Ser46-Phosphorylated MARCKS Is a Marker of Neurite Degeneration at the Pre-aggregation Stage in PD/DLB Pathology

Comprehensive phosphoproteomic analyses of postmortem human AD and DLB brains. A, Occipital and temporal tips of pathologically pure AD (five females) and DLB (5 females) brains along with age-matched normal controls (5 females) were used for comprehensive phosphoproteome analyses as described previously (Tagawa et al., 2015). Independent phospho-sites were compared between AD and DLB. B, Comparison of changes at four different phosphorylation sites in MARCKS [Ser27/Ser27, Ser46/Ser46, Ser145/Ser138, and Thr150/Thr143 (human/mouse)] between human DLB and AD patients. (See also Fig. 1-1.)

Immunohistochemistry of human DLB brains. A, Costaining of pSer46-MARCKS with MAP2. Low and high magnification of temporal lobes from non-neurologic disease control patients (5 females) and human DLB patients (5 females). Images were acquired on an Olympus FV1200 IX83 confocal microscopy. All bar graphs indicate averages and SEM. In each patient, quantitative analyses of signal intensities (mean pixel intensities) were performed in 10 visual fields (100 × 100 μm) randomly selected from the corresponding area. Statistical analyses were performed with Student’s t test; **, p < 0.01. B, Staining of pSer129-α-Syn revealed multiple cytoplasmic inclusions (Lewy bodies) in the same patient group. C, Costaining of ubiquitin with pSer129-α-Syn. D, Costaining of pSer46-MARCKS with pSer129-α-Syn. E, Western blotting analysis of temporal lobes from non-DLB control patients and DLB patients with antibody against pSer46-MARCKS, total MARCKS, and β-actin. Graph shows the quantitative result of pSer46-MARCKS from 5 patients and 5 non-neurologic disease controls. The band intensity was normalized against β-actin. Statistical analyses were performed with Student’s t test; *, p < 0.05.

Chronological changes of pSer46-MARCKS in human α-Syn-BAC-Tg/GBA-hetero-KO mice. pSer46-MARCKS and pSer129-α-Syn were costained in human normal α-Syn-BAC-Tg/glucocerebrosidase (GBA)-hetero-KO mice at 1, 6, and 24 months of age (3 male mice at each time point). Images were acquired by Olympus FV1200 IX83 confocal microscopy. All bar graphs show average and SEM. Three mice were used for each group, and quantitative analyses of signal intensities (mean pixel intensities) were performed in 10 visual fields (100 × 100 μm) of each mouse, randomly selected from the brain region. Statistical analyses were performed with two-way ANOVA followed by Student’s t test; *, p < 0.05; **, p < 0.01. The increase in phosphorylation was first detected in olfactory bulb and frontal cortex, followed by the temporal and occipital cortices. Signal intensities of pSer46-MARCKS increased rapidly in the temporal and occipital regions and became most prominent among multiple brain regions. (See also Figs. 3-1, 3-2, and 3-3.)

Costaining of pSer46-MARCKS and pSer129-α-Syn in olfactory bulb of human α-Syn-BAC-Tg/GBA-hetero-KO mice. A–C, High magnification of olfactory bulb of human α-Syn-BAC-Tg/GBA-hetero-KO mice costained with antibodies against pSer46-MARCKS and pSer129-α-Syn. Cytoplasmic staining of pSer46-MARCKS (white arrow) and dot-like stains of pSer129-α-Syn (asterisk) were detected from 1 month of age, whereas cytoplasmic aggregates of pSer129-α-Syn were detected only at 24 months. D, Ubiquitin was costained as dot-like structures or cytoplasmic aggregates in a subset of cells (yellow arrow), whereas pSer129-α-Syn–positive/ubiquitin-negative dots or aggregates were also observed.

Immunohistochemistry of the parietal lobe of α-Syn-BAC-Tg/GBA-hetero-KO mice. A, pSer46-MARCKS and pSer129-α-Syn were costained in external and internal pyramidal cell layers at 24 months of age. pSer46-MARCKS was stained in both apical dendrites and cell bodies, whereas pSer129-α-Syn was stained in cytoplasmic aggregates. B, Parietal cortex tissues from human α-Syn-BAC-Tg/GBA-hetero-KO mice were costained for pSer129-α-Syn and pSer46-MARCKS or ubiquitin at 1, 6, and 24 months of age. Costaining patterns similar to those in the olfactory bulb were confirmed in the parietal lobe.

Western blot analysis of α-Syn-BAC-Tg/GBA-hetero-KO mice at multiple time points. Left panels show Western blots of whole cortex (3 males at each time point) with antibodies against pSer46-MARCKS, pSer129-α-Syn, and ubiquitin. Right graphs show quantitative analyses of three independent blots for pSer46-MARCKS, pSer129-α-Syn, and ubiquitin; band intensities were normalized against β-actin. Statistical analyses were performed with two-way ANOVA followed by Student’s t test; *, p < 0.05; **, p < 0.01. (See also Fig. 6-1.)

Interaction between MARCKS and α-Syn. A, Immunoprecipitates by anti-pSer46-MARCKS or anti-pSer129-α-Syn antibody from brain samples of α-Syn-BAC-Tg/GBA-hetero-KO mice at 1, 6, and 24 months were blotted with anti-pSer129-α-Syn or anti-pSer46-MARCKS antibody, respectively. B, The similar coprecipitations from temporal lobe cortexes of human DLB patients were examined.