A Northwestern blotting approach for studying iron regulatory element-binding proteins

At least two proteins binding to iron regulatory elements (IRE) in mRNA are known, designated as iron regulatory proteins (IRP) 1 and 2. Their binding activity is widely studied by electrophoretic mobility shift assays (EMSA), which resolves one or two bands depending on the species. We used Northwestern blotting to resolve this EMSA complex into four components, and identified two other IRE-binding peptides present in HepG2 cell extracts. We designate these six peptide bands A to F on Northwestern blots, ranging in apparent molecular weight from 111 to 37 kDa. Band C is lost when cells are preloaded with iron or when leupeptin (but not several other protease inhibitors) is included in the extraction buffer. Band E is also lost with leupeptin but increases with iron loading. Binding of all bands is sensitive to iron in vitro. Two-dimensional electrophoresis reveals additional processing, especially indicating charge variants of band C. Northwestern bands A and B both react with an antibody to IRP-1 on parallel Western blots. We conclude that cellular processing can produce multiple IRE-binding species that may be involved in a more complex regulation of iron metabolism than generally appreciated. The Northwestern approach should facilitate studies of processing and binding requirements of proteins and peptides that recognize the IRE sequence. (Mol Cell Biochem 268: 67–74, 2005)     

β-Sheet-breaking peptides inhibit the fibrillation of human α-synuclein

α-Synuclein is the major components of the intracellular protein-aggregates, found in the dopaminergic neurons of Parkinson’s disease patients. Previously, we screened for α-synuclein substitution mutants that prevent fibril formation of both wild-type and Parkinson’s disease-linked α-synuclein variants. In the present study, we show that short synthetic peptides derived from these mutant sequences not only prevented α-synuclein fibrillation but also dissolved preformed α-synuclein aggregates in vitro. The hexapeptide PGVTAV, which was the shortest peptide that retained the ability to block α-synuclein fibrillation, may serve as a lead compound for the development of therapeutics for Parkinson’s disease.     

Neurodegeneration in mnd2 mutant mice is not prevented by parkin transgene

Parkinson’s disease (PD) is a common neurodegenerative disorder. The motor neuron degeneration 2 mutant (mnd2) mouse is considered to be an animal model of PD, and exhibits striatal neuron loss, severe muscle wasting, weight loss and death before 40 days of age. We found for the first time that parkin expression was decreased in the mnd2 mouse brain. Since parkin is a crucial protein for PD, the neurodegenerative disorder in mnd2 mice may be caused by parkin protein loss. We therefore examined whether compensation of parkin protein prevents neurodegenerative disorders in mnd2 mice by generating parkin-transgenic (parkin-Tg) mnd2 mice. However, both parkin-Tg mnd2 mice and mnd2 mice were smaller than wild type mice. In muscle strength and survival rate, parkin-Tg mnd2 mice showed similar values to mnd2 mice. Our data suggest that repression of parkin protein does not play a major role in neurodegeneration of mnd2 mice and administration of parkin protein does not rescue mnd2 mice.     

Aggregation-defective α-synuclein mutants inhibit the fibrillation of Parkinson’s disease-linked α-synuclein variants

α-Synuclein comprises the fibrillar core of Lewy bodies, which is one of the histologically defining lesions of Parkinson’s disease. Previously, we screened for α-synuclein substitution mutants that do not form fibrils. For preventative or therapeutic uses, it is essential to suppress the oligomerization/fibrillation of the wild-type and PD-linked α-synuclein proteins. Here we have examined the effects of fibrillation-retarded α-synuclein mutants on fibril formation by wild-type and PD-linked α-synuclein molecules. Six self-aggregation-defective α-synuclein mutants completely inhibit the fibrillation of both wild-type and Parkinson’s disease-linked α-synuclein variants. These results suggest future applications for gene therapy: the transplantation of a fibrillation-blocking mutant α-synuclein gene into individuals who carry an early-onset PD-associated α-synuclein allele. Short synthetic peptides derived from these mutant sequences may also serve as a lead compound for the development of therapeutics for Parkinson’s disease.     

