by these agents, aging as a percent of phosphorylated AChE was not reduced. Thus, their major effect was in reducing the percent AChE phosphorylation, which consequently resulted in reduction of total aged AChE. The finding that quaternary ammonium compounds protect against phosphorylation is consonant with the proposed presence of the active site of AChE in an aromatic gorge.     

Inhibitors of catalase-amyloid interactions protect cells from beta-amyloid-induced oxidative stress and toxicity

Compelling evidence shows a strong correlation between accumulation of neurotoxic β-amyloid (Aβ) peptides and oxidative stress in the brains of patients afflicted with Alzheimer disease (AD). One hypothesis for this correlation involves the direct and harmful interaction of aggregated Aβ peptides with enzymes responsible for maintaining normal, cellular levels of reactive oxygen species (ROS). Identification of specific, destructive interactions of Aβ peptides with cellular anti-oxidant enzymes would represent an important step toward understanding the pathogenicity of Aβ peptides in AD. This report demonstrates that exposure of human neuroblastoma cells to cytotoxic preparations of aggregated Aβ peptides results in significant intracellular co-localization of Aβ with catalase, an anti-oxidant enzyme responsible for catalyzing the degradation of the ROS intermediate hydrogen peroxide (H(2)O(2)). These catalase-Aβ interactions deactivate catalase, resulting in increased cellular levels of H(2)O(2). Furthermore, small molecule inhibitors of catalase-amyloid interactions protect the hydrogen peroxide-degrading activity of catalase in Aβ-rich environments, leading to reduction of the co-localization of catalase and Aβ in cells, inhibition of Aβ-induced increases in cellular levels of H(2)O(2), and reduction of the toxicity of Aβ peptides. These studies, thus, provide evidence for the important role of intracellular catalase-amyloid interactions in Aβ-induced oxidative stress and propose a novel molecular strategy to inhibit such harmful interactions in AD.     

Novel histone-like DNA-binding proteins in the nucleoid from the acidothermophillic archaebacterium Sulfolobus acidocaldarius that protect DNA against thermal denaturation

DNA of acidothermophilic archaebacterium Sulfolobus acidocaldarius has a base composition of about 40 mol% G + C content. A low intracellular salt concentration has been inferred for this organism. These features and the high optimal temperature of growth (75°C) would have a destabilising effect on the helical structure of the intracellular DNA. Hence, the nucleoid of this organism has been isolated in order to analyse its proteins composition and to identify any protein factors responsible for stabilisation of the organism's DNA at its growth temperature. The acid-soluble fraction of the nucleoid contains four low-molecular-weight basic proteins. The four proteins have been purified to homogeneity and antibodies to these proteins have been raised in rabbits. Immunodiffusion results suggest that the proteins are antigenically distinct. Three proteins (A, C and C′) stabilise different double-stranded DNA during thermal denaturation and increase T_m of DNA by about 25 C°. These proteins are referred to as helix-stabilising nucleoid proteins (HSNP). Protein B (referred to a DNA-binding nucleoid protein, DBNP-B) does not show helix-stabilising effect. None of the four proteins stabilises double-stranded RNA. The four proteins bind to native and denatured DNA to different extents as measured by DNA-cellulose chromatography and [³H]DNA binding by filtration. We suggest, based on the DNA binding, histone-like and helix-stabilising properties, that the intracellular function of these proteins is to prevent strand separation of DNA at the optimal temperature of growth (75°C).     

Neuromelanin can protect against iron-mediated oxidative damage in system modeling iron overload of brain aging and Parkinson's disease

In Parkinson's disease (PD), dopamine neurons containing neuromelanin selectively degenerate. Neuromelanin binds iron and accumulates in aging. Iron accumulates in reactive form during aging, PD, and is involved in neurodegeneration. It is not clear how the interaction of neuromelanin and iron can be protective or toxic by modulating redox processes. Here, we investigated the interaction of neuromelanin from human substantia nigra with iron in the presence of ascorbic acid, dopamine, and hydrogen peroxide. We observed that neuromelanin blocks hydroxyl radical production by Fenton's reaction, in a dose-dependent manner. Neuromelanin also inhibited the iron-mediated oxidation of ascorbic acid, thus sparing this major antioxidant molecule in brain. The protective effect of neuromelanin on ascorbate oxidation occurs even in conditions of iron overload into neuromelanin. The blockade of iron into a stable iron–neuromelanin complex prevents dopamine oxidation, inhibiting the formation of neurotoxic dopamine quinones. The above processes occur intraneuronally in aging and PD, thus showing that neuromelanin is neuroprotective. The iron–neuromelanin complex is completely decomposed by hydrogen peroxide and its degradation rate increases with the amount of iron bound to neuromelanin. This occurs in PD when extraneuronal iron–neuromelanin is phagocytosed by microglia and iron–neuromelanin degradation releases reactive/toxic iron.     

Evidence for familial factors that protect against dementia and outweigh the effect of increasing age.

A positive family history is associated with increased risk for dementia. It is not known whether a negative family history with long-lived relatives predicts a reduced risk for dementia. We studied the survival rate and the occurrence of dementia in 232 parents and siblings of 43 optimally healthy individuals > or = 84 years of age and compared them with 233 parents and siblings of 51 random controls and 499 parents and siblings of 88 Alzheimer disease (AD) patients. Prevalence of dementia after age 60 years was .031 for the relatives of healthy elderly, .066 for the relatives of random controls, and .217 for the relatives of AD patients. The cumulative incidence of dementia by age 85 years was estimated as .041 (+/- .019) for the relatives of healthy elderly individuals, .102 (+/- .038) for the relatives of random controls, and .360 (+/- .037) for the relatives of AD patients. Hazard-ratio estimates suggest that the risk of dementia for the relatives of healthy elderly is 3 times lower than the risk for the relatives of random controls (P < .03) and is 11 times lower than the risk for the relatives of AD patients (P < .00005). An analysis of age at death indicated that the relatives of healthy elderly and the relatives of AD patients had a longer life span than did the relatives of random controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of skeletal muscle aging: insights from Drosophila and mammalian models

A characteristic feature of aged humans and other mammals is the debilitating, progressive loss of skeletal muscle function and mass that is known as sarcopenia. Age-related muscle dysfunction occurs to an even greater extent during the relatively short lifespan of the fruit fly Drosophila melanogaster. Studies in model organisms indicate that sarcopenia is driven by a combination of muscle tissue extrinsic and intrinsic factors, and that it fundamentally differs from the rapid atrophy of muscles observed following disuse and fasting. Extrinsic changes in innervation, stem cell function and endocrine regulation of muscle homeostasis contribute to muscle aging. In addition, organelle dysfunction and compromised protein homeostasis are among the primary intrinsic causes. Some of these age-related changes can in turn contribute to the induction of compensatory stress responses that have a protective role during muscle aging. In this Review, we outline how studies in Drosophila and mammalian model organisms can each provide distinct advantages to facilitate the understanding of this complex multifactorial condition and how they can be used to identify suitable therapies.