Polyubiquitinylation Profile in Down Syndrome Brain Before and After the Development of Alzheimer Neuropathology

Abstract

Aims: Among the putative mechanisms proposed to be common factors in Down syndrome (DS) and Alzheimer's disease (AD) neuropathology, deficits in protein quality control (PQC) have emerged as a unifying mechanism of neurodegeneration. Considering that disturbance of protein degradation systems is present in DS and that oxidized/misfolded proteins require polyubiquitinylation for degradation via the ubiquitin proteasome system, this study investigated if dysregulation of protein polyubiquitinylation is associated with AD neurodegeneration in DS.

Results: Postmortem brains from DS cases before and after development of AD neuropathology and age-matched controls were analyzed. By selectively isolating polyubiquitinated proteins, we were able to identify specific proteins with an altered pattern of polyubiquitinylation as a function of age. Interestingly, we found that oxidation is coupled with polyubiquitinylation for most proteins mainly involved in PQC and energy metabolism.

Innovation: This is the first study showing alteration of the polyubiquitinylation profile as a function of aging in DS brain compared with healthy controls. Understanding the onset of the altered ubiquitome profile in DS brain may contribute to identification of key molecular regulators of age-associated cognitive decline.

Conclusions: Disturbance of the polyubiquitinylation machinery may be a key feature of aging and neurodegeneration. In DS, age-associated deficits of the proteolytic system may further exacerbate the accumulation of oxidized/misfolded/polyubiquitinated proteins, which is not efficiently degraded and may become harmful to neurons and contribute to AD neuropathology. Antioxid. Redox Signal. 26, 280-298.

Keywords: Alzheimer disease; Down syndrome; proteasome; proteomics; trisomy21; ubiquitin.

FIG. 1.

Isolation of endogenously polyubiquitinated protein complexes by use of the ubiquitin enrichment kit. A workflow for isolating endogenously ubiquitinylated protein complexes for proteomic analysis is shown. (A) Ubiquitinylated proteins were isolated from brain homogenates by IP. IP fraction (BF) and the supernatant (NBF) were loaded on an SDS-PAGE gel and stained for total protein expression (A1) and subsequently blotted on nitrocellulose membrane and stained with a polyclonal anti-Ub antibody (anti-Ub Ab) to determine enrichment efficiency (A2). (B) The IP fractions (BF and NBF) were further processed with the enrichment kit (Thermo) containing the polyubiquitin affinity resin (Poly-Ub AR). BF, NBF, and FT were loaded in SDS page (B1) and blotted with antiubiquitin antibody (B2). (C) Heat denaturation of high-affinity resin using high temperature (60°) was used to destroy the bond between resin and poly-Ub antibody. Poly-Ub was detected by performing a Western blot (agarose-only control; C1). BF, bound fraction; FT, flow-through; IP, immunoprecipitation; NBF, nonbound fraction; Poly-Ub, polymeric Ub; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; Ub, ubiquitin.

FIG. 2.

Poly-Ub levels. (A) Total levels of poly-Ub-bound proteins in DS (n = 6/group) and DS/AD (n = 6/group) cases compared with their age-matched controls (n = 6/group). (B, C) Increased poly-Ub of two types of ubiquitin chains [K63 (B) and K48 (C)] in both DS (n = 6/group) and DS/AD (n = 6/group) cases compared with their age-matched controls (n = 6/group). Representative bands are shown and protein levels (A−C, upper bands) were normalized per total protein load, named total load (A−C, lower bands). Densitometric values are shown as percentage of Ctr Y set as 100%. Mean ± SEM (DS vs. Ctr y *p < 0.05; DS/AD vs. DS **p < 0.01; DS/AD vs. Ctr O ***p < 0.001; DS vs. Ctr Y and DS/AD vs. DS [for K48] *p < 0.05; DS vs. Ctr Y, DS/AD vs. Ctr O, and DS/AD vs. DS [for K63] **p < 0.01; Ctr O vs. Ctr Y ^#p < 0.05 one-way ANOVA). AD, Alzheimer's disease; ANOVA, analysis of variance; DS, Down syndrome; DS/AD, Down syndrome with Alzheimer's disease; SEM, standard error of the mean.

FIG. 3.

Proteomic profile of representative 2D blots. (A) Representative SYPRO Ruby-stained expression gel from a DS case. (B) SYPRO Ruby-stained polyubiquitinated gel in a DS case. SYPRO Ruby-stained gel images were obtained using a Chemidoc MP System at different times of exposure for the expression of a poly-Ub profile. 2D, two-dimensional.

FIG. 4.