SIRT2 interferes with autophagy-mediated degradation of protein aggregates in neuronal cells under proteasome inhibition

Abnormal protein aggregates have been suggested as a common pathogenesis of many neurodegenerative diseases. Two well-known protein degradation pathways are responsible for protein homeostasis by balancing protein biosynthesis and degradative processes: the ubiquitin–proteasome system (UPS) and autophagy-lysosomal system. UPS serves as the primary route for degradation of short-lived proteins, but large-size protein aggregates cannot be degraded by UPS. Autophagy is a unique cellular process that facilitates degradation of bulky protein aggregates by lysosome. Recent studies have demonstrated that autophagy plays a crucial role in the pathogenesis of neurodegenerative diseases characterized by abnormal protein accumulation, suggesting that regulation of autophagy may be a valuable therapeutic strategy for the treatment of various neurodegenerative diseases. Sirtuin-2 (SIRT2) is a class III histone deacetylase that is expressed abundantly in aging brain tissue. Here, we report that SIRT2 increases protein accumulation in murine cholinergic SN56 cells and human neuroblastoma SH-SY5Y cells under proteasome inhibition. Overexpression of SIRT2 inhibits lysosome-mediated autophagic turnover by interfering with aggresome formation and also makes cells more vulnerable to accumulated protein-mediated cytotoxicity by MG132 and amyloid beta. Moreover, MG132-induced accumulation of ubiquitinated proteins and p62 as well as cytotoxicity are attenuated in siRNA-mediated SIRT2-silencing cells. Taken together, these results suggest that regulation of SIRT2 could be a good therapeutic target for a range of neurodegenerative diseases by regulating autophagic flux.     

Identification of chemicals to inhibit the kinase activity of leucine-rich repeat kinase 2 (LRRK2), a Parkinson's disease-associated protein

Parkinson’s disease (PD) is a late-onset neurodegenerative disease which occurs at more than 1% in populations aging 65-years and over. Recently, leucine-rich repeat kinase 2 (LRRK2) has been identified as a causative gene for autosomal dominantly inherited familial PD cases. LRRK2 G2019S which is a prevalent mutant found in familial PD patients with LRRK2 mutations, exhibited kinase activity stronger than that of the wild type, suggesting the LRRK2 kinase inhibitor as a potential PD therapeutics. To develop such therapeutics, we initially screened a small chemical library and selected compound 1, whose IC₅₀ is about 13.2 μM. To develop better inhibitors, we tested five of the compound 1 derivatives and found a slightly better inhibitor, compound 4, whose IC₅₀ is 4.1 μM. The cell-based assay showed that these two chemicals inhibited oxidative stress-induced neurotoxicity caused by over-expression of a PD-specific LRRK2 mutant, G2019S. In addition, the structural analysis of compound 4 suggested hydrogen bond interactions between compound 4 and Ala 1950 residue in the backbone of the ATP binding pocket of LRRK2 kinas domain. Therefore, compound 4 may be a promising lead compound to further develop a PD therapeutics based on LRRK2 kinase inhibition.     

Evolutionary aspects of the synuclein super-family and sub-families based on large-scale phylogenetic and group-discrimination analysis

Over the last decade, many genetic studies have suggested that the synucleins, which are small, natively unfolded proteins, are closely related to Parkinson’s disease and cancer. Less is known about the molecular basis of this role. A comprehensive analysis of the evolutionary path of the synuclein protein family may reveal the relationship between evolutionarily conserved residues and protein function or structure. The phylogeny of 252 unique synuclein sequences from 73 organisms suggests that gamma-synuclein is the common ancestor of alpha- and beta-synuclein. Although all three sub-families remain highly conserved, especially at the N-terminal, nearly 15% of the residues in each sub family clearly diverged during evolution, providing crucial guidance for investigations of the different properties of the members of the superfamily. His50 is found to be an alpha-specific conserved residue (91%) and, based on mutagenesis, evolutionarily developed a secondary copper binding site in the alpha synuclein family. Surprisingly, this site is located between two well-known polymorphisms of alpha-synuclein, E46K and A53T, which are linked to early-onset Parkinson’s disease, suggesting that the mutation-induced impairment of copper binding could be a mechanism responsible for alpha-synuclein aggregation.     

