Increased expression of the proapoptotic presenilin associated protein is involved in neuronal tangle formation in human brain

Abstract

Presenilin-associated protein (PSAP) is a mitochondrial proapoptotic protein as established in cell biology studies. It remains unknown whether it involves in neurodegenerative diseases. Here, we explored PASP expression in adult and aged human brains and its alteration relative to Alzheimer-disease (AD)-type neuropathology. In pathology-free brains, light PASP immunoreactivity (IR) occurred among largely principal neurons in the cerebrum and subcortical structures. In the brains with AD pathology, enhanced PSAP IR occurred in neuronal and neuritic profiles with a tangle-like appearance, with PSAP and pTau protein levels elevated in neocortical lysates relative to control. Neuronal/neuritic profiles with enhanced PSAP IR partially colocalized with pTau, but invariably with Amylo-Glo labelled tangles. The neuronal somata with enhanced PASP IR also showed diminished IR for casein kinase 1 delta (Ck1δ), a marker of granulovacuolar degeneration; and diminished IR for sortilin, which is normally expressed in membrane and intracellular protein sorting/trafficking organelles. In old 3xTg-AD mice with β-amyloid and pTau pathologies developed in the brain, PSAP IR in the cerebral sections exhibited no difference relative to wildtype mice. These findings indicate that PSAP upregulation is involved in the course of tangle formation especially in the human brain during aging and in AD pathogenesis.

Keywords: Amyloid plaques; Brain aging; Dystrophic neurites; Neuronal death; Neuronal tangles; Tauopathy.

Fig. 1

Presenilin-associated protein (PSAP) immunoreactivity. (A) shows the low magnification view, with the framed areas enlarged sequentially as indicated. PSAP IR appears neuropil-like in the cerebral cortex and cellular layers of the hippocampal formation. (B-B3, C-C3) Relatively large-sized pyramidal neurons show the highest IR, especially in those located in deep layers III and V. (D-D3; E-E3) The pyramidal neurons in the layer II islands of the entorhinal cortex and the subicular and hippocampal subregions as well as the hilar mossy cells in the dentate gyrus are also clearly labeled. CA1-CA3: Ammon’s horn subregions; DG: dentate gyrus; FG: fusiform gyrus; GCL: granule cell layer: Hi: hilus; ITG: inferior temporal gyrus; I-VI: cortical layers I to VI; ML: molecular layer; MTG: middle temporal gyrus; PHG: parahippocampal gyrus; Pro-S: prosubiculum; Pre-S: presubiculum; Sub: subiculum; s.o.: stratum oriens; s.p.: stratum pyramidale; s.r.: stratum radiatum, WM: white matter. Scale bars are as indicated.

Fig. 6

Double immunofluorescent characterization of presenilin-associated protein (PSAP), and casein kinase 1δ (CK1δ) in pAD/AD human brains. (A) The low power view of double-labeled temporal lobe paraffin section with DAPI counterstain, with framed areas enlarged as other panel sets (B-E). The PSAP-enriched neuronal somata (pointed by arrows) and dystrophic neurites (open arrowheads) rarely colocalize with CK1δ IR, whereas CK1δ labeled granulovacuolar degeneration (GVD) bodies (pointed by white arrowheads) are present in other neuronal somata with light PSAP IR. (F) The numbers of PSAP positive neurons with and without CK1δ labeled GVD bodies quantified in the parahippocampal neocortex and subicular to CA1 pyramidal layers from four brains, with the ratios indicated (mean ± SD, n = 4, two-tailed unpaired t-test). CA1-CA3: Ammon’s horn subregions; DG: dentate gyrus; PHG: parahippocampal gyrus; Pro-S: prosubiculum; Pre-S: presubiculum; Sub: subiculum. Antibodies and fluorescence channels, and scale bars, are as indicated in the image panels.

