Association of region-specific hippocampal reduction of neurogranin with inflammasome proteins in post mortem brains of Alzheimer's disease

Abstract

Introduction: Neurogranin (Ng) is considered a biomarker for synaptic dysfunction in Alzheimer's disease (AD). In contrast, the inflammasome complex has been shown to exacerbate AD pathology.

Methods: We investigated the protein expression, morphological differences of Ng, and correlated Ng to hyperphosphorylated tau in the post mortem brains of 17 AD cases and 17 age- and sex-matched controls. In addition, we correlated the Ng expression with two different epitopes of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC).

Results: We show a reduction of Ng immunopositive neurons and morphological differences in AD compared to controls. Ng immunostaining was negatively correlated with neurofibrillary tangles, humanized anti-ASC (IC100) positive neurons and anti-ASC positive microglia, in AD.

Discussion: The finding of a negative correlation between Ng and ASC speck protein expression in post mortem brains of AD suggests that the activation of inflammasome/ASC speck pathway may play an important role in synaptic degeneration in AD.

Highlights: We show the role that neurogranin plays on post-synaptic signaling in specific hippocampal regions.We demonstrate that there could be clinical implications of using neurogranin as a biomarker for dementia.We describe the loss of plasticity and neuronal scaffolding proteins in the present of AD pathology.We show the response of neuroinflammation when tau proteins phosphorylate in hippocampal neurons.We show that there is a potential therapeutic target for the inflammasome, and future studies may show that IC100, a humanized monoclonal antibody directed against ASC, may slow the progression of neurodegeneration.

Keywords: Alzheimer's disease; axon; inflammasome; microglia; neurogranin.

FIGURE 1

Examples of immunoreactivity of mouse‐anti‐tau (AT‐8), mouse‐anti‐microtubule‐associated protein 2 (MAP2), and mouse‐anti‐neurogranin (Ng) in normal control and in a case with intermediate Alzheimer's disease (AD) in a section from the hippocampal region cornu ammonis (CA2). The control (A) shows sparse apical primary dendritic staining of tau in the pyramidal neurons; in the AD case there are moderate (E) amounts of staining spreading into the apical dendrite. The MAP2 staining was seen in both groups, but the processes are well defined in the control (B) compared to cases of intermediate AD (F). Ng protein expression differs between the groups in pyramidal neurons, as this appears denser and more concentrated around the nucleus of pyramidal neurons in the intermediate AD cases (G), whereas in the control cases the staining throughout the apical dendritic processes (C). No significant Pearson's correlation, in the controls, was seen between the number of Ng2 positive neurons and the number of neurofibrillary tangles (NFTs) defined with phosphorylated tau (pTau; D), whereas a significant negative correlation between Ng and NFTs was seen in the intermediate AD cases (H). Scale bars = 10 μm (A, B, C, E, F, G)

FIGURE 2

Photomicrographs of the immunostaining seen in the cornu ammonis (CA3) hippocampal region. Mouse‐anti‐phosphorylated tau (pTau; AT‐8), mouse‐anti‐microtubule‐associated protein 2 (MAP2), and mouse‐anti‐neurogranin (Ng) is identified in controls and intermediate Alzheimer's disease (AD). In (A), control cases, there is a sparse density staining of the neurofibrillary tangles (NFTs) in the pyramidal neurons, in the intermediate AD case (D) there is a frequent amount of staining that spreads the dendritic processes. The MAP2 staining was seen in both the control (B) and intermediate AD cases (E). Extracellular and intracellular Ng2 expression is seen the controls (C), but in the intermediate AD group, the expression is seen in the pyramidal neuronal bodies (F). Pearson's correlation analysis did not reveal any significant relationship between the number of Ng2‐positive neurons and the number of NFTs defined with pTau in the control group (G), whereas there was a significant negative correlation between Ng and NFTs in the intermediate AD cases (H). Scale bars = 30 μm (A, B, C, E, F, G)

FIGURE 3

Pearson's correlation analysis of Ng and NFTs (AT‐8) in the CA1 hippocampal region and surrounding structures. The correlation analysis did not reveal any significant relationship between the number of Ng‐positive neurons and the number of NFTs defined with pTau in the control group in the entorhinal cortex (A), the subiculum (C), the CA1 (E), and the dentate gyrus (G). The intermediate AD cases showed a significant negative correlation between Ng and NFTs in the entorhinal cortex (B) and the CA1 hippocampal region (F), and in the subiculum (D) but not in the dentate gyrus (H). AD, Alzheimer's disease; CA, cornu ammonis; NFTs, neurofibrillary tangles; Ng, neurogranin; pTau, hyperphosphorylated tau

FIGURE 4

Pearson's correlation analysis of Ng and humanized anti‐ASC (IC100) in the hippocampal subfields. An example of double‐labeling immunostaining of Ng (green) and 1C100 (red) of controls (A) in intermediate AD (B). In the controls, the correlation analysis did not reveal any significant relationship between the number of Ng positive neurons and the number of IC100 ‐positive neurons (C, and E). The intermediate AD cases showed a significant negative correlation between Ng and IC100 in the CA1 (D), and the CA2 (F) hippocampal regions. AD, Alzheimer's disease; ASC, apoptosis‐associated speck‐like protein containing a caspase recruitment domain; CA, cornu ammonis; Ng, neurogranin. Scale bars = 30 μm (A) and (B)

FIGURE 5

Pearson's correlation analysis of Ng and mouse anti‐ASC proteins seen in the hippocampal subfields. A photomicrograph of Ng (green), 1C100 (red), and mouse anti‐ASC (purple) are seen in control (A, Ai, and Aii) and the intermediate AD cases (B, Bi, and Bii). In the controls, the correlation analysis did not reveal any significant relationship between the number of Ng‐positive neurons and the number of ASC‐positive microglia in CA1 and CA2 (C and E). The intermediate AD cases showed a significant negative correlation between Ng counts and ASC positive microglia in the CA1 (C) and in the CA2 (F) hippocampal regions. AD, Alzheimer's disease; ASC, apoptosis‐associated speck‐like protein containing a caspase recruitment domain; CA, cornu ammonis; Mo, mouse; Ng, neurogranin (Ng). Scale bars = 20 μm (A), (Ai), (Aii), and (B), (Bi), (Bii)