The effect of prolonged formalin fixation on the expression of proteins in human brain tissues

Wu, Xiaochang; Deng, Congcong; Su, Yan-Yan; Zhang, Chaoyi; Chen, Ming‐Hui; Kai, Tetsuya; Wu, Houwei; Xu, Shaochun

doi:10.1016/j.acthis.2022.151879

Cited by 6 publications

(15 citation statements)

References 32 publications

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“…Delayed autopsy and prolonged fixation—which requires harsh conditions for antigen retrieval—are reported to have limited impact on highly-aggregated proteins such as Aβ and pTau ( 148 ) but can obscure signals for less aggregated proteins. ( 149 , 150 , 151 ) Thus, use of rapidly-autopsied, and uniformly and minimally-fixed brain specimens is an important strength of the present study. Postmortem specimens spanning the clinicopathological spectrum of sAD were used to approximate the spatiotemporal sequence of NFT progression.…”

Section: Discussionmentioning

confidence: 98%

ApoER2-Dab1 disruption as the origin of pTau-related neurodegeneration in sporadic Alzheimer's disease

Ramsden

Zamora

Horowitz

et al. 2023

Preprint

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BACKGROUND Sporadic Alzheimer’s disease (sAD) is not a global brain disease. Specific regions, layers and neurons degenerate early while others remain untouched even in advanced disease. The prevailing model used to explain this selective neurodegeneration—prion-like Tau spread—has key limitations and is not easily integrated with other defining sAD features. Instead, we propose that in humans Tau hyperphosphorylation occurs locally via disruption in ApoER2-Dab1 signaling and thus the presence of ApoER2 in neuronal membranes confers vulnerability to degeneration. Further, we propose that disruption of the Reelin/ApoE/ApoJ-ApoER2-Dab1-P85α-LIMK1-Tau-PSD95 (RAAAD-P-LTP) pathway induces deficits in memory and cognition by impeding neuronal lipoprotein internalization and destabilizing actin, microtubules, and synapses. This new model is based in part on our recent finding that ApoER2-Dab1 disruption is evident in entorhinal-hippocampal terminal zones in sAD. Here, we hypothesized that neurons that degenerate in the earliest stages of sAD (1) strongly express ApoER2 and (2) show evidence of ApoER2-Dab1 disruption through co-accumulation of multiple RAAAD-P-LTP components. METHODS We applied in situ hybridization and immunohistochemistry to characterize ApoER2 expression and accumulation of RAAAD-P-LTP components in five regions that are prone to early pTau pathology in 64 rapidly autopsied cases spanning the clinicopathological spectrum of sAD. RESULTS We found that: (1) selectively vulnerable neuron populations strongly express ApoER2; (2) numerous RAAAD-P-LTP pathway components accumulate in neuritic plaques and abnormal neurons; and (3) RAAAD-P-LTP components were higher in MCI and sAD cases and correlated with histological progression and cognitive deficits. Multiplex-IHC revealed that Dab1, pP85αTyr607, pLIMK1Thr508, pTau and pPSD95Thr19 accumulated together within dystrophic dendrites and soma of ApoER2-expressing neurons in the vicinity of ApoE/ApoJ-enriched extracellular plaques. These observations provide evidence for molecular derangements that can be traced back to ApoER2-Dab1 disruption, in each of the sampled regions, layers, and neuron populations that are prone to early pTau pathology. CONCLUSION Findings support the RAAAD-P-LTP hypothesis, a unifying model that implicates dendritic ApoER2-Dab1 disruption as the major driver of both pTau accumulation and neurodegeneration in sAD. This model provides a new conceptual framework to explain why specific neurons degenerate and identifies RAAAD-P-LTP pathway components as potential mechanism-based biomarkers and therapeutic targets for sAD.

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Section: Discussionmentioning

confidence: 98%

ApoER2-Dab1 disruption as the origin of pTau-related neurodegeneration in sporadic Alzheimer's disease

Ramsden

Zamora

Horowitz

et al. 2023

Preprint

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“…Delayed autopsy and prolonged fixation are reported to have limited impact on highly-aggregated proteins such as Aβ and pTau [ 129 ] but can obscure signals for less aggregated proteins [ 7 , 66 , 163 ]. Thus, use of rapidly-autopsied specimens that underwent uniform, time-limited (48 h) fixation, is an important strength of the present study.…”

Section: Discussionmentioning

confidence: 99%

ApoER2-Dab1 disruption as the origin of pTau-associated neurodegeneration in sporadic Alzheimer’s disease