Proteomic profile of representative 2D gels with proteins differentially polyubiquitinated in Ctr Y and Ctr O cases versus DS and DS/AD cases. The comparison (A) is shown for DS versus Ctr Y; (B) between DS/AD and DS; (C) between DS/AD and Ctr O; and (D) between Ctr O and Ctr Y. The spot numbers are reported in Table 2.

FIG. 5.

UCH-L1. (A, B) All samples (n = 6/group) were immunoprecipitated with anti-UCH-L1. Immunoprecipitated proteins were separated on SDS-PAGE, transferred on nitrocellulose membranes, and probed with anti-poly-UbK63 (A, upper bands) and anti-poly-UbK48 (B, upper bands). (A) Shows increased levels of poly-UbK63 bound to UCH-L1 in DS and DS/AD compared with their matched controls. (B) Shows an increase in poly-UbK48 bound to UCH-L1 in DS/AD compared with DS. All the IP experiments were normalized on the total amount of UCH-L1 (indicated as Expr.; A, B, lower bands). Representative bands are shown. Densitometric values are shown as percentage of Ctr Y set as 100%. Mean ± SEM (DS vs. Ctr Y [for K63], DS/AD vs. DS [for K48] *p < 0.05; DS/AD vs. Ctr O [for K63] **p < 0.01 one-way ANOVA). UCH-L1, ubiquitin carboxyl-terminal hydrolase isozyme L1.

FIG. 6.

Gelsolin. (A, B) All samples (n = 6/group) were immunoprecipitated with anti-gelsolin. Immunoprecipitated proteins were separated on SDS-PAGE, transferred on nitrocellulose membranes, and probed with anti-poly-UbK63 (A, upper bands) and anti-poly-UbK48 (B, upper bands). (A) Shows increased level of poly-UbK63 bound to gelsolin in DS compared with Ctr Y, an opposite trend for poly-UbK48 bound to gelsolin in DS compared with Ctr Y is shown (B). All the IP experiments were normalized on the total amount of gelsolin (indicated as Expr.; A, B, lower bands). Representative bands are shown. Densitometric values are shown as percentage of Ctr Y set as 100%. Mean ± SEM (DS vs. Ctr Y [for K63] **p < 0.01; DS vs. Ctr Y [for K48] *p < 0.05 one-way ANOVA).

FIG. 7.

Ubiquilin-1. (A) Expression levels of ubiquilin-1 are significantly decreased in DS (n = 6/group) and DS/AD (n = 6/group) specimens compared with their age-matched controls (n = 6/group). (B, C) All samples (n = 6/group) were immunoprecipitated with anti-ubiquilin-1. Immunoprecipitated proteins were separated on SDS-PAGE, transferred on nitrocellulose membranes, and probed with anti-poly-UbK63 (B) and anti-poly-UbK48 (C). (B) Shows a decrease in poly-Ub K63 bound to ubiquilin-1 (indicated as Poly-UbK63, first bands) in DS/AD compared with DS, in contrast poly-UbK48 bound to Ubiquilin-1 (indicated as Poly-UbK48, second bands) displays an opposite trend compared with poly-Ub K63 (C). All the IP experiments were normalized on the total amount of ubiquilin-1 (indicated as Expr.; third bands). In the upper side of the figure, representative bands are shown. Densitometric values are shown as percentage of Ctr Y set as 100%. Mean ± SEM (DS vs. Ctr Y and DS/AD vs. Ctr O *p < 0.05; DS/AD vs. Ctr O [K48] *p < 0.05; DS/AD vs. DS [K48] *p < 0.05; DS/AD vs. DS [K63] **p < 0.01 one-way ANOVA).

FIG. 8.

Proteasome activities. Chymotrypsin-like (A), trypsin-like (B), and caspase-like (C) proteasome activities are reduced in DS and DS/AD compared with their age-matched controls. Proteasome function was evaluated by enzymatic assay in Ctr Y and Ctr O cases (n = 6/group), DS with and without AD cases (n = 6/group). Fluorescence intensity (Arbitrary Units, AU) as percentage of Ctr Y set as 100%. Mean ± SEM (DS vs. Ctr y and DS/AD vs. Ctr O *p < 0.05; DS vs. Ctr Y **p < 0.01 one-way ANOVA).

FIG. 9.

Pie chart representing polyubiquitinated proteins grouped according to their function. The table shows the list of MS/MS-identified proteins with increased poly-Ub among the different groups of analysis. MS, mass spectrometry.

FIG. 10.

Overlap between oxidative modification and polyubiquitinylation of selected proteins in Down syndrome.