laser-microdissection-based-protocol-for-lc-msms-analysis-proteomic

Laser Microdissection-Based Protocol for the LC-MS/MS Analysis of the Proteomic Profile of Neuromelanin Granules

Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in dopaminergic neurons of the substantia nigra pars compacta. ...

Novel effective treatment is urgently needed for sporadic Alzheimer's disease (sAD). M30 ([5-(N-methyl-N-propargylaminomethyl)-8-hydroxyquinoline]) and HLA-20 (5-{4-propargylpiperazin-1-ylmethyl}-8-hydroxyquinoline) are brain permeable, iron chelating compounds with antioxidant activity, showing also neuroprotective activity in animal models of neurodegeneration.Weaimed to explore their therapeutic potential in non-transgenic (non-Tg) rat model of sAD developed by intracerebroventricular administration of streptozotocin (STZ-icv).

Multi-target iron-chelators improve memory loss in a rat model of sporadic Alzheimer's disease.

Neuromelanin is a complex polymer pigment found primarily in the dopaminergic neurons of human substantia nigra. Neuromelanin pigment is stored in granules including a protein matrix and lipid droplets. Neuromelanin granules are yet only partially characterised regarding their structure and function. To clarify the exact function of neuromelanin granules in humans, their enrichment and in-depth characterization from human substantia nigra is necessary. Previously published global proteome studies of neuromelanin granules in human substantia nigra required high tissue amounts. Due to the limited availability of human brain tissue we established a new method based on laser microdissection combined with mass spectrometry for the isolation and analysis of neuromelanin granules. With this method it is possible for the first time to isolate a sufficient amount of neuromelanin granules for global proteomics analysis from ten 10 μm tissue sections. In total 1,000 proteins were identified associated with neuromelanin granules. More than 68% of those proteins were also identified in previously performed studies. Our results confirm and further extend previously described findings, supporting the connection of neuromelanin granules to iron homeostasis and lysosomes or endosomes. Hence, this method is suitable for the donor specific enrichment and proteomic analysis of neuromelanin granules.

Proteomic characterization of neuromelanin granules isolated from human substantia nigra by laser-microdissection.

Prof. Dr. Kurt Jellinger: an appreciation.

This article was originally published with the wrong title. The correct title is: Abstracts of the WASAD Congress 2017, in conjunction with the Collaborative Research Centers SFB-TRR 58, Fear, Anxiety, Anxiety Disorders 14-16 September, Würzburg, Germany.

Erratum to: Abstracts of the WASAD Congress 2017, in conjunction with the Collaborative Research Centers SFB-TRR 58, Fear, Anxiety, Anxiety Disorders 14-16 September, Würzburg, Germany.

Toshiharu (Toshi) Nagatsu: an appreciation.

Premortem, postmortem, and storage conditions are parameters that can influence the quality and interpretation of data from studies of postmortem tissue. While many neurochemicals in the brain are relatively stable for several hours after death if stored at 4°C, the postmortem delay nevertheless becomes an important variable when examining the disease state because neurochemical levels may change with extended postmortem delay. Moreover, in the postmortem brain, neurochemical levels may also play a key role in determining the diagnosis. This is particularly true for some neurodegenerative disorders where many of the clinical features of the disease are not exclusive to one illness. It is therefore imperative to employ brain tissue of the highest quality from both nondiseased (control) and diseased brain tissue to ascertain the specific molecular and genetic mechanisms particular to the disease pathogenesis. Consequently, it would be very useful if specific markers could be employed to demonstrate and determine the quality of postmortem brain tissue that is suitable for such studies. In this chapter, the following neurochemical markers are critically reviewed as potential candidates to assess the quality of postmortem brain tissue: tryptophan levels, glutathione levels (and glutathione metabolic enzymes), enzymatic activities (glutamate decarboxylase, phosphofructokinase-1), epigenetic enzymes (acetyltransferase, methyltransferase), and tissue pH. In conclusion, the neurochemical tryptophan appears to be the most suitable candidate for assessing the integrity and quality of postmortem brain tissue. However, to optimize the quality of the samples, neuropathologic diagnostic characterization must also be employed in the interpretation and understanding of the data generated. It would also be judicious to consider as many premortem and postmortem conditions as possible as they can also affect the genetic and molecular integrity of the brain tissue.

Neurochemical markers as potential indicators of postmortem tissue quality.

Introduction to the special issue on monoamine oxidase A and B: eternally enigmatic isoenzymes.

Journal of Neural Transmission: a scientific journal devoted since 1950 to the translation of neuroscience into clinical practice.

Prof. Dr. Moussa B.H. Youdim: an appreciation on the occasion of his 80th birthday.

Gerald Stern (1930-2018).

