Role of Alterations in Protein Kinase p38γ in the Pathogenesis of the Synaptic Pathology in Dementia With Lewy Bodies and α-Synuclein Transgenic Models

Abstract

Progressive accumulation of the pre-synaptic protein α-synuclein (α-syn) has been strongly associated with the pathogenesis of neurodegenerative disorders of the aging population such as Alzheimer's disease (AD), Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy. While the precise mechanisms are not fully understood, alterations in kinase pathways including that of mitogen activated protein kinase (MAPK) p38 have been proposed to play a role. In AD, p38α activation has been linked to neuro-inflammation while alterations in p38γ have been associated with tau phosphorylation. Although p38 has been studied in AD, less is known about its role in DLB/PD and other α-synucleinopathies. For this purpose, we investigated the expression of the p38 family in brains from α-syn overexpressing transgenic mice (α-syn Tg: Line 61) and patients with DLB/PD. Immunohistochemical analysis revealed that in healthy human controls and non-Tg mice, p38α associated with neurons and astroglial cells and p38γ localized to pre-synaptic terminals. In DLB and α-syn Tg brains, however, p38α levels were increased in astroglial cells while p38γ immunostaining was redistributed from the synaptic terminals to the neuronal cell bodies. Double immunolabeling further showed that p38γ colocalized with α-syn aggregates in DLB patients, and immunoblot and qPCR analysis confirmed the increased levels of p38α and p38γ. α1-syntrophin, a synaptic target of p38γ, was present in the neuropil and some neuronal cell bodies in human controls and non-Tg mice. In DLB and and Tg mice, however, α1-syntrophin was decreased in the neuropil and instead colocalized with α-syn in intra-neuronal inclusions. In agreement with these findings, in vitro studies showed that α-syn co-immunoprecipitates with p38γ, but not p38α. These results suggest that α-syn might interfere with the p38γ pathway and play a role in the mechanisms of synaptic dysfunction in DLB/PD.

Keywords: MAPK; dementia with Lewy bodies; p38gamma; synaptic pathology; synucleinopathies; α-synuclein.

FIGURE 1

Immunohistochemical analysis of the distribution of p38α and p38γ in DLB and control brains. Vibratome sections from the frontal cortex were immunolabeled with antibodies against p38α and p38γ and developed with DAB. (A) Left: representative low power bright field microscopic images (200×) (scale bar = 40 μm) of human brains from healthy controls (top) and DLB patients (bottom) immunostained with a p38α, middle: enlarged images (630×) of glial cells from the overview panel (*) (scale bar = 10 μm), right: enlarged images (630×) of neuropil and blood vessels from the overview panel (**) (scale bar = 10 μm). (B,C) Number of p38α positive cells per 0.1 mm² and overall optical density of the neuropil. (D) Left: representative low power bright field microscopic images (200×) (scale bar = 40 μm) of healthy controls (top) and DLB patients (bottom) immunostained with a p38γ, middle: enlarged images (630×) of neuronal cells (represented as N in control) from the overview panel (*) (scale bar = 10 μm), right: enlarged images (630×) of neuropil pre-synaptic terminals (**) (scale bar = 10 μm). (E,F) Number of p38γ positive cells per 0.1 mm² and overall optical density of the neuropil. n = 8 for control and n = 12 for DLB (**p < 0.05, ***p < 0.001).

FIGURE 2

Double immunohistochemical analysis of p38γ cellular localization in DLB and control brains. Vibratome sections from the frontal cortex of human control and DLB cases were double immunofluorescence labeled with antibodies against p38γ (red) and neuronal and glial cell markers (green) and analyzed with Apotome II mounted in a Carl Zeiss AxioImager Z1 microscope. Far right (detail): enlarged image of the area contained in the dotted square. (A) p38γ (red) and the pre-synaptic marker synaptophysin (green), colocalization in yellow; (B) image analysis showing high colocalization of p38γ to synapses; (C) p38γ (red) and the neuronal marker NeuN (green); (D) image analysis showing increased localization of p38γ to neurons in DLB; (E) p38γ (red) and the astroglial cell marker GFAP (green); (F) image analysis showing minimal localization of p38γ to astroglia in human brains; (G) p38γ (red) and α-synuclein (green); (H) image analysis showing high colocalization of p38γ to Lewy bodies in DLB cases; (I)α1-syntrophin (red) and the pre-synaptic marker synaptophysin (green), colocalization in yellow; (J) image analysis showing low colocalization of α1-syntrophin to pre-synaptic site in control and DLB cases; (K)α1-syntrophin (red) and α-synuclein (green); (L) image analysis showing high localization of α1-syntrophin and α-syn in DLB. Scale bars are 10 μm in the standard panels and 5 μm in the zoomed panels. Image analysis represents% colocalization between the two markers. n = 8 for control and n = 12 for DLB (***p < 0.001). Image analysis of p38γ and synaptic proteins in DLB and control brains. The single labeled channels from above were further analyzed to calculate the percent area of the neuropil occupied by (M) synaptophysin; (N) α-syn; (O) p38γ; and (P) α1-syntrophin fluorescence in healthy control and DLB patients. (Q) Linear regression analysis was performed to ascertain the correlation coefficient between the levels of p38γ in the neuropil (% area) and synaptophysin, α-syn, and α1-syntrophin in control and DLB cases. n = 8 for control and n = 12 for DLB (*p < 0.05, ***p < 0.001).

