Brain banks: benefits,limitations and cautions concerning the use of post-mortem brain tissue for molecular studies

Brain banks are facilities providing an interface between generous donation of nervous tissues and research laboratories devoted to increase our understanding of the diseases of the nervous system, discover new diagnostic targets, and develop new strategies. Considering this crucial role, it is important to learn about the suitabilities, limitations and proper handling of individual brain samples for particular studies. Several factors may interfere with preservation of DNA, RNA, proteins and lipids, and, therefore, special care must be taken first to detect sub-optimally preserved tissues and second to provide adequate material for each specific purpose. Basic aspects related with DNA, RNA and protein preservation include agonal state, post-mortem delay, temperature of storage and procedures of tissue preservation. Examination of DNA and RNA preservation is best done by using bioanalyzer technologies instead of less sensitive methods such as agarose gels. Adequate RNA preservation is mandatory in RNA microarray studies and adequate controls are necessary for proper PCR validation. Like for RNA, the preservation of proteins is not homogeneous since some molecules are more vulnerable than others. This aspect is crucial in the study of proteins including expression levels and possible post-translational modifications. Similarly, the reliability of functional and enzymatic studies in human post-mortem brain largely depends on protein preservation. Much less is known about other aspects, such as the effects of putative deleterious factors on epigenetic events such as methylation of CpGs in gene promoters, nucleosome preservation, histone modifications, and conservation of microRNA species. Most brains are appropriate for morphological approaches but not all brains are useful for certain biochemical and molecular studies.     

Issues of banking breast cancer cells to generate mammospheres

Identification of breast cancer stem cells, within the context of the tumor tissue, requires an efficient and standardized method to preserve the functional features of living cells. Although isolating cancer stem cells can be laborious and time-consuming, minimal processing may be advantageous for the banking of specimens from which cultures are not immediately needed. Homogenization of banking procedures will result in a reliable network of biorepositories for cooperative studies and several research groups are focusing on the issues of tissue banks for translational medicine. Most tissue banks collect and freeze unprocessed cancer specimens, which cannot therefore be used to generate viable cells. We discuss the principal issues of biobanking breast cancer living cells and protocols for mammospheres formation from single cell suspension of tumor cells.     

多巴胺能神经元线粒体的氧化损伤与帕金森病

帕金森病(Parkinson’s disease,PD)的一个主要病理特征就是中脑黑质多巴胺能神经元的丧失,目前研究认为该病理变化与多种因素有关,包括蛋白质异常积聚、泛素蛋白酶体系统功能异常、神经炎症、线粒体损伤和氧化应激。在帕金森病人和动物模型中,中脑黑质有着明显的氧化改变。帕金森病的遗传和环境因素均会作用于线粒体,尤其对线粒体呼吸链复合体I有着抑制作用,造成线粒体损伤,产生活性氧(ROS)。活性氧的大量产生造成脂类、蛋白质和DNA的氧化,从而加剧多巴胺能神经元的线粒体和细胞损伤。多巴胺代谢过程中会产生活性氧,该自身代谢特点决定了多巴胺能神经元存在有较高的氧化应激,易受环境因素的影响。因而,线粒体的氧化损伤在帕金森病病理发生中起着重要作用。     

Shotguns in the front line: phosphoproteomics in plants

The emergence of 'shotgun proteomics' has paved the way for high-throughput proteome analysis, by which thousands of proteins can be identified simultaneously from complex samples. Although the shotgun approach has the potential to monitor many different post-translational modifications, further technological development is needed to enrich each post-translational 'modificome'. Large-scale in vivo phosphorylation site mapping, so-called shotgun phosphoproteomics, has become feasible in various organisms, including plants, owing to recent technological breakthroughs. Shotgun phosphoproteomics is not a mature technology, but progress has been rapid. In this review, we highlight the scope and limitations of current methods, and some key technological issues in this field.     

Multiple sclerosis: an important role for post-translational modifications of myelin basic protein in pathogenesis

Myelin basic protein (MBP) represents a candidate autoantigen in multiple sclerosis (MS). We isolated MBP from normal and MS human white matter and purified six components (charge isomers) to compare the post-translational modifications on each. The sites and extent of methylation, deimination, and phosphorylation were documented for all tryptic peptides by mass spectrometry. We found that mono and dimethylated arginine 107 was increased in MS samples; deimination of arginine occurred at a number of sites and was elevated in MS; phosphorylation was observed in 10 peptides in normal samples but was greatly reduced or absent in most peptides from MS samples. Data obtained with MBP isolated from fresh brain obtained from a spontaneously demyelinating mouse model supported the view that the changes observed in human brain were probably related to pathogenesis of demyelination, i.e. we found decreased phosphorylation and decreased amounts of glycogen synthesis kinase in brain homogenates using specific antibodies. This study represents the first to define post-translational modifications in demyelinating disease and suggest an important role in pathogenesis.     