Misfolding of the protein α-synuclein contributes to the formation of the intracellular inclusion, Lewy bodies. Although these structures are not exclusive to Parkinson's disease, nevertheless, their presence in the substantia nigra is mandatory for the pathological diagnosis of the disorder. Therefore, there must be a focus on the pathological mechanisms responsible for Lewy body generation. Recent studies have suggested that α-synuclein has the potential to operate as the enzyme ferrireductase. Perhaps in the early diseased state, overexpression or mutation of alpha-synuclein/ferrireductase invokes the dyshomeostasis of iron (III)/(II) only, while in advanced stages, accumulation of iron in particular areas of the brain follows. Furthermore, the loss of an important iron chelator, neuromelanin (due to dopaminergic neuronal death), may then result in the release and increase in unbound free iron. Iron could generate reactive oxygen species, which could instigate a torrent of cellular deleterious processes. In addition, loss of energy supply may contribute to the alteration in activity of enzymes involved in the mitochondrial respiratory chain and would, therefore, confer a vulnerability to the dopaminergic neurons in the substantia nigra. Therefore, the ferrireductase alpha-synuclein may hold the key for major pathology of Parkinson's disease. In conclusion, we hypothesize that environmentally or genetically overexpressed and/or mutated α-synuclein/ferrireductase causes iron dyshomeostasis without increase of free iron concentration in the early phases of PD, while increased iron concentration accompanied by iron dyshomeostasis is a marker for progressed PD stages. It is essential to elucidate these degenerative mechanisms, so as to provide effective therapeutic treatment to halt or delay the progression of the illness already in the early phase of PD. The development of iron chelators seems to be a reasonable approach.

The role of alpha-synuclein as ferrireductase in neurodegeneration associated with Parkinson's disease.

Parkinson's disease (PD) etiology is heterogeneous, genetic, and multi-factorial, resulting in a varied disease from a mild slow progression to a more severe rapid progression. Prognostic information on the nature of the patient's disease at diagnosis aids the physician in counseling patients on treatment options and life planning. In a cohort of PD patients from the PPMI study, the relative gene expression levels of SKP1A, UBE2K, ALDH1A1, PSMC4, HSPA8 and LAMB2 were measured in baseline blood samples by real-time quantitative PCR. At baseline PD patients were up to 2 years from diagnosis, H&Y scale ≤ 2 and PD treatment naïve. PD-Prediction algorithm comprised of ALDH1A1, LAMB2, UBE2K, SKP1A and age was created by logistic regression for predicting progression to ≤ 70% Modified Schwab and England Activities of Daily Living (S&E-ADL). In relation to patients negative for PD-Prediction (n = 180), patients positive (n = 30) for Cutoff-1 (at 82% specificity, 80.0% sensitivity) had positive hazard ratio (HR+) of 10.6 (95% CI, 2.2-50.1), and positive (n = 23) for Cutoff-2 (at 93% specificity, 47% sensitivity) had HR+ of 17.1 (95% CI, 3.2-89.9) to progress to ≤ 70% S&E-ADL within 3 years (P value < 0.0001). Likewise, patients positive for PD-Prediction Cutoff-1 (n = 49) had HR+ 4.3 (95% CI, 1.6-11.6) for faster time to H&Y 3 in relation to patients negative (n = 170) for PD-Prediction (P value = 0.0002). Our findings show an algorithm that seems to predict fast PD progression and may potentially be used as a tool to assist the physician in choosing an optimal treatment plan, improving the patient's quality of life and overall health outcome.

Creation of a gene expression classifier for predicting Parkinson's disease rate of progression.

The pathological hallmark of Parkinson's disease (PD) is the loss of neuromelanin-containing dopaminergic neurons within the substantia nigra pars compacta (SNpc). Additionally, numerous studies indicate an altered synaptic function during disease progression. To gain new insights into the molecular processes underlying the alteration of synaptic function in PD, a proteomic study was performed. Therefore, synaptosomes were isolated by density gradient centrifugation from SNpc tissue of individuals at advanced PD stages (N = 5) as well as control subjects free of pathology (N = 5) followed by mass spectrometry-based analysis. In total, 362 proteins were identified and assigned to the synaptosomal core proteome. This core proteome comprised all proteins expressed within the synapses without regard to data analysis software, gender, age, or disease. The differential analysis between control subjects and PD cases revealed that CD9 antigen was overrepresented and fourteen proteins, among them Thymidine kinase 2 (TK2), mitochondrial, 39S ribosomal protein L37, neurolysin, and Methionine-tRNA ligase (MARS2) were underrepresented in PD suggesting an alteration in mitochondrial translation within synaptosomes.

Proteomic Characterization of Synaptosomes from Human Substantia Nigra Indicates Altered Mitochondrial Translation in Parkinson's Disease.