Fig. 2

Presenilin-associated protein (PSAP) IR in the temporal lobe regions in a probable Alzheimer’s disease (pAD) human brain. (A) Low magnification view, with the framed areas enlarged sequentially as indicated. PSAP IR is apparently enhanced in a subpopulation of pyramidal neurons in the temporal neocortex (B-B2, C-C2) and subicular and CA subregions (D, D1), and mossy cells in the dentate gyrus (D, D2). These darkly labeled neurons show a tangle-like morphological appearance. Heavily labeled neuritic processes (as pointed by arrows) are also present in the cortical and hippocampal regions. CA1-CA3: Ammon’s horn subregions; DG: dentate gyrus; FG: fusiform gyrus; GCL: granule cell layer: Hi: hilus; ITG: inferior temporal gyrus; I-VI: cortical layers I to VI; ML: molecular layer; MTG: middle temporal gyrus; PHG: parahippocampal gyrus; Pro-S: prosubiculum; Pre-S: presubiculum; Sub: subiculum; s.o.: stratum oriens; s.p.: stratum pyramidale; s.r.: stratum radiatum, WM: white matter. Scale bars are as indicated.

Fig. 3

Comparative assessment of presenilin-associated protein (PSAP) and phosphorylated tau (pTau) immunolabeling in paraffin sections and their protein elevations in pAD/AD human cortical lysates. (A-A4, B-B4) Micrograph panels show PSAP (A-A4) and pTau (B-B4) immunolabeling with hematoxylin counterstain in adjacent temporal lobe sections from an AD case. PSAP and pTau labeled somata (arrows) and neurites (hallowed arrows) show similar morphological features. The nuclei in the somata with heavy tangle-like products appear to be lost, dislocated near the cell border, or shrunken (A1-A4, B1-B4). PSAP-labeled neurites are mostly elongated, while pTau-labeled neurites appear elongated (hallowed arrows) as well as globular (arrowheads). (C) show the immunoblotted PSAP (assayed with 10% SDS-PAGE gel) and pTau (AT8, assayed with 10% SDS-PAGE gel). (D) show the bands of total tau (TAU-5, assayed with 12% SDS-PAGE), and Aβ products blotted with the antibody D12B2 (assayed with 15% SDS-PAGE gel. (E) The full-length protein and clearage fragment of the β-amyloid precursor protein (APP) and Aβ productions blotted with the 6E10 antibody (assayed 12% SDS-PAGE gel). Immunoblots from a set of cortical lysates from four pAD/AD cases and four neuropathology-free controls (CTL) are presented as indicated. Original western blot images were shown in Additional file 1. (F) The bar/dot graphs show the densitometric data and statistics from the two groups as indicated (mean ± SD, n = 8/group, two-tailed unpaired t-test). Arrowheads point to the putative monomer Aβ productions. CA1-CA3: Ammon’s horn subregions; DG: dentate gyrus; FG: fusiform gyrus; GCL: granule cell layer: Hi: hilus; PHG: parahippocampal gyrus; Pro-S: prosubiculum; Pre-S: presubiculum; Sub: subiculum; s.p.: stratum pyramidale; s.r.: stratum radiatum, WM: white matter.

Fig. 4

Double immunofluorescent characterization of presenilin-associated protein (PSAP) and phosphorylated tau (pTau) (AT8 antibody) in pAD/AD human brain sections. (A) The low power view of immunofluorescently stained temporal lobe paraffin section with DAPI counterstain (from an AD case), with framed areas enlarged as other panel sets (B-E). Fluorescent markers are as indicated. PSAP and pTau immunofluorescence are partially colocalized in neuronal somata and dystrophic neurites (open arrows). Thus, neuronal somata singly labeled for PSAP (pointed by arrows), singly labeled for pTau (pointed by empty arrows), and double-labeled (pointed by arrowheads), are present in the same microscopic field. A diminished DAPI labeling is seen among many PSAP labeled neurons. (F) The numbers of the single- and double-labeled neurons based on quantification in the parahippocampal neocortex from four brains, with cell type ratios indicated (mean ± SD, n = 4, one-way ANOVA together with Bonferroni post hoc test). CA1-CA3: Ammon’s horn subregions; DG: dentate gyrus; Ent: entorhinal cortex; PHG: parahippocampal gyrus; Pro-S: prosubiculum; Pre-S: presubiculum; Sub: Subiculum; WM: white matter. Antibodies and fluorescence channels, and scale bars, are as indicated in the image panels.