Levetiracetam treatment ameliorates LRRK2 pathological mutant phenotype

Mutations in leucine‐rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease (PD). The LRRK2 physiological and pathological function is still debated. However, different experimental evidence based on LRRK2 cellular localization and LRRK2 protein interactors suggests that LRRK2 may be part and regulate a protein network modulating vesicle dynamics/trafficking. Interestingly, the synaptic vesicle protein SV2A is part of this protein complex. Importantly, SV2A is the binding site of the levetiracetam (LEV), a compound largely used in human therapy for epilepsy treatment. The binding of LEV to SV2A reduces the neuronal firing by the modulation of vesicle trafficking although by an unclear molecular mechanism. In this short communication, we have analysed the interaction between the LRRK2 and SV2A pathways by LEV treatment. Interestingly, LEV significantly counteracts the effect of LRRK2 G2019S pathological mutant expression in three different cellular experimental models. Our data strongly suggest that LEV treatment may have a neuroprotective effect on LRRK2 pathological mutant toxicity and that LEV repositioning could be a viable compound for PD treatment.     

Methylcobalamin promotes proliferation and migration and inhibits apoptosis of C2C12 cells via the Erk1/2 signaling pathway

Methylcobalamin (MeCbl) is a vitamin B12 analog that has some positive effects on peripheral nervous disorders. Although some previous studies revealed the effects of MeCbl on neurons, its effect on the muscle, which is the final target of motoneuron axons, remains to be elucidated. This study aimed to determine the effect of MeCbl on the muscle. We found that MeCbl promoted the proliferation and migration of C2C12 myoblasts in vitro and that these effects are mediated by the Erk1/2 signaling pathway without affecting the activity of the Akt signaling pathway. We also demonstrated that MeCbl inhibits C2C12 cell apoptosis during differentiation. Our results suggest that MeCbl has beneficial effects on the muscle in vitro. MeCbl administration may provide a novel therapeutic approach for muscle injury or degenerating muscle after denervation.     

The hexapeptide PGVTAV suppresses neurotoxicity of human α-synuclein aggregates

In Parkinson’s disease patients, α-synuclein is the major component of the intracellular protein aggregates found in dopaminergic neurons. Previously, short synthetic α-synuclein-derived peptides have been shown to not only prevent α-synuclein fibrillation but also dissolve preformed α-synuclein aggregates in vitro. The hexapeptide PGVTAV was the shortest peptide that retained the ability to block α-synuclein fibrillation. For preventative or therapeutic effectiveness, a treatment must suppress the neurotoxicity of α-synuclein aggregates and remain stable in plasma. The present study shows that specific peptides can protect neuronal cells from α-synuclein aggregation-induced cell death. The β-sheet-breaking hexapeptide PGVTAV remained intact in human plasma for longer than one day, suggesting that it may be a candidate for the development of therapeutics to treat Parkinson’s disease.     

Methamphetamine binds to α-synuclein and causes a conformational change which can be detected by nanopore analysis

α-Synuclein is an intrinsically disordered protein of 140 amino acids which is abundant in dopaminergic neurons. Misfolding and aggregation of α-synuclein leads to the formation of Lewy bodies inside the neurons which is the hallmark of Parkinson’s disease and related dementias. Here we show by nanopore analysis that the recreational drug, methamphetamine, binds to the N-terminus of α-synuclein and causes a conformational change which cannot be detected by circular dichroism spectroscopy. The results suggest a mechanism for the psychoactivity of methamphetamine as well as an increased incidence of Parkinson’s disease amongst users of the drug.     