The risk of Parkinson's disease increases with age. However, the etiology of the illness remains obscure. It appears highly likely that the neurodegenerative processes involve an array of elements that influence each other. In addition, genetic, endogenous, or exogenous toxins need to be considered as viable partners to the cellular degeneration. There is compelling evidence that indicate the key involvement of modified α-synuclein (Lewy bodies) at the very core of the pathogenesis of the disease. The accumulation of misfolded α-synuclein may be a consequence of some genetic defect or/and a failure of the protein clearance system. Importantly, α-synuclein pathology appears to be a common denominator for many cellular deleterious events such as oxidative stress, mitochondrial dysfunction, dopamine synaptic dysregulation, iron dyshomeostasis, and neuroinflammation. These factors probably employ a common apoptotic/or autophagic route in the final stages to execute cell death. The misfolded α-synuclein inclusions skillfully trigger or navigate these processes and thus amplify the dopamine neuron fatalities. Although the process of neuroinflammation may represent a secondary event, nevertheless, it executes a fundamental role in neurodegeneration. Some viral infections produce parkinsonism and exhibit similar characteristic neuropathological changes such as a modest brain dopamine deficit and α-synuclein pathology. Thus, viral infections may heighten the risk of developing PD. Alternatively, α-synuclein pathology may induce a dysfunctional immune system. Thus, sporadic Parkinson's disease is caused by multifactorial trigger factors and metabolic disturbances, which need to be considered for the development of potential drugs in the disorder.

The Nigral Coup in Parkinson's Disease by α-Synuclein and Its Associated Rebels.

Kurt Jellinger, Doyen of international neuropathology.

Galactose, a ubiquitous monosaccharide with incompletely understood physiology is often exploited for inducing oxidative-stress mediated aging in animals. Recent research demonstrates that galactose can conserve cellular function during periods of starvation and prevent/alleviate cognitive deficits in a rat model of sporadic Alzheimer's disease. The present aim is to examine the acute effects of oral galactose on the redox regulatory network (RRN).

Article	Year
Multi-target iron-chelators improve memory loss in a rat model of sporadic Alzheimer's disease. Life sciences\| PubMed ID: 26159898	2015
Proteomic characterization of neuromelanin granules isolated from human substantia nigra by laser-microdissection. Scientific reports\| PubMed ID: 27841354	2016
Prof. Dr. Kurt Jellinger: an appreciation. Journal of neural transmission (Vienna, Austria : 1996)\| PubMed ID: 28508292	2017
Erratum to: Abstracts of the WASAD Congress 2017, in conjunction with the Collaborative Research Centers SFB-TRR 58, Fear, Anxiety, Anxiety Disorders 14-16 September, Würzburg, Germany. Journal of neural transmission (Vienna, Austria : 1996)\| PubMed ID: 28929176	2017
Toshiharu (Toshi) Nagatsu: an appreciation. Journal of neural transmission (Vienna, Austria : 1996)\| PubMed ID: 29260324	2018
Neurochemical markers as potential indicators of postmortem tissue quality. Handbook of clinical neurology\| PubMed ID: 29496135	2018
Introduction to the special issue on monoamine oxidase A and B: eternally enigmatic isoenzymes. Journal of neural transmission (Vienna, Austria : 1996)\| PubMed ID: 30259129	2018
Journal of Neural Transmission: a scientific journal devoted since 1950 to the translation of neuroscience into clinical practice. Journal of neural transmission (Vienna, Austria : 1996)\| PubMed ID: 30911821	2019
Prof. Dr. Moussa B.H. Youdim: an appreciation on the occasion of his 80th birthday. Journal of neural transmission (Vienna, Austria : 1996)\| PubMed ID: 32016605	2020
Gerald Stern (1930-2018). Journal of neural transmission (Vienna, Austria : 1996)\| PubMed ID: 32219618	2020
The role of alpha-synuclein as ferrireductase in neurodegeneration associated with Parkinson's disease. Journal of neural transmission (Vienna, Austria : 1996)\| PubMed ID: 32318880	2020
Creation of a gene expression classifier for predicting Parkinson's disease rate of progression. Journal of neural transmission (Vienna, Austria : 1996)\| PubMed ID: 32385576	2020
Proteomic Characterization of Synaptosomes from Human Substantia Nigra Indicates Altered Mitochondrial Translation in Parkinson's Disease. Cells\| PubMed ID: 33276480	2020
The Nigral Coup in Parkinson's Disease by α-Synuclein and Its Associated Rebels. Cells\| PubMed ID: 33803185	2021
Kurt Jellinger, Doyen of international neuropathology. Journal of neural transmission (Vienna, Austria : 1996)\| PubMed ID: 34420118	2021
Is Galactose a Hormetic Sugar? An Exploratory Study of the Rat Hippocampal Redox Regulatory Network. Molecular nutrition & food research\| PubMed ID: 34453395	2021

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