A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein

Background

Alpha-synuclein (asyn) has been shown to play an important role in the neuropathology of Parkinson’s disease (PD). In the diseased brain, classic intraneuronal inclusions called Lewy bodies contain abnormal formations of asyn protein which is mostly phosphorylated at serine 129 (pS129 asyn). This suggests that post-translational modifications may play a role in the pathogenic process. To date, several uniplex assays have been developed in order to quantify asyn not only in the brain but also in cerebrospinal fluid and blood samples in order to correlate asyn levels to disease severity and progression. Notably, only four assays have been established to measure pS129 asyn specifically and none provide simultaneous readout of the total and pS129 species. Therefore, we developed a sensitive high-throughput duplex assay quantifying total and pS129 human asyn (h-asyn) in the same well hence improving accuracy as well as saving time, consumables and samples.

Results

Using our newly established duplex assay we measured total and pS129 h-asyn in vitro showing that polo-like kinase 2 (PLK2) can phosphorylate asyn up to 41 % in HEK293 cells and in vivo the same kinase phosphorylated h-asyn up to 17 % in rat ventral midbrain neurons. Interestingly, no increase in phosphorylation was observed when PLK2 and h-asyn were co-expressed in rat striatal neurons. Furthermore, using this assay we investigated h-asyn levels in brain tissue samples from patients with PD as well as PD dementia and found significant differences in pS129 h-asyn levels not only between disease tissue and healthy control samples but also between the two distinct disease states especially in hippocampal tissue samples.

Conclusions

These results demonstrate that our duplex assay for simultaneous quantification is a useful tool to study h-asyn phosphorylation events in biospecimens and will be helpful in studies investigating the precise causative link between post-translational modification of h-asyn and PD pathology.

     

Prolyl-isomerase Pin1 accumulates in lewy bodies of parkinson disease and facilitates formation of alpha-synuclein inclusions

Parkinson disease (PD) is a relatively common neurodegenerative disorder that is characterized by the loss of dopaminergic neurons and by the formation of Lewy bodies (LBs), which are cytoplasmic inclusions containing aggregates of alpha-synuclein. Although certain post-translational modifications of alpha-synuclein and its related proteins are implicated in the genesis of LBs, the specific molecular mechanisms that both regulate these processes and initiate subsequent inclusion body formation are not yet well understood. We demonstrate in our current study, however, that the prolyl-isomerase Pin1 localizes to the LBs in PD brain tissue and thereby enhances the formation of alpha-synuclein immunoreactive inclusions. Immunohistochemical analysis of brain tissue from PD patients revealed that Pin1 localizes to 50-60% of the LBs that show an intense halo pattern resembling that of alpha-synuclein. By utilizing a cellular model of alpha-synuclein aggregation, we also demonstrate that, whereas Pin1 overexpression facilitates the formation of alpha-synuclein inclusions, dominant-negative Pin1 expression significantly suppresses this process. Consistent with these observations, Pin1 overexpression enhances the protein half-life and insolubility of alpha-synuclein. Finally, we show that Pin1 binds synphilin-1, an alpha-synuclein partner, via its Ser-211-Pro and Ser-215-Pro motifs, and enhances its interaction with alpha-synuclein, thus likely facilitating the formation of alpha-synuclein inclusions. These results indicate that Pin1-mediated prolyl-isomerization plays a pivotal role in a post-translational modification pathway for alpha-synuclein aggregation and in the resultant Lewy body formations in PD.     

Production of serpins using yeast expression systems

Serpins occupy a unique niche in the field of biology. As more of them are discovered, the need to produce sufficient quantities of each to aid experimental and therapeutic research increases. Yeast expression systems are well suited for the production of recombinant serpins. The genetics of many yeast species is well understood and readily manipulated to induce the targeted over-production of many different serpins. In addition, protease-deficient strains of certain species are available and a few species carry out post-translational modifications resembling those of humans. Yeasts are easy to grow and multiply readily in simple culture media hence the cost of production is low, while the scale of production can be small or large. The disadvantages are the inability of most yeast(s) to perform complex post-translational modifications and a lower product yield of secreted protein compared to intracellular protein production. However, for the intracellular production of serpins, in particular the clade B serpins that do not have complex post-translational modifications, yeast expression systems should be among the first systems considered.     