Actein inhibits the Na+-K+-ATPase and enhances the growth inhibitory effect of digitoxin on human breast cancer cells

The Na⁺-K⁺-ATPase is a known target of cardiac glycosides such as digitoxin and ouabain. We determined that the enzyme also is a target of the structurally-related triterpene glycoside actein, present in the herb black cohosh. Actein’s inhibition of Na⁺-K⁺-ATPase activity was less potent than that of digitoxin, but actein potentiated digitoxin’s inhibitory effect on Na⁺-K⁺-ATPase activity and MDA-MB-453 breast cancer cell growth. We observed different degrees of signal amplification for the two compounds. Actein’s inhibitory effect on ATPase activity was amplified 2-fold for cell growth inhibition, whereas digitoxin’s signal was amplified 20-fold. Actein induced a biphasic response in proteins downstream of ATPase: low dose and short duration of treatment upregulated NF-κB promoter activity, p-ERK, p-Akt and cyclin D1 protein levels, whereas higher doses and longer exposure inhibited these activities. Actein and digitoxin may be a useful synergistic combination for cancer chemoprevention and/or therapy.     

Enhanced Histochemical Detection of Iron in Paraffin Sections of Mouse Central Nervous System Tissue: Application in the APP/PS1 Mouse Model of Alzheimer’s Disease

Histochemical methods of detecting iron in the rodent brain result mainly in the labeling of oligodendrocytes, but as all cells utilize iron, this observation suggests that much of the iron in the central nervous system goes undetected. Paraffin embedding of tissue is a standard procedure that is used to prepare sections for microscopic analysis. In the present study, we questioned whether we could modify the iron histochemical procedure to enable a greater detection of iron in paraffin sections. Indeed, various modifications led to the widespread labeling of iron in mouse brain tissue (for instance, labeling of neurons and neuropil). Sites of focal concentrations, such as cytoplasmic punctate or nucleolar staining, were also observed. The modified procedures were applied to paraffin sections of a mouse model (APP/PS1) of Alzheimer’s disease. Iron was revealed in the plaque core and rim. The plaque rim had a fibrillary or granular appearance, and it frequently contained iron-labeled cells. Further analysis indicated that the iron was tightly associated with the core of the plaque, but less so with the rim. In conclusion, modifications to the histochemical staining revealed new insights into the deposition of iron in the central nervous system. In theory, the approach should be transferrable to organs besides the brain and to other species, and the underlying principles should be incorporable into a variety of staining methods.     

Methylenetetrahydrofolate reductase C677T and A1298C polymorphisms and variations of homocysteine concentrations in patients with Behcet's disease

Background

Behcet's disease (BD) is a chronic, relapsing, multi-systemic inflammatory disorder of unknown causes. This disease is mainly characterized by mucocutaneous, ocular, vascular, and central nervous system manifestations. The aim of this study is to investigate the associations between C677T and A1298C polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene and the plasma homocysteine (Hcy), folate, and B12 levels in a relatively large cohort of Tunisian patients with BD.

Methods

The study included 142 patients with BD and 172 healthy controls. The C677T and A1298C polymorphisms were genotyped using PCR-RFLP. Serum Hcy level was determined using a fluorescence polarization immunoassay. Serum folate and vitamin B12 levels were measured by electrochemiluminescence immunoassay.

Results

Genotype and allele frequencies of the two studied MTHFR polymorphisms did not show any significant differences among BD patients compared to controls. Patient carriers of the 677TT variant and the 677 T allele displayed significantly higher Hcy concentration. Moreover, no significant association was found between neither A1298C polymorphism nor the C allele and Hcy, folate, and B12 levels. In multivariate analyses, we reported that 677 T allele, male gender, and creatinine level were independent risk factors for hyperhomocysteinemia (HHC).

Conclusions

In the present study, we report the absence of any significant differences between genotype and allele frequencies for both studied polymorphisms among BD patients compared to healthy controls. Besides, we showed that the T allele of MTHFR C677T polymorphism influenced the Hcy level which is an independent risk factor for HHC in Tunisian BD patients.     

