Isolation of Amyloid Plaques and Neurofibrillary Tangles from Archived Alzheimer’s Disease Tissue Using Laser-Capture Microdissection for Downstream Proteomics

Drummond, Eleanor; Nayak, Shruti; Pires, Geoffrey; Ueberheide, Beatrix; Wısnıewskı, Thomas

doi:10.1007/978-1-4939-7558-7_18

Cited by 41 publications

(41 citation statements)

References 14 publications

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“…With the use of laser capture microscopy and proteomics techniques, analyses of micro-dissected regions are feasible and could identify key neuroinflammatory factors associated with AD that are not represented when larger dissected pieces of brain tissue are analyzed. The feasibility of this approach has been demonstrated in proteomic analyses of micro-dissected plaques and tangles from AD brains [49,50]. microglial P2RY12 with ticagrelor, an antagonist, reduced ischemic damage by microglia by reducing their migration to sites of injury [47].…”

Section: Discussionmentioning

confidence: 99%

Patterns of Expression of Purinergic Receptor P2RY12, a Putative Marker for Non-Activated Microglia, in Aged and Alzheimer’s Disease Brains

Walker

Tang

Mendsaikhan

et al. 2020

IJMS

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Neuroinflammation is considered a key pathological process in neurodegenerative diseases of aging, including Alzheimer’s disease (AD). Many studies have defined phenotypes of reactive microglia, the brain-resident macrophages, with different antigenic markers to identify those potentially causing inflammatory damage. We took an alternative approach with the goal of characterizing the distribution of purinergic receptor P2RY12-positive microglia, a marker previously defined as identifying homeostatic or non-activated microglia. We examined the expression of P2RY12 by dual-color light and fluorescence immunohistochemistry using sections of middle temporal gyrus from AD, high plaque and low plaque non-demented cases in relation to amyloid beta (Aβ) plaques and phosphorylated tau, markers of pathology, and HLA-DR, IBA-1, CD68, and progranulin, microglial phenotype markers. In low plaque cases, P2RY12-positive microglia mostly had non-activated morphologies, while the morphologies of P2RY12-positive microglia in AD brains were highly variable, suggesting its expression could encompass a wider range of phenotypes than originally hypothesized. P2RY12 expression by microglia differed depending on the types of plaques or tangles they were associated with. Areas of inflammation characterized by lack of P2RY12-positive microglia around mature plaques could be observed, but many diffuse plaques showed colocalization with P2RY12-positive microglia. Based on these results, P2RY12 expression by microglia should not be considered solely a marker of resting microglia as P2RY12 immunoreactivity was identifying microglia positive for CD68, progranulin and to a limited extent HLA-DR, markers of activation.

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Section: Discussionmentioning

confidence: 99%

Patterns of Expression of Purinergic Receptor P2RY12, a Putative Marker for Non-Activated Microglia, in Aged and Alzheimer’s Disease Brains

Walker

Tang

Mendsaikhan

et al. 2020

IJMS

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“…We sampled brain tissue from formalin-fixed paraffin embedded (FFPE) blocks of hippocampus or superior frontal gyrus collected and processed for autopsy neuropathology. LCM was performed using our published protocol [31][32][33]. Briefly, 8µm-thick FFPE sections containing hippocampus or frontal superior gyrus were collected onto LCM PET Frame Slides (Leica).…”

Section: Laser-capture Microdissection (Lcm)mentioning

confidence: 99%

“…The samples were subsequently digested with 300ng of sequencing grade modified trypsin (Promega) overnight at RT with gentle shaking. After digestion samples were acidified with trifluoroacetic acid to a pH of 2 and peptides were desalted using POROS R2 C18 beads as previously described [31][32][33]. The samples were resuspended in 0.5% acetic acid and stored at -80C until further analysis.…”

