A manual multiplex immunofluorescence method for investigating neurodegenerative diseases

Ehrenberg, Alexander J.; Morales, Dulce Ovando; Piergies, Antonia M.H.; Li, Song Hua; Tejedor, Jorge Santos; Mladinov, Mihovil; Mulder, Jan; Grinberg, Lea T.

doi:10.1016/j.jneumeth.2020.108708

Cited by 13 publications

(22 citation statements)

References 52 publications

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“…The way we defined cell type subpopulations independently of disease progression allowed us to compare gene expression between different cell type subpopulations within individuals while controlling for differences among individuals; this is more robust than comparing gene expression in a given subpopulation across groups of individuals, which can be influenced by differences in confounding factors between the groups. Lastly, by validating our findings using a novel multiplex immunofluorescence approach that enables probing a higher number of antibodies simultaneously 43 , we could quantify the relative abundance of excitatory neurons and RORB+ neurons and also demonstrate that RORB+ excitatory neurons were preferentially affected by neurofibrillary inclusions. A limitation of our study is that we only included male APOE ε3/ε3 individuals in the snRNAseq analysis.…”

Section: Discussionsupporting

confidence: 58%

“…6, see Methods). We used multiplex immunofluorescence 43 to label cells (DAPI), excitatory neurons (TBR1), RORB+ neurons, and phospho-tau neuronal inclusions (CP-13, Ser 202). We failed to find statistically significant changes in the proportion of excitatory neurons overall (TBR1+ cells among all cells) across disease progression (Fig.…”

Section: Validation Of the Selective Vulnerability Of Rorb-expressingmentioning

confidence: 99%

“…A quality control slide was used to verify the efficacy of the antibody stripping process. A detailed description of the method is provided in Ehrenberg et al 43 Sections were scanned using a Zeiss AxioScan Slide Scanner. For generating the images shown in Fig.…”

Section: Functional Annotation Of Differentially Expressed Genes In Gmentioning

confidence: 99%

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Molecular characterization of selectively vulnerable neurons in Alzheimer’s Disease

Leng

Eser

et al. 2020

Preprint

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Alzheimer's disease (AD) is characterized by the selective vulnerability of specific neuronal populations, the molecular signatures of which are largely unknown. To identify and characterize selectively vulnerable neuronal populations, we used single-nucleus RNA sequencing to profile the caudal entorhinal cortex and the superior frontal gyrus -brain regions where neurofibrillary inclusions and neuronal loss occur early and late in AD, respectively -from individuals spanning the neuropathological progression of AD. We identified RORB as a marker of selectively vulnerable excitatory neurons in the entorhinal cortex, and subsequently validated their depletion and selective susceptibility to neurofibrillary inclusions during disease progression using quantitative neuropathological methods. We also discovered an astrocyte subpopulation, likely representing reactive astrocytes, characterized by decreased expression of genes involved in homeostatic functions. Our characterization of selectively vulnerable neurons in AD paves the way for future mechanistic studies of selective vulnerability and potential therapeutic strategies for enhancing neuronal resilience. MAIN TEXTSelective vulnerability is a fundamental feature of neurodegenerative diseases, in which different neuronal populations show a gradient of susceptibility to degeneration 1, 2 . Selective vulnerability at the network level has been extensively explored in Alzheimer's disease (AD) 3-5 , currently the leading cause of dementia and lacking in effective therapies. However, little is known about the mechanisms underlying selective vulnerability at the cellular level in AD, which could provide insight into disease mechanisms and lead to therapeutic strategies.The entorhinal cortex (EC), an allocortex, is one of the first cortical brain regions to exhibit neuronal loss in AD 6 . Neurons in the external EC layers, especially in layer II (also known as alpha clusters of the lamina principalis externa, abbreviated "Pre-alpha") 7 , accumulate taupositive neurofibrillary changes and die early on in the course of AD 8-13 . However, these selectively vulnerable neurons have yet to be characterized extensively at the molecular level. Furthermore, it is unknown whether there are differences in vulnerability among subpopulations of these neurons. Although rodent models of AD have offered some insights [14][15][16] , the human brain has unique features with regard to cellular physiology, composition and aging [17][18][19] , limiting the extrapoloation of findings from animal models to address selective vulnerability.Previous studies have combined laser capture microdissection with microarray analysis of gene expression 20, 21 to characterize EC neurons in AD, but focused on disease-related changes in gene expression, rather than selective vulnerability. More recently, single-nucleus RNA-sequencing (snRNA-seq) has enabled large-scale characterization of transcriptomic profiles of individual cells from post-mortem human brain tissue 22, 23 . However, snRNA-seq studies of AD p...