The epigenetic effects of amyloid-β1-40 on global DNA and neprilysin genes in murine cerebral endothelial cells

Amyloid-β (Aβ) is the core component of senile plaques, which are the pathological markers for Alzheimer’s disease and cerebral amyloid angiopathy. DNA methylation/demethylation plays a crucial role in gene regulation and could also be responsible for presentation of senescence. Oxidative stress, which may be induced by Aβ, is thought to be an important contributor of DNA hyper-methylation; however, contradicting this is the fact that global DNA hypo-methylation has been found in aging brains. It therefore remains largely unknown as to whether Aβ does in fact cause DNA methylation/demethylation. Neprilysin (NEP) is one of the enzymes responsible for Aβ degradation, with its expression decreasing in both Alzheimer and aging brains. Using high-performance liquid chromatography (HPLC), we explore whether Aβ is responsible for alteration of the global DNA methylation status on a murine cerebral endothelial cells model, and also use methylation-specific PCR (MSPCR) to examine whether DNA methylation status is altered on the NEP promoter region. We find that Aβ reduces global DNA methylation whilst increasing NEP DNA methylation and further suppressing the NEP expression in mRNA and protein levels. Our results support that Aβ induces epigenetic effects, implying that DNA methylation may be part of a vicious cycle involving the reduction in NEP expression along with a resultant increase in Aβ accumulation, and that Aβ may induce global DNA hypo-methylation.     

Loss of PINK1 function decreases PP2A activity and promotes autophagy in dopaminergic cells and a murine model

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder. Mutations in PTEN-induced kinase 1 (PINK1) are a frequent cause of recessive PD. Autophagy, a pathway for clearance of protein aggregates or impaired organelles, is a newly identified mechanism for PD development. However, it is still unclear what molecules regulate autophagy in PINK1-silenced cells. Here we report that autophagosome formation is promoted in the early phase in response to PINK1 gene silencing by lentivirus transfer vectors expressed in mouse striatum. Reduced PP2A activity and increased phosphorylation of PP2A at Y307 (inactive form of PP2A) were observed in PINK1-knockdown dopaminergic cells and striatum tissues. Treatment with C2-ceramide (an agonist of PP2A) reduced autophagy levels in PINK1-silenced MN9D cells, which suggests that PP2A plays an important role in the PINK1-knockdown-induced autophagic pathway. Furthermore, phosphorylation of Bcl-2 at S87 increased in PINK1-silenced cells and was negatively regulated by additional treatment with C2-ceramide, which indicates that Bcl-2 may be downstream of PP2A inactivation in response to PINK1 dysfunction. Immunoprecipitation also revealed dissociation of the Bcl-2/Beclin1 complex in PINK1-silenced cells, which was reversed by additional treatment with C2-ceramide, and correlated with changes in level of autophagy and S87 phosphorylation of Bcl-2. Finally, Western blots for cleaved caspase-9 and flow cytometry results for active caspase-3 revealed that PP2A inactivation is involved in the protective effect of autophagy on PINK1-silenced cells. Our findings show that downregulation of PP2A activity in PINK1-silenced cells promotes the protective effect of autophagy through phosphorylation of Bcl-2 at S87 and blockage of the caspase pathway. These results may have implications for identifying the mechanism of PD.     

Multitargeted drugs discovery: balancing anti-amyloid and anticholinesterase capacity in a single chemical entity

Memoquin (1) is a lead compound multitargeted against Alzheimer’s disease (AD). It is an AChE inhibitor, free-radical scavenger, and inhibitor of amyloid-β (Aβ) aggregation. A new series of 1 derivatives was designed and synthesized by linking its 2,5-diamino-benzoquinone core with motifs that are present in the structure of known amyloid binding agents like curcumin, the benzofuran derivative SKF64346, or the benzothiazole bearing compounds KHG21834 and BTA-1. The weaker AChE inhibitory potencies and the concomitant nearly equipotent anti-amyloid activities of the new compounds with respect to 1 resulted in a more balanced biological profile against both targets. Selected compounds turned out to be effective Aβ aggregation inhibitors in a cell-based assay. By properly combining two or more distinct pharmacological properties in a molecule, we can achieve greater effectiveness compared to single-targeted drugs for investigating AD.     

Parkin:一种作用极其广泛的E3泛素连接酶

Parkin,又名PARK2,自发现初始便与帕金森病(Parkinson's disease,PD)密切相关.Parkin被认为是一种神经保护性基因.随着对其结构的深入了解,揭开了作为E3泛素连接酶的面纱.Parkin参与调控细胞周期、线粒体动态平衡和能量代谢等细胞进程,并与许多疾病息息相关,甚至在同一通路中发挥完全相...