Proteomics in Parkinson’s disease: current trends,translational snags and future possibilities

Proteomic technologies are widely used to understand the molecular mechanism of Parkinson’s disease (PD) and to develop biomarkers for its early diagnosis. The differential expression patterns of brain, cerebrospinal fluid and blood proteins of patients or chemically induced animal models are used to identify protein fingerprints for developing diagnostic and therapeutic strategies for PD. A number of differentially expressed proteins associated with energy metabolism, oxidative stress, signal transduction, electron transport and detoxification pathways are identified using proteomic strategies. Proteomics immensely contributed to the detection of qualitative and quantitative changes of expressed proteins and their post-translational modifications. An update on proteomics-driven research for developing early biomarkers and understanding the molecular aspects of PD, along with their translational snags, challenges and future possibilities, are discussed in this review.     

The 20S proteasome isolated from Alzheimer's disease brain shows post-translational modifications but unchanged proteolytic activity

Chronic neurodegenerative diseases are characterized by the accumulation of aggregated protein species, and functional impairment of the ubiquitin proteasome system has been hypothesized to contribute to neuronal cell loss. Decreased proteolytic activity of the 20S proteasome has been shown postmortem in crude brain lysates from Alzheimer's disease (AD) patients. In the present study, we demonstrate, however, that catalytic activity of the 20S proteasome increases during chromatographic purification from AD brains as compared with age-matched controls. By two-dimensional difference gel electrophoresis we detected pI shifts in several proteasome subunits in AD samples pointing to differential post-translational modifications. Moreover, we identified N-terminal acetylation and dephosphorylation of subunit alpha7 in AD by tandem mass spectrometry. Thus, reduced peptidase activity in AD brain extracts is not an intrinsic property of the 20S proteasome, but may be resulting from the presence of endogenous inhibitory proteins or substrates. Post-translational modifications of non-catalytic subunits in situ may contribute to the trend towards enhanced hydrolytic activity of the isolated 20S proteasome after removal of the endogenous inhibitors.     

Post-translation modification of proteins; methodologies and applications in plant sciences

Proteins have the potential to undergo a variety of post-translational modifications and the different methods available to study these cellular processes has advanced rapidly with the continuing development of proteomic technologies. In this review we aim to detail five major post-translational modifications (phosphorylation, glycosylaion, lipid modification, ubiquitination and redox-related modifications), elaborate on the techniques that have been developed for their analysis and briefly discuss the study of these modifications in selected areas of plant science.     

A survey of DNA diagnostic laboratories regarding DNA banking.

This article reports the findings of a survey of 148 academically based and commercial DNA diagnostic labs regarding DNA banking (defined as the storage of individual DNA samples in some form with identifiers for later retrieval). The population surveyed consisted of all laboratories listed with HELIX, a national directory of DNA diagnostic labs that includes a fairly comprehensive listing of clinical service labs as well as a large number of research labs. The survey was concerned primarily with the legal and ethical issues that the long-term storage of DNA may raise. The survey inquired into the respondents' policies and procedures concerning (1) the extent of DNA banking and of interest in developing DNA banking in academia and industry and (2) the degree to which DNA banks had developed written internal policies and/or a written depositor's agreement (a signed document defining the rights and obligations of the person from whom the sample was taken and the bank) designed to anticipate or prevent some of the ethical and legal problems that can arise from the long-term retention of DNA. Our research suggests that (1) the activity of DNA banking is growing, particularly in the academic setting, and (2) most academically based DNA banks lack written internal policies, written depositor's agreements, or other relevant documentation regarding important aspects of this activity.     

Molecular profiling of experimental Parkinson's disease: direct analysis of peptides and proteins on brain tissue sections by MALDI mass spectrometry

Direct molecular profiling of biological samples using matrix-assisted laser desorption ionization mass spectrometry is a powerful tool for identifying phenotypic markers. In this report, protein profiling was used for the first time to generate peptide and protein profiles of brain tissue sections obtained from experimental Parkinson's disease (unilaterally 6-hydroxydopamine treated rats). The mass spectrometer was used to map the peptide and protein expression directly on 12 microm tissue sections in mass-to-charge (m/z) values, providing the capability of mapping specific molecules of the original sample, that is, localization, intensity and m/z ratio. Several protein expression profile differences were found in the dopamine depleted side of the brain when compared to the corresponding intact side, for example, calmodulin, cytochrome c, and cytochrome c oxidase. An increased ratio of post-translational modifications such as acetylations were found in the striatum of proteins in the dopamine depleted side of the brain. These modifications were decreased after subchronic administration of L-Dopa. The present study shows that unique protein profiles can be obtained in specific brain regions (and subregions) directly on brain tissue sections and allows for the study of complex biochemical processes such as those occurring in experimental Parkinson's disease.     