Ramsden,

Zamora,

Horowitz

et al. 2023

acta neuropathol commun

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In sporadic Alzheimer’s disease (sAD) specific regions, layers and neurons accumulate hyperphosphorylated Tau (pTau) and degenerate early while others remain unaffected even in advanced disease. ApoER2-Dab1 signaling suppresses Tau phosphorylation as part of a four-arm pathway that regulates lipoprotein internalization and the integrity of actin, microtubules, and synapses; however, the role of this pathway in sAD pathogenesis is not fully understood. We previously showed that multiple ApoER2-Dab1 pathway components including ApoE, Reelin, ApoER2, Dab1, pP85αTyr607, pLIMK1Thr508, pTauSer202/Thr205 and pPSD95Thr19 accumulate together within entorhinal-hippocampal terminal zones in sAD, and proposed a unifying hypothesis wherein disruption of this pathway underlies multiple aspects of sAD pathogenesis. However, it is not yet known whether ApoER2-Dab1 disruption can help explain the origin(s) and early progression of pTau pathology in sAD. In the present study, we applied in situ hybridization and immunohistochemistry (IHC) to characterize ApoER2 expression and accumulation of ApoER2-Dab1 pathway components in five regions known to develop early pTau pathology in 64 rapidly autopsied cases spanning the clinicopathological spectrum of sAD. We found that (1) these selectively vulnerable neuron populations strongly express ApoER2; and (2) multiple ApoER2-Dab1 components representing all four arms of this pathway accumulate in abnormal neurons and neuritic plaques in mild cognitive impairment (MCI) and sAD cases and correlate with histological progression and cognitive deficits. Multiplex-IHC revealed that Dab1, pP85αTyr607, pLIMK1Thr508, pTauSer202/Thr205 and pPSD95Thr19 accumulate together within many of the same ApoER2-expressing neurons and in the immediate vicinity of ApoE/ApoJ-enriched extracellular plaques. Collective findings reveal that pTau is only one of many ApoER2-Dab1 pathway components that accumulate in multiple neuroanatomical sites in the earliest stages of sAD and provide support for the concept that ApoER2-Dab1 disruption drives pTau-associated neurodegeneration in human sAD.

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“…These studies showed that the influence of prolonged post-fixation on IHC and/or HC staining was either negative or unaltered. However, the time spans of post-fixation for the examined group versus the control group from these prior studies were either very short (weeks or months) (Webster et al, 2009;Pikkarainen et al, 2010;Wu et al, 2022) or rather wide (a group with mixed post-fixation times from 1 to 20 years) (Alrafiah and Alshali, 2019). Furthermore, the detailed information on post-fixation times for presented data were missing and no quantitative analysis was performed (Alrafiah and Alshali, 2019), so it was not possible to systematically correlate the qualitative data with the post-fixation times.…”

Section: Introductionmentioning

confidence: 99%

Differential effects of prolonged post-fixation on immunohistochemical and histochemical staining for postmortem human brains

Ma,

Frigon,

Maranzano

et al. 2024

Preprint

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Immunohistochemical (IHC) and histochemical (HC) staining is widely used for human brains postfixed in formalin for years and decades. Understanding the effect of prolonged post-fixation, postmortem interval (PMI) and age on these staining procedures is important for interpreting their outcomes, thus improving diagnosis and research of brain disorders. In this study, we performed both IHC and HC staining for prefrontal cortex (PFC) of postmortem human brains postfixed for 1, 5, 10, 15 and 20 years. A negative correlation was detected between the intensity of neuron marker NeuN, microglia marker Iba1, cresyl violet and Luxol fast blue staining versus post-fixation times. By contrast, a positive correlation was seen between the intensity of astrocyte marker GFAP and H&E staining versus post-fixation times. No correlation was found between most of these parameters versus age or PMI. These data suggest that prolonged post-fixation exerts both positive and negative effects, but age and PMI have limited effects, on these IHC and HC parameters. Hence these differential changes need to be considered in the interpretation of the results when using tissues with prolonged post-fixation. It is recommended to perform IHC and HC staining for human brains with similar post-fixation time to offset its impact on downstream analyses.

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The effect of prolonged formalin fixation on the expression of proteins in human brain tissues

Cited by 6 publications

References 32 publications

ApoER2-Dab1 disruption as the origin of pTau-related neurodegeneration in sporadic Alzheimer's disease

ApoER2-Dab1 disruption as the origin of pTau-related neurodegeneration in sporadic Alzheimer's disease

ApoER2-Dab1 disruption as the origin of pTau-associated neurodegeneration in sporadic Alzheimer’s disease

Differential effects of prolonged post-fixation on immunohistochemical and histochemical staining for postmortem human brains

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