Laser microdissection for spatial proteomics of postmortem brain tissue

Quantitative data of the proteome is highly valuable for providing unbiased information on protein expression changes related to disease or identifying related biomarkers. In brain diseases the affected area can be small and pathogenic events can be related to a specific cell type in an otherwise heterogeneous tissue type. An emerging approach dedicated to analysing this type of samples is laser microdissection (LMD) combined with LC-MS/MS into a single workflow. In this chapter, we describe two LMD methods for isolating tissue at the level of a small area and individual cells suitable for subsequent LC-MS/MS analysis

Laser microdissection for spatial proteomics of postmortem brain tissue

Quantitative data of the proteome is highly valuable for providing unbiased information on protein expression changes related to disease or identifying related biomarkers. In brain diseases the affected area can be small and pathogenic events can be related to a specific cell type in an otherwise heterogeneous tissue type. An emerging approach dedicated to analysing this type of samples is laser microdissection (LMD) combined with LC-MS/MS into a single workflow. In this chapter, we describe two LMD methods for isolating tissue at the level of a small area and individual cells suitable for subsequent LC-MS/MS analysis

A laser microdissection–liquid chromatography–tandem mass spectrometry workflow for post-mortem analysis of brain tissue

Improved speed and sensitivity of mass spectrometry allow the simultaneous quantification of high numbers of proteins from increasingly smaller quantities of tissue sample. Quantitative data of the proteome is highly valuable for providing unbiased information on, for example, protein expression changes related to disease or identifying related biomarkers. In brain diseases the affected area can be small and pathogenic events can be related to a specific cell type in an otherwise heterogeneous tissue type. An emerging approach dedicated to analyzing this type of samples is laser micro-dissection (LMD) combined with LC-MS/MS into a single workflow. In this chapter, we describe different options for isolating tissue suitable for LC-MS/MS analysis

A Laser Microdissection-Liquid Chromatography-Tandem Mass Spectrometry Workflow for Post-mortem Analysis of Brain Tissue

Improved speed and sensitivity of mass spectrometry allow the simultaneous quantification of high numbers of proteins from increasingly smaller quantities of tissue sample. Quantitative data of the proteome is highly valuable for providing unbiased information on, for example, protein expression changes related to disease or identifying related biomarkers. In brain diseases the affected area can be small and pathogenic events can be related to a specific cell type in an otherwise heterogeneous tissue type. An emerging approach dedicated to analyzing this type of samples is laser micro-dissection (LMD) combined with LC-MS/MS into a single workflow. In this chapter, we describe different options for isolating tissue suitable for LC-MS/MS analysis

The proteome of granulovacuolar degeneration and neurofibrillary tangles in Alzheimer’s disease

Granulovacuolar degeneration (GVD) is a common feature in Alzheimer’s disease (AD). The occurrence of GVD is closely associated with that of neurofibrillary tangles (NFTs) and GVD is even considered to be a pre-NFT stage in the disease process of AD. Currently, the composition of GVD bodies, the mechanisms associated with GVD and how GVD exactly relates to NFTs is not well understood. By combining immunohistochemistry (IHC) and laser microdissection (LMD) we isolated neurons with GVD and those bearing tangles separately from human post-mortem AD hippocampus (n = 12) using their typical markers casein kinase (CK)1δ and phosphorylated tau (AT8). Control neurons were isolated from cognitively healthy cases (n = 12). 3000 neurons per sample were used for proteome analysis by label free LC–MS/MS. In total 2596 proteins were quantified across samples and a significant change in abundance of 115 proteins in GVD and 197 in tangle bearing neurons was observed compared to control neurons. With IHC the presence of PPIA, TOMM34, HSP70, CHMP1A, TPPP and VXN was confirmed in GVD containing neurons. We found multiple proteins localizing specifically to the GVD bodies, with VXN and TOMM34 being the most prominent new protein markers for GVD bodies. In general, protein groups related to protein folding, proteasomal function, the endolysosomal pathway, microtubule and cytoskeletal related function, RNA processing and glycolysis were found to be changed in GVD neurons. In addition to these protein groups, tangle bearing neurons show a decrease in ribosomal proteins, as well as in various proteins related to protein folding. This study, for the first time, provides a comprehensive human based quantitative assessment of protein abundances in GVD and tangle bearing neurons. In line with previous functional data showing that tau pathology induces GVD, our data support the model that GVD is part of a pre-NFT stage representing a phase in which proteostasis and cellular homeostasis is disrupted. Elucidating the molecular mechanisms and cellular processes affected in GVD and its relation to the presence of tau pathology is highly relevant for the identification of new drug targets for therapy