SwePep, a database designed for endogenous peptides and mass spectrometry

A new database, SwePep, specifically designed for endogenous peptides, has been constructed to significantly speed up the identification process from complex tissue samples utilizing mass spectrometry. In the identification process the experimental peptide masses are compared with the peptide masses stored in the database both with and without possible post-translational modifications. This intermediate identification step is fast and singles out peptides that are potential endogenous peptides and can later be confirmed with tandem mass spectrometry data. Successful applications of this methodology are presented. The SwePep database is a relational database developed using MySql and Java. The database contains 4180 annotated endogenous peptides from different tissues originating from 394 different species as well as 50 novel peptides from brain tissue identified in our laboratory. Information about the peptides, including mass, isoelectric point, sequence, and precursor protein, is also stored in the database. This new approach holds great potential for removing the bottleneck that occurs during the identification process in the field of peptidomics. The SwePep database is available to the public.     

Forensic DNA data banking by state crime laboratories.

This article reports the results of a survey of the responsible crime laboratories in the first 19 states with legislation establishing forensic DNA data banks. The survey inquired into the labs' policies and procedures regarding the collection, storage, and analysis of samples; the retention of samples and data; search protocols; access to samples and data by third parties; and related matters. The research suggests that (1) the number of samples collected from convicted offenders for DNA data banking has far surpassed the number that have been analyzed; (2) data banks have already been used in a small but growing number of cases, to locate suspects and to identify associations between unresolved cases; (3) crime labs currently plan to retain indefinitely the samples collected for their data banks; and (4) the nature and extent of security safeguards that crime labs have implemented for their data banks vary among states. The recently enacted DNA Identification Act (1994) will provide $40 million in federal matching grants to states for DNA analysis activities, so long as states comply with specified quality-assurance standards, submit to external proficiency testing, and limit access to DNA information. Although these additional funds should help to ease some sample backlogs, it remains unclear how labs will allocate the funds, as between analyzing samples for their data banks and testing evidence samples in cases without suspects. The DNA Identification Act provides penalties for the disclosure or obtaining of DNA data held by data banks that participate in CODIS, the FBI's evolving national network of DNA data banks, but individual crime labs must also develop stringent internal safeguards to prevent breaches of data-bank security.     

Use of a simple method to isolate intact RNA from partially hydratedSelaginella lepidophylla plants

Isolation of high-quality RNA is a necessary step in gene expression analysis. Although many methods can be used to isolate RNA from plants where contamination of preparations with complex carbohydrates or phenolic compounds is a problem, the application of these methods toSelaginella lepidophylla tissues has failed to obtain good-quality RNA. Here we introduce 2 modifications to the method developed by Chomczynski and Sacchi (1987), generating a simple and rapid method that allows the isolation of intact RNA fromS. lepidophylla-dehydrated tissues. Although the introduced modifications are not new, their addition proved to be decisive for success in RNA isolation. Quality of the RNA obtained was evaluated by electrophoresis in agarose and by 3 different PCR-based techniques—RT-PCR, RNA differential display, and synthesis of a cDNA library.     

Palmitoylation and polymerization of hepatitis C virus NS4B protein

Hepatitis C Virus (HCV) NS4B protein induces a specialized membrane structure which may serve as the replication platform for HCV RNA replication. In the present study, we demonstrated that NS4B has lipid modifications (palmitoylation) on two cysteine residues (cysteines 257 and 261) at the C-terminal end. Site-specific mutagenesis of these cysteine residues on individual NS4B proteins and on an HCV subgenomic replicon showed that the lipid modifications, particularly of Cys261, are important for protein-protein interaction in the formation of the HCV RNA replication complex. We further demonstrated that NS4B can undergo polymerization. The main polymerization determinants were mapped in the N-terminal cytosolic domain of NS4B protein; however, the lipid modifications on the C terminus also facilitate the polymerization process. The lipid modification and the polymerization activity could be two properties of NS4B important for its induction of the specialized membrane structure involved in viral RNA replication.     

Acetyl-CoA regulates lipid metabolism and histone acetylation modification in cancer

Acetyl-CoA, as an important molecule, not only participates in multiple intracellular metabolic reactions, but also affects the post-translational modification of proteins, playing a key role in the metabolic activity and epigenetic inheritance of cells. Cancer cells require extensive lipid metabolism to fuel for their growth, while also require histone acetylation modifications to increase the expression of cancer-promoting genes. As a raw material for de novo lipid synthesis and histone acetylation, acetyl-CoA has a major impact on lipid metabolism and histone acetylation in cancer. More importantly, in cancer, acetyl-CoA connects lipid metabolism with histone acetylation, forming a more complex regulatory mechanism that influences cancer growth, proliferation, metastasis.