Ubiquilin-1 regulates amyloid precursor protein maturation and degradation by stimulating K63-linked polyubiquitination of lysine 688

Abstract

The pathogenesis of Alzheimer's disease (AD) is associated with proteolytic processing of the amyloid precursor protein (APP) to an amyloidogenic peptide termed Aβ. Although mutations in APP and the secretase enzymes that mediate its processing are known to result in familial forms of AD, the mechanisms underlying the more common sporadic forms of the disease are still unclear. Evidence suggests that the susceptibility of APP to amyloidogenic processing is related to its intracellular localization, and that secretase-independent degradation may prevent the formation of cytotoxic peptide fragments. Recently, single nucleotide polymorphisms in the UBQLN1 gene have been linked to late-onset AD, and its protein product, ubiquilin-1, may regulate the maturation of full-length APP. Here we show that ubiquilin-1 inhibits the maturation of APP by sequestering it in the early secretory pathway, primarily within the Golgi apparatus. This sequestration significantly delayed the proteolytic processing of APP by secretases and the proteasome. These effects were mediated by ubiquilin-1-stimulated K63-linked polyubiquitination of lysine 688 in the APP intracellular domain. Our results reveal the mechanistic basis by which ubiquilin-1 regulates APP maturation, with important consequences for the pathogenesis of late-onset AD.

Fig. 1.

Ubiquilin-1 inhibits APP maturation. (A) Degradation rate of mature (M) and immature (Im) levels of endogenous APP in PC12 cells. Cells were treated with CHX or vehicle and collected at the indicated time points (in minutes). (B) Quantification of three separate experiments as in A represented as the mean ± SEM. (C) Degradation of APP in cells expressing ubiquilin-1 as in A. (D) Quantification from three separate experiments as in B. P values are shown. In A and C, ubiquilin expression was determined by blotting for the myc epitope present on the N terminus of the protein. (E) Subcellular fractionation and endogenous APP distribution in PC12 cells overexpressing ubiquilin-1 or empty-vector transfected cells. Cells were treated with CHX for 2 h, and lysates were prepared and subjected to differential centrifugation. Mature and immature APP bands are identified with an arrow. The asterisk (*) indicates an unidentified reactive band that has faster mobility than immature APP, possibly full-length APP that has not undergone N-linked glycosylation. 10K, 10,000 × g pellet; 100K, 100,000 × g pellet. Equal protein loading was determined by examining endogenous levels of c-Myc (Lower). (F) Biotinylation experiments to examine cell-surface expression of endogenous APP in cells expressing ubiquilin-1 or transfected with empty vector. WCL, whole-cell lysate; PM, plasma membrane; non-PM, nonplasma membrane. (G) Quantification of PM and non-PM APP levels pooled from four experiments.

Fig. 2.

Ubiquilin-1 inhibits APP–GFP trafficking and secretase processing. (A) Schematic representation of the membrane topology of the APP–GFP construct used in this study. Secretase processing is predicted to release AICD–GFP from the membrane into the cytosol (resulting in diffuse fluorescence). (B) Fluorescent images showing APP–GFP distribution in NGF-differentiated PC12 cells before and after 40 min of CHX treatment in cells not expressing ubiquilin-1 (Upper) and cells expressing ubiquilin-1 (Lower). See also Movies S1 and S2. (C) Expression of mature APP–GFP, immature APP–GFP, and AICD–GFP in PC12 cells after CHX treatment. Optical-density AICD–GFP expression levels and the ratio of AICD–GFP to full-length APP expression are given below the blot. Note increasing ratio in APP–GFP-expressing cells, which is eliminated in cells overexpressing ubiquilin-1. (D) Quantification of the change in fluorescence from punctate to diffuse over time (punctate/diffuse index) in single PC12 cells. A reduction in this index indicates more homogeneous (i.e., diffuse) distribution of fluorescence. Solid line indicates no change. The ubiquilin-1 RNAi oligonucleotides have been described and validated elsewhere (18). (E) Punctate/diffuse index of APP–GFP in primary rat cortical neurons as in C. Similar results were seen in three independent experiments.

Fig. 3.

Ubiquilin-1 colocalizes with and retains APP–GFP in the Golgi apparatus. (A) Confocal fluorescent images of APP–GFP localization in GlcNAc-T1–mCherry-expressing PC12 cells treated with CHX for 45 min with or without ubiquilin-1 overexpression, as indicated. (B) APP–GFP and GlcNAc-T1–mCherry colocalization without (Upper) or with (Lower) ubiquilin-1 overexpression as determined by the normalized mean deviation product (nMDP) (21) in living cells treated with CHX at time 0. (Upper Left and Lower Left) Raw, wide-field fluorescent image at time 0; other panels are nMDP values. nMDP panels are pseudocolored such that low values are cool colors, and high values are warmer colors (see scale bar). (C) Quantification of colocalization (I_corr) after 40 min CHX treatment. Data are presented as the mean ± SEM of 10 separate cells.

Fig. 4.

Ubiquilin-1 inhibits nonlysosomal degradation of APP. (A) Time course of APP degradation after addition of cycloheximide in the presence or absence of the proteasomal inhibitor MG132 (Upper). (Lower) Quantification of APP band intensities in an experiment representative of three separate determinations. (B) Time course of APP degradation after cycloheximide addition in the absence or presence of lysosomal inhibitor NH₄Cl (Upper). (Lower) Quantification of band intensities in an experiment representative of three separate determinations. Tubulin is used as a loading control.

Fig. 5.

Ubiquilin-1 regulates APP trafficking and degradation through K63-linked polyubiquitination of lysine residue 688. (A) Ubiquitination of endogenous immunoprecipitated APP as determined by linkage-specific antibodies in the presence or absence of MG132 in cells coexpressing HA-ubiquitin (HA-Ubq) plus ubiquilin-1 or a control vector as indicated at the top. (B) Ubiquitination of endogenous APP in the presence of MG132 in cells coexpressing wild-type or mutant HA-ubiquitin constructs in which all lysine residues are mutated to arginine except K63 or K48. (C) Ubiquitination of overexpressed wild-type APP or a mutant in which lysine 688 is mutated to arginine (K688R). All lanes were preincubated with MG132. (D) Subcellular distribution of wild-type overexpressed APP in the presence or absence of ubiquilin-1 expression and the effects of CHX treatment as indicated. Expression of the ER-resident inositol 1,4,5-trisphosphate receptor type 1 (IP₃R-1) is used as a loading control. (E) Subcellular distribution of K688R APP in the presence or absence of ubiquilin-1 expression and the effects of CHX treatment. (F) Confocal fluorescence images showing the subcellular distribution of wild-type and K688R APP–GFP before and 45 min after CHX treatment.