Section: Label-free Quantitative Mass Spectrometry (Ms) Proteomicsmentioning

confidence: 99%

Proteomic Differences in the Hippocampus and Cortex of Epilepsy Brain Tissue

Pires

Leitner

Drummond

et al. 2020

Preprint

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Epilepsy is a common neurological disorder affecting over 70 million people worldwide, with a high rate of pharmaco-resistance, diverse comorbidities including progressive cognitive and behavioral disorders, and increased mortality from direct (e.g., Sudden Unexpected Death in Epilepsy [SUDEP], accidents, drowning) or indirect effects of seizures and therapies. Extensive research with animal models and human studies provides limited insights into the mechanisms underlying seizures and epileptogenesis, and these have not translated into significant reductions in pharmaco-resistance, morbidities or mortality. To help define changes in molecular signaling networks associated with epilepsy, we examined the proteome of brain samples from epilepsy and control cases. Label-free quantitative mass spectrometry (MS) was performed on the hippocampal CA1-3 region, frontal cortex, and dentate gyrus microdissected from epilepsy and control cases (n=14/group). Epilepsy cases had significant differences in the expression of 777 proteins in the hippocampal CA1-3 region, 296 proteins in the frontal cortex, and 49 proteins in the dentate gyrus in comparison to control cases. Network analysis showed that proteins involved in protein synthesis, mitochondrial function, G-protein signaling, and synaptic plasticity were particularly altered in epilepsy. While protein differences were most pronounced in the hippocampus, similar changes were observed in other brain regions indicating broad proteomic abnormalities in epilepsy. Among the most significantly altered proteins, G-protein Subunit Beta 1 (GNB1) was one of the most significantly decreased proteins in epilepsy in all regions studied, highlighting the importance of G-protein subunit signaling and G-protein–coupled receptors (GPCRs) in epilepsy. Our results provide insights into the molecular mechanisms underlying epilepsy, which may allow for novel targeted therapeutic strategies.

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“…This study is an example of the powerful and informative nature of unbiased localized proteomics studies, which permit the discovery of novel disease related proteins present within neuropathological features through unbiased analysis [12][13][14][15]. It is important to recognize that SCRN1 was only brought to our attention as a novel AD associated protein because of the localized nature of our original proteomics study that identified SCRN1 in dystrophic neurites in amyloid plaques [12].…”

Section: Discussionmentioning

confidence: 99%

O4‐01‐06: Secernin‐1 Is a Novel Phosphorylated Tau Binding Protein That Accumulates in Alzheimer's Disease and Not in Other Tauopathies

Pires

Prelli

Halliday

et al. 2019

Alzheimer's & Dementia

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We recently identified Secernin-1 (SCRN1) as a novel amyloid plaque associated protein using localized proteomics. Immunohistochemistry studies confirmed that SCRN1 was present in plaque-associated dystrophic neurites and also revealed distinct and abundant co-localization with neurofibrillary tangles (NFTs). Little is known about the physiological function of SCRN1 and its role in Alzheimer's disease (AD) and other neurodegenerative diseases has not been studied. Therefore, we performed a comprehensive study of SCRN1 distribution in neurodegenerative diseases. Immunohistochemistry was used to map SCRN1 accumulation throughout the progression of AD in a cohort of 58 patients with a range of NFT pathology (Abundant NFT, n = 21; Moderate NFT, n = 22; Low/No NFT, n = 15), who were clinically diagnosed as having AD, mild cognitive impairment or normal cognition. SCRN1 accumulation was also examined in two cases with both Frontotemporal Lobar Degeneration (FTLD)-Tau and ADrelated neuropathology, cases of Down Syndrome (DS) with AD (n = 5), one case of hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) and other non-AD tauopathies including: primary age-related tauopathy (PART, [n = 5]), Corticobasal Degeneration (CBD, [n = 5]), Progressive Supranuclear Palsy (PSP, [n = 5]) and Pick's disease (PiD, [n = 4]). Immunohistochemistry showed that SCRN1 was a neuronal protein that abundantly accumulated in NFTs and plaque-associated dystrophic neurites throughout the progression of AD. Quantification of SCRN1 immunohistochemistry confirmed that SCRN1 preferentially accumulated in NFTs in comparison to surrounding non-tangle containing neurons at both early and late stages of AD. Similar results were observed in DS with AD and PART. However, SCRN1 did not co-localize with phosphorylated tau inclusions in CBD, PSP or PiD. Coimmunoprecipitation revealed that SCRN1 interacted with phosphorylated tau in human AD brain tissue. Together, these results suggest that SCRN1 is uniquely associated with tau pathology in AD, DS and PART. As such, SCRN1 has potential as a novel therapeutic target and could serve as a useful biomarker to distinguish AD from other tauopathies.

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Isolation of Amyloid Plaques and Neurofibrillary Tangles from Archived Alzheimer’s Disease Tissue Using Laser-Capture Microdissection for Downstream Proteomics

Cited by 41 publications

References 14 publications

Patterns of Expression of Purinergic Receptor P2RY12, a Putative Marker for Non-Activated Microglia, in Aged and Alzheimer’s Disease Brains

Patterns of Expression of Purinergic Receptor P2RY12, a Putative Marker for Non-Activated Microglia, in Aged and Alzheimer’s Disease Brains

Proteomic Differences in the Hippocampus and Cortex of Epilepsy Brain Tissue

O4‐01‐06: Secernin‐1 Is a Novel Phosphorylated Tau Binding Protein That Accumulates in Alzheimer's Disease and Not in Other Tauopathies

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