FIGURE 3

Immunoblot and RNA analysis for p38α and γ in DLB and control brains. Human frontal cortex samples were separated into cytosolic and membrane fractions by extracting sequentially with Tris and PDGF buffer and probed with p38α, p38γ, and β-actin. (A,B) Representative immunoblot images for (A) cytosolic and (B) membrane fractions probed with p38α, p38γ, and β-actin. In each panel, left six lanes show healthy controls and right eight lanes show DLB patients. (C,D) Image analysis of the cytosolic fraction immunoblot for p38α and p38γ, respectively. (E,F) Image analysis of the membrane fraction immunoblot for p38α and p38γ, respectively. (G,H) Real time qPCR for p38α and p38γ mRNA, respectively. n = 6 for control and n = 8 for DLB (*p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 4

Immunohistochemical analysis of the distribution of p38γ in brains in non-Tg and α-syn Tg mouse brains. Saggital vibratome sections from complete right hemibrains were immunolabeled with an antibody against p38γ and developed with DAB. (A) Panels with a solid outline show representative low power bright field microscopic images (200×) (scale bar = 100 μm) of non-Tg (top) and α-syn Tg (bottom) mouse brains immunostained with p38γ; panels with a dotted outline show enlarged images (630×) (scale bar = 20 μm) of the indicated region of interest. (B–G) Number of p38γ-positive cells per 1 mm² of the frontal cortex (B), caudal cortex (C), hippocampus (D), striatum (E), thalamus, (F) and mid brain (G). n = 6 per group (*p < 0.05, ***p < 0.001).

FIGURE 5

Double immunohistochemical analysis of p38γ cellular localization in non-Tg and α-syn Tg mouse brains. Vibratome sections of murine brains were double immunofluorescence labeled and analyzed with Apotome II mounted in a Carl Zeiss AxioImager Z1 microscope. Far right (detail): enlarged image of the area contained in the dotted square. (A) p38γ (red) and the pre-synaptic marker synaptophysin (green), colocalization between the two markers is in yellow; (B) image analysis showing high colocalization of p38γ to synapses in non-Tg mice; (C) p38γ (red) and the neuronal marker NeuN (green); (D) image analysis showing increased localization of p38γ to neurons in α-syn Tg mice; (E) p38γ (red) and the astroglial cell marker GFAP (green); (F) image analysis showing minimal localization of p38γ to astroglia in mouse brains; (G) p38γ (red) and α-syn (green); (H) image analysis showing high colocalization of p38γ to α-syn aggregates in the cytoplasm (Lewy body-like) in α-syn Tg mice. (I) α1-syntrophin (red) and pre-synaptic marker synaptophysin (green); (J) image analysis showing low colocalization of α1-syntrophin to pre-synaptic site in mice brains; (K) α1-syntrophin (red) and α-synuclein (green); (L) image analysis showing high localization of α1-syntrophin with α-syn in α-syn Tg mice; Scale bars are 10 μm in the standard panels and 5 μm in the zoomed panels. n = 6 per group (^∗∗p < 0.01, ^∗∗∗p < 0.001). Image analysis of p38γ and synaptic proteins in α-syn Tg mouse brains. The single labeled channels from above were further analyzed to calculate the% area of the neuropil occupied by (M) synaptophysin, (N) α-syn, (O) p38γ, and (P) α1-syntrophin immunofluorescence in non-Tg and Tg mice. (Q) Linear regression analysis was performed to ascertain the correlation coefficient between the levels of p38γ in the neuropil (% area) and synaptophysin, α-syn, and α1-syntrophin in non-Tg and Tg mice. n = 6 per group (**p < 0.01, ***p < 0.001).

FIGURE 6

Immunoblot and RNA analysis in non-Tg and α-syn Tg mouse brains. Mouse hemibrains were separated into cytosolic and membrane fractions by extracting sequentially with Tris and PDGF buffer and probed with p38α, p38γ, α1-syntrophin, α-syn, and β-actin. (A,B) Representative immunoblot images for (A) cytosolic and (B) membrane fractions probed with p38α, p38γ, α1-syntrophin, α-synuclein, and β-actin. In each panel, left five lanes show non-Tg mice and right lanes show α-syn Tg mice. (C,D) Image analysis of cytosolic and membrane fractions probed for p38α, respectively; (E,F) image analysis of cytosolic and membrane fractions probed for p38γ, respectively; (G,H) image analysis of cytosolic and membrane fractions probed for α-1 syntrophin, respectively (I,J) image analysis of cytosolic and membrane fractions probed for α-synuclein, respectively; (K,L) real time quantitative PCR analysis showed no significant differences in p38α (K) and p38γ (L) RNA expression between non-Tg and α-syn Tg mice. n = 5 per group (*p < 0.05).

FIGURE 7

Immunoprecipitation analysis between p38 and α-synuclein in a neuronal cell line. B103 neuroblastoma cells were transfected with pcDNA3, pcDNA-human-α-synuclein, pcDNA3-Flag-p38α, and pcDNA3-Flag-p38γ and analyzed by immunoblotting and immunocytochemistry. (A) After 48 h of transfection, cell lysates were analyzed by western blot and probed with an antibody against Flag to detect p38α, p38γ, and α-syn (Syn1); (B) levels of p38α, p38γ and α-syn were determined by densitometric quantification. (C) Co-immunoprecipitation of p38α, p38γ, and α-syn. p38α and p38γ were pulled down from transfected cell lysates and analyzed by western blot with an antibody against pathological α-syn (LB509) showing the interaction between α-syn and p38γ. (D) Representative images from double immunostaining for α-syn (red)/p38α (green) and α-syn (red)/p38γ (green). Strong colocalization (yellow) was observed in neuronal cells co-expressing α-syn and p38γ. Scale bar is 10 μm.