Aging, Alzheimer's, and APOE genotype influence the expression and neuronal distribution patterns of microtubule motor protein dynactin-P50

Abstract

Reports from neural cell cultures and experimental animal studies provide evidence of age- and disease-related changes in retrograde transport of spent or misfolded proteins destined for degradation or recycling. However, few studies address these issues in human brain from those who either age without dementia and overt neuropathology, or succumb to Alzheimer's; especially as such propensity may be influenced by APOE genotype. We studied the expression and distribution of the dynein subunit dynactin-P50, the β amyloid precursor protein (βAPP), and hyperphosphorylated tau (P-tau) in tissues and tissue sections of brains from non-demented, neuropathology-free patients and from Alzheimer patients, with either APOE ε3,3 or APOE ε4,4. We found that advanced age in patients without dementia or neuropathological change was associated with coordinated increases in dynactin-P50 and βAPP in neurons in pyramidal layers of the hippocampus. In contrast, in Alzheimer's, βAPP and dynactin were significantly reduced. Furthermore, the dynactin-P50 and βAPP that was present was located primarily in dystrophic neurites in Aβ plaques. Tissues from Alzheimer patients with APOE ε3,3 had less P-tau, more βAPP, dynactin-P50, and synaptophysin than did tissues from Alzheimer patients carrying APOE ε4,4. It is logical to conclude, then, that as neurons age successfully, there is coordination between retrograde delivery and maintenance and repair, as well as between retrograde delivery and degradation and/or recycling of spent proteins. The buildup of proteins slated for repair, synaptic viability, transport, and re-cycling in neuron soma and dystrophic neurites suggest a loss of this coordination in Alzheimer neurons. Inheritance of APOE ε3,3 rather than APOE ε4,4, is associated with neuronal resilience, suggestive of better repair capabilities, more synapses, more efficient transport, and less hyperphosphorylation of tau. We conclude that even in disease the ε3 allele is neuroprotective.

Keywords: APOE genotype; Alzheimer; P-tau; aging; dynactin-P50; motor proteins; synaptophysin; βAPP.

Figure 1

Examples of immunofluorescent images used in neuritic plaque color overlays and for grayscale quantification. Separate channel images were taken and overlaid, and colocalization was assessed based on color combinations, e.g., Red + Green = Yellow. Red represents hyperphosphorylated tau (AT8) and green represents dynactin-P50 (DynP50).

Figure 2

Age-related changes in expression of dynactinP-50 (DynP50) and β-amyloid precursor protein (βAPP). Dynactin-P50 and βAPP were detected by immunofluorescence in tissue sections from hippocampus at the level of the lateral geniculate nucleus from 6 neurologically normal individuals across an age span of 16–80 years. (A) Representative immunofluorescent images from the six individuals; blue represents DAPI staining of cellular DNA, red represents dynactinP-50, green represents βAPP, and yellow represents colocalization of dynactinP-50 and βAPP. Images were digitized at 20× magnification. Scale bar = 20 μm. Quantitation of dynactinP-50 (B) and βAPP (C) immunofluorescence intensity was obtained by thresholding gray-scale images and integrating pixels as described in Materials and Methods section. Values reflect the mean of 6 images per specimen.

Figure 3

Comparison of dynactin-P50 (DynP50) expression between neurologically normal controls (AMC) and Alzheimer (AD) patients. (A) DynactinP-50 (green) in tissue sections from the hippocampal cell layer. Images were digitized at 20× magnification. Blue represents DAPI staining of cellular DNA. (B) Quantification of DynP50 in AD (n = 4) vs. neurologically normal control (AMC, n = 4). Values reflect the mean of 30 images of the pyramidal cell layer for AD and AMC. Significance determined by Wilcoxon-Mann-Whitney Rank Sum Test, with ^*denoting p ≤ 0.05. Data reported as group mean with error bars denoting SEM.

Figure 4

βAPP levels are reduced in Alzheimer's (AD), and dynactin-P50 (DynP50) and βAPP localization is disrupted. Dynactin-P50 (green) and βAPP (red) were detected by immunofluorescence in tissue sections from hippocampus. (A) Total APP intensity is decreased in AD cases. Blue represents DAPI staining of cellular DNA. Images were digitized at 40× magnification. Scale bar = 10 μm. (B) Overall βAPP fluorescence intensity is diminished in AD compared to neurologically normal controls (AMC). Significance determined by Wilcoxon-Mann-Whitney Rank Sum Test, with ^*denoting p ≤ 0.05. Data reported as group mean with error bars denoting SEM.

Figure 5

Dynactin-P50 (DynP50), βAPP, and hyperphosphorylated tau in neurofibrillary tangles (NFT) localization in AD and neurologically normal controls (AMC). (A) Dynactin-P50 (red), βAPP (green), and NFT (blue) were detected by immunofluorescence in hippocampal tissue of AD and neurologically normal individuals (AMC). In AMC (top row), note the absence of neurofibrillary tangles, and increased dynactin-P50 and βAPP compared to AD (middle and bottom rows). Further, DynP50 and βAPP appear brighter in somas of AMC vs. somas of AD without NFTs (white arrows). Dynactin-P50, APP, and NFT immunoreactivity was observed in anuclear bulbous neurites (yellow arrows). AD somas containing NFTs showed the highest immunoreactivity in AD for dynactin-P50, and βAPP, implying possible aggregation of all three antigens (red arrows). (B) Duolink proximity ligation assay (PLA) detects immunogens within 40 nm distance in tissue sections. Image shows neuritic plaque enriched with dynactin-P50 and P-tau in close proximity.

Figure 6

Comparison of tissue levels of dynactin-P50 (DynP50), P-tau, and synaptophysin (Syn) in Alzheimer patients with APOE ε3,3 vs. ε4,4. (A) Dynactin-P50 (green) was less in AD patients with APOE ε4,4, than in those with ε3,3, while (B) P-tau was increased in ε4,4. Representative images. (C) Fluorescence intensity quantification of P-tau and dynactin-P50 levels in AD patients with ε3,3 vs. ε4,4. (D) Steady state protein levels, relative to that of actin, of P-tau in Alzheimer patients with ε3,3 were lower than levels in those with ε4,4, while synaptophysin and dynactin-P50 levels were higher in ε3,3 compared with ε4,4. (E) Western blot comparison of protein levels in AD vs. Control, and between AD ε3,3 vs. AD ε4,4. Actin was used as loading control. Lanes in gray boxes demonstrate a patient with APOE ε3,3 genotype with similar levels of proteins to those in a patient with APOE ε4,4 genotype, suggestive of the possible presence of Alzheimer risk factors aside from APOE genotype. Wilcoxon-Mann-Whitney Rank Sum Test was used for statistical significance, with ^*denoting p ≤ 0.05 and ^**denoting p ≤ 0.01. Data reported as group mean with error bars denoting SEM.