Proteomics analysis identifies new markers associated with capillary cerebral amyloid angiopathy in Alzheimer’s disease

Abstract Alzheimer’s disease (AD) is characterized by amyloid beta (Aβ) deposits as plaques in the parenchyma and in the walls of cortical and leptomeningeal blood vessels of the brain called cerebral amyloid angiopathy (CAA). It is suggested that CAA type-1, which refers to amyloid deposition in both capillaries and larger vessels, adds to the symptomatic manifestation of AD and correlates with disease severity. Currently, CAA cannot be diagnosed pre-mortem and disease mechanisms involved in CAA are elusive. To obtain insight in the disease mechanism of CAA and to identify marker proteins specifically associated with CAA we performed a laser dissection microscopy assisted mass spectrometry analysis of post-mortem human brain tissue of (I) AD cases with only amyloid deposits in the brain parenchyma and no vascular related amyloid, (II) AD cases with severe CAA type-1 and no or low numbers of parenchymal amyloid deposits and (III) cognitively healthy controls without amyloid deposits. By contrasting the quantitative proteomics data between the three groups, 29 potential CAA-selective proteins were identified. A selection of these proteins was analysed by immunoblotting and immunohistochemistry to confirm regulation and to determine protein localization and their relation to brain pathology. In addition, specificity of these markers in relation to other small vessel diseases including prion CAA, CADASIL, CARASAL and hypertension related small vessel disease was assessed using immunohistochemistry. Increased levels of clusterin (CLU), apolipoprotein E (APOE) and serum amyloid P-component (APCS) were observed in AD cases with CAA. In addition, we identified norrin (NDP) and collagen alpha-2(VI) (COL6A2) as highly selective markers that are clearly present in CAA yet virtually absent in relation to parenchymal amyloid plaque pathology. NDP showed the highest specificity to CAA when compared to other small vessel diseases. The specific changes in the proteome of CAA provide new insight in the pathogenesis and yields valuable selective biomarkers for the diagnosis of CAA

Distinct cell type-specific protein signatures in GRN and MAPT genetic subtypes of frontotemporal dementia

Frontotemporal dementia is characterized by progressive atrophy of frontal and/or temporal cortices at an early age of onset. The disorder shows considerable clinical, pathological, and genetic heterogeneity. Here we investigated the proteomic signatures of frontal and temporal cortex from brains with frontotemporal dementia due to GRN and MAPT mutations to identify the key cell types and molecular pathways in their pathophysiology. We compared patients with mutations in the GRN gene (n = 9) or with mutations in the MAPT gene (n = 13) with non-demented controls (n = 11). Using quantitative proteomic analysis on laser-dissected tissues we identified brain region-specific protein signatures for both genetic subtypes. Using published single cell RNA expression data resources we deduced the involvement of major brain cell types in driving these different protein signatures. Subsequent gene ontology analysis identified distinct genetic subtype- and cell type-specific biological processes. For the GRN subtype, we observed a distinct role for immune processes related to endothelial cells and for mitochondrial dysregulation in neurons. For the MAPT subtype, we observed distinct involvement of dysregulated RNA processing, oligodendrocyte dysfunction, and axonal impairments. Comparison with an in-house protein signature of Alzheimer’s disease brains indicated that the observed alterations in RNA processing and oligodendrocyte function are distinct for the frontotemporal dementia MAPT subtype. Taken together, our results indicate the involvement of different brain cell types and biological mechanisms in genetic subtypes of frontotemporal dementia. Furthermore, we demonstrate that comparison of proteomic profiles of different disease entities can separate general neurodegenerative processes from disease-specific pathways, which may aid the development of disease subtype-specific treatment strategies