Fig. 5

Triple fluorescent characterization of presenilin-associated protein (PSAP), phosphorylated tau (pTau) and Amylo-Glo labeling in pAD/AD human brain sections. (A) The low power view of triple-labeled temporal lobe paraffin section, with framed areas enlarged. (B) Neuritic plaques (hallowed triangles) in the molecular layer of the dentate gyrus. A great majority of the dystrophic neurites appear triple-labeled, with the Amylo-Glo also staining extracellular amyloid deposits. (C, D) Varied colocalization patterns of PSAP, pTau and Amylo-Glo among somal profiles, which can be identified as five groups: PSAP/AT8/Amylo-Glo triple-labeled (pointed by white arrows), PSAP/Amylo-Glo double-labeled (pointed by white arrowheads), AT8/Amylo-Glo double- labeled (hallowed white arrows), Amylo-Glo single labeled (hollowed arrowheads) and AT8 single labeled (two-tailed arrows). (D) The numbers of the five types of neuronal somata were quantified in four brains, with the calculated cell type ratios indicated (mean ± SD, n = 4, one-way ANOVA together with Bonferroni post hoc test). CA1-CA3: Ammon’s horn subregions; DG: dentate gyrus; Ent: entorhinal cortex; GCL: granule cell layer; Hi: hilus; ML: molecular layer; PHG: parahippocampal gyrus; Pro-S: prosubiculum; Pre-S: presubiculum; Sub: subiculum. Antibodies and fluorescence channels, and scale bars, are as indicated in the image panels.

Fig. 7

Double immunofluorescent characterization of presenilin-associated protein (PSAP) and sortilin in pAD/AD human brains. (A) Section map, with enlarged areas show that the majority of PSAP-enriched neuronal somata (pointed by arrows) are not colocalized with sortilin immunoreactivity across the regions (B-E), while a few of cells colocalized somata observed (pointed by arrowhead). No sortilin colocalization is seen in PSAP-labeled dystrophic neurites (C, open arrows). The DAPI nuclear stain in the PSAP enriched neurons appears reduced or lost. (F) The numbers of PSAP-positive neurons with and without sortilin labeling counted in the parahippocampal neocortex and subicular to CA1 pyramidal layers, with the ratios calculated (mean ± SD, n = 4, two-tailed unpaired t-test). CA1-CA3: Ammon’s horn subregions; DG: dentate gyrus; Ent: entorhinal cortex; PHG: parahippocampal gyrus; Pro-S: prosubiculum; Pre-S: presubiculum; Sub: subiculum. Antibodies and fluorescence channels, and scale bars, are as indicated in the image panels.

Fig. 8

Comparative assessment of presenilin-associated protein (PSAP), phosphorylated tau (pTau) and β-amyloid (Aβ) IR in 3xTg-AD and C57BL/6J mouse forebrain. Light PSAP immunolabeling is present in the cerebral cortex and hippocampal formation, localized to the somata and dendrites of pyramidal neurons, which is similar between the 3xTg-AD (A-A3) and C57BL/6J (D-D2) brain sections. pTau IR is distinct in cortical and hippocampal pyramidal neurons in the transgenics (B-B3), which is absent in the C57BL/6J mouse brain section (E-E2). Aβ deposition is seen only in the transgenics, most evident in the subicular and CA1 areas (C-C3; F-F1). CA1 and CA2: Ammon’s horn subregions; DG: dentate gyrus; III-VI: cortical layers; WM: white matter. Scale bars are indicated.