Screening of Hsps using double-label FIHC with different anti-Hsp and anti-α-syn antibodies visualized by Alexa Fluor 594 or Texas Red and Alexa Fluor 488, respectively, shows co-localization of a subset of Hsps examined in LBs (a–g) and GCIs (h–n). Yellow color represents co-localization of Hsp and α-syn. Hsp27 (b), Hsp40 (c), Hsc70 (e), and Hsp90 (g) are located in LBs, whereas αB-crystallin (h) and Hsp90 (n) are located in GCIs and threads. Specificity of Hsp90 antibodies (o), 9D2 (lanes 1 and 2), and AC88 (lanes 3 and 4) is shown in HS fraction of human (lanes 1 and 3) and mouse (lanes 2 and 4) brain homogenates. Rat 9D2 does not recognize mouse Hsp90 (lane 2). Photomicrographs in p–x show modest Hsp90 IR in neurons of normal brain (p), which also is seen in α-synucleinopathy brains, in addition to more intense Hsp90 IR in LBs (arrowhead) of the PD SN (q), DLB amygdala (r), and GCIs (arrow) in the MSA pons (s). Little or no iHsp90 IR is seen in neurofibrillary tangles of AD (t, double arrowheads) and DLB (r). t: IHC profile with an antibody against Hsp90 (9D2) or tau (AT8) (inset) from adjacent sections in the CA1 region of AD hippocampus are presented for comparison. It is apparent that Hsp90 IR is reduced in neurofibrillary tangles. u: Modest Hsp90 IR is noted in ubiquitin inclusions in the hippocampus of the FTD-MND brain. Inset shows ubiquitin IR in inclusions on the adjacent section. v and w show double-label IHC with horseradish peroxidase-DAB and β-galactosidase-X-gal to illustrate co-localization of iHsp90 IR (brown) with α-syn IR (blue) in LBs (arrowhead) in the midbrain of PD (v) as well as GCIs (arrow) in the pons of MSA (w). x: No CHIP IR was found on GCI in the pons of MSA, whereas the adjacent section indicates descent α-syn IR inclusions (inset). Scale bars, 10 μm.

Double-label FIHC confirms Hsp90 IR (Texas Red) co-localizes with α-syn IR (Alexa Fluor 488) in LBs of PD amygdala (arrowhead in a–c) and GCIs in the MSA pons (d–f). g–j: Representative images from triple-label FIHC of DLB amygdala shows differential co-localization of Hsp90 with α-syn (arrow) rather than tau-positive inclusions (arrowhead) in these preparations. Even when tau IR is located in iHsp90 IR cells, the subcellular localization of these proteins is normally discordant (asterisk). k: Semiquantitative analysis reveals the number of iHsp90 IR with α-syn- or tau IR lesions including intracytoplasmic inclusions, dystrophic neurites, and spheroids. l: iHsp90 co-localizes with α-syn to a significantly greater extent (P < 0.01) than tau in disease lesions. Asterisks indicate statistically significant difference. P < 0.05. Scale bars, 10 μm (a–j).

Triple-label FIHC micrographs of iHsp90 (Texas Red), ubiquitin (Alexa Fluor 488), and α-syn (AMCA) of DLB amygdala (a–d) and MSA pons (e–h) indicate that iHsp90 co-localizes preferentially with ubiquitinated α-syn lesions (arrow), whereas a subset of α-syn-positive profiles are ubiquitin-negative (arrowhead). i and j: Data from semiquantitative analyses of triple-label FIHC studies of amygdala from DLB brain (n = 6 in i) and pons of MSA brains (n = 6 in j) showing that a similar percentage (∼30 to 45%) of the total number α-syn lesions (neurites plus perikaryal inclusions) are iHsp90− (α-syn + Hsp90) and ubiquitin IR (α-syn + Ubiq), whereas >90% of perikaryal α-syn inclusion lesions are iHsp90− (α-syn + Hsp90) and ubiquitin IR (α-syn + Ubiq). There were no significant differences between the number of Hsp90-positive and ubiquitin-positive α-syn inclusions. Scale bars, 10 μm (a–h).