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Section: Discussionsupporting

confidence: 58%

Section: Validation Of the Selective Vulnerability Of Rorb-expressingmentioning

confidence: 99%

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Molecular characterization of selectively vulnerable neurons in Alzheimer’s Disease

Leng

Eser

et al. 2020

Preprint

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“…To enable multiplex staining using antibodies raised in the same species (rabbit), we chose to use a 3-plex TSA strategy [ 49 ], where antibodies were added one at a time, and insoluble tyramide signal amplification (TSA) was used for visualisation followed by heating and inactivation of the first antibody. This was then followed by the second antibody and a different TSA fluorophore (TSA-Plus; PerkinElmer).…”

Section: Methodsmentioning

confidence: 99%

TGFBR3L—An Uncharacterised Pituitary Specific Membrane Protein Detected in the Gonadotroph Cells in Non-Neoplastic and Tumour Tissue

Kolnes

Olarescu

Mitsios

et al. 2020

Cancers

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Here, we report the investigation of transforming growth factor beta-receptor 3 like (TGFBR3L), an uncharacterised pituitary specific membrane protein, in non-neoplastic anterior pituitary gland and pituitary neuroendocrine tumours. A polyclonal antibody produced within the Human Protein Atlas project (HPA074356) was used for TGFBR3L staining and combined with SF1 and FSH for a 3-plex fluorescent protocol, providing more details about the cell lineage specificity of TGFBR3L expression. A cohort of 230 pituitary neuroendocrine tumours were analysed. In a subgroup of previously characterised gonadotroph tumours, correlation with expression of FSH/LH, E-cadherin, oestrogen (ER) and somatostatin receptors (SSTR) was explored. TGFBR3L showed membranous immunolabeling and was found to be gonadotroph cell lineage-specific, verified by co-expression with SF1 and FSH/LH staining in both tumour and non-neoplastic anterior pituitary tissues. TGFBR3L immunoreactivity was observed in gonadotroph tumours only and demonstrated intra-tumour heterogeneity with a perivascular location. TGFBR3L immunostaining correlated positively to both FSH (R = 0.290) and LH (R = 0.390) immunostaining, and SSTR3 (R = 0.315). TGFBR3L correlated inversely to membranous E-cadherin staining (R = −0.351) and oestrogen receptor β mRNA (R = −0.274). In conclusion, TGFBR3L is a novel pituitary gland specific protein, located in the membrane of gonadotroph cells in non-neoplastic anterior pituitary gland and in a subset of gonadotroph pituitary tumours.

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“…We developed a 5-plex immunofluorescence (IF) protocol [31], with novel monoclonal antibodies (mAbs) recognizing caspase cleavage sites on tau, and quantitative analyses in tissue microarray (TMA) blocks containing well-characterized postmortem brain tissue from two cortical areas of common tauopathies and healthy controls to investigate the following questions: 1) Is there evidence of aCasp-6 in non-AD tauopathies? 2) Besides D421, is the accumulation of caspase-6 tr-tau fragments present in AD and other tauopathies?…”

Section: Discussionmentioning

confidence: 99%

Caspase-6-cleaved tau is relevant in Alzheimer’s disease but not in other tauopathies: diagnostic and therapeutic implications

Theofilas

Piergies

et al. 2021

Preprint

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AimTau truncation (tr-tau) by active caspase-6 (aCasp-6) generates toxic tau fragments prone to self-aggregation. Yet, the relationship between aCasp-6, different forms of tr-tau, and hyperphosphorylated tau (p-tau) accumulation in human brains with Alzheimer’s disease (AD) and other tauopathies remains unclear.MethodsWe generated two neoepitope monoclonal antibodies against tr-tau sites (D402 and D13) targeted by aCasp-6. Then, we used 5-plex immunofluorescence to quantify the neuronal and astroglial burden of aCasp-6, tr-tau, p-tau, and their co-occurrence in healthy controls, AD, and primary tauopathies.ResultsCasp-6 activation was strong in AD, followed by Pick’s disease (PiD), but almost absent in 4-repeat (4R) tauopathies. Tr-tau neuronal burden was much higher in AD than in 4R tauopathies, and disproportionally higher when normalizing by p-tau pathology. Tr-tau astrogliopathy was detected in low numbers in 4R tauopathies. Unexpectedly, half of the tr-tau positive neurons in AD lacked p-tau aggregates.ConclusionsEarly modulation of aCasp-6 with consequent amortization of tr-tau pathology is a promising therapeutic strategy in AD and possibly PiD, but it is unlikely to benefit 4R tauopathies. The large percentage of tr-tau neurons lacking p-tau suggests that not all tau pathology and vulnerable neurons are detected by conventional p-tau Ser 202 antibody and, that AD, has distinct mechanisms of tangle formation. Therapeutic strategies against tr-tau pathology could modulate tau abnormalities in AD. The disproportional higher burden of tr-tau in AD supports the evaluation of biofluid biomarkers against N-terminus tr-tau to detect AD and differentiate it from 4R tauopathies at a single patient level.

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A manual multiplex immunofluorescence method for investigating neurodegenerative diseases

Cited by 13 publications

References 52 publications

Molecular characterization of selectively vulnerable neurons in Alzheimer’s Disease

Molecular characterization of selectively vulnerable neurons in Alzheimer’s Disease

TGFBR3L—An Uncharacterised Pituitary Specific Membrane Protein Detected in the Gonadotroph Cells in Non-Neoplastic and Tumour Tissue

Caspase-6-cleaved tau is relevant in Alzheimer’s disease but not in other tauopathies: diagnostic and therapeutic implications

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