Representative images from Western blot analyses (WB) of HS, RIPA, and FA samples of cingulate cortex from normal (norm), PD (PD), DLB brains (DLB), and pons of MSA brain (MSA). Equal amounts of sample were analyzed by SDS-polyacrylamide gel electrophoresis. These results show that the FA fraction from PD, DLB, and MSA brains harbors accumulations of oligomeric α-syn species, while variably increased amounts of Hsp90 and Hsc70, but not Hsp70, are seen in FA fractions of all α-synucleinopathy disease brains. Additionally, some pathological accumulations of Hsp40 as well as Hsp27 and αB-crystallin were noted in PD/DLB and MSA brains. Polyubiquitinated protein species in the FA fraction were more apparent in the disease brains than normal brain.

A: Effects of MG-132 and lactacystin (LC) on cultured cells. In oligodendrocyte primary culture (a and b), exposure to MG-132 (1 μmol/L) for 3, 5, 7, and 18 hours leads to the appearance of polyubiquitinated proteins after 18 hours, with slight up-regulation of Hsp90 levels (a). By 18 hours of incubation of different doses of MG-132 (1, 5, 10 μmol/L) or LC at one (10 μmol/L) dose there is a sharp induction of ubiquitinated proteins, accompanied by up-regulation of Hsp90 protein and mRNA level (b, bottom). Similar results are seen in OLN (c) cells transfected with tau40 plus α-syn and N2A cells (d) transfected with α-syn although the levels of tubulin and α-syn remain unchanged. Bar graphs indicate quantitation of variable Hsp90 protein levels due to MG132 treatment in respective cell types, confirming up-regulation of the protein level in all cases. B: Co-immunoprecipitation analysis using OLN-tau40-α-syn cells revealed noticeable protein-protein interactions between α-syn and Hsp90 as well as Hsc70 (bound), but not between α-syn and Hsp70 or Hsp40 (unbound). The protein complex of interest was isolated from the OLN cell homogenate using either no antibody (no Ab), α-syn Ab, Hsp90, Hsc70, Hsp70, or Hsp40 (IP Ab) and detected by SNL1 (α-syn) or AC88 (Hsp90) by Western blot (WB Ab). Input represents original material.

In the brainstem of a 3-month-old control non-Tg mouse (a–c), M7 Tg mouse (data not shown), and asymptomatic α-syn (M83) Tg mouse (d–f), there are no α-syn lesions and modest Hsp90 IR. However, in older symptomatic M83 Tg mouse (9 months old), numerous α-syn inclusions are formed, which are frequently associated with iHsp90 IR (g–i, arrow) and ubiquitin IR (j–l, arrow). Western blots of non-Tg (lanes 1 to 3 in m–o) and M83Tg mouse (lanes 4 to 6 in m–o) show accumulation of α-syn (m), Hsp90, and Hsc70, but not Hsp70, Hsp40, or αB-crystallin (n) and polyubiquitinated (o) proteins (likely including α-syn) exclusively in the FA fraction (lane 6) of 9-month-old symptomatic M83 Tg mouse brain, but not in the 3-month-old non-Tg mouse brain (m–o) or 9-month-old non-Tg mouse (data not shown). Scale bars, 10 μm (a–l).

Double-immunoelectron microscopy reveals a close association of Hsp90 and α-syn in filamentous LBs in dopaminergic neurons of the SN in PD brain (a, c, e). α-Syn-positive (10 nm gold, arrow) filaments in the LB are also Hsp90-positive (18 nm gold, arrowhead), whereas unaffected dopaminergic neurons in the same SN section (f) reveal infrequent Hsp90 labeling (arrowhead). Image e is a high-power view of the inset in c, from the center of a LB. PG, neuronal pigment; N, nucleus.