Assessment of proteasome impairment and accumulation/aggregation of ubiquitinated proteins in neuronal cultures

Abstract

The ubiquitin/proteasome pathway (UPP) is the major proteolytic quality control system in cells and involves tightly regulated removal of unwanted proteins and retention of those that are essential. In addition to its function in normal protein degradation, the UPP plays a critical role in the quality control process by degrading mutated or abnormally folded proteins. The proteolytic component of the UPP is a multiprotein complex known as the proteasome. Many factors, including the aging process, can cause proteasome impairment leading to formation of abnormal ubiquitin-protein aggregates that are found in most progressive neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. In this chapter, we describe protocols to measure proteasome activity, evaluate its state of assembly, and assess the accumulation and aggregation of ubiquitinated proteins in two types of neuronal cultures: human neuroblastoma cells and rat primary cortical cultures. These protocols can be used with different types of neuronal cultures to estimate proteasome activity and the levels and aggregation of ubiquitinated proteins. In addition, they can be used to identify compounds potentially capable of preventing a decline in proteasome activity and formation of ubiquitin-protein aggregates associated with neurodegeneration.

Fig. 1

In-gel assay for proteasome activity and assembly in rat E18 primary cortical neuronal cultures. Crude extracts were prepared from control cultures (−) or cultures treated with 15 μM PGJ2 for 16 h (+). Cleared lysates (40 μg/sample) were subjected to nondenaturing gel electrophoresis. In (a) the proteasomal chymotrypsin-like activity was measured with Suc-LLVY-AMC by the in-gel assay. In (b and c) 26S and 20S proteasomes were detected by immunoblotting with an anti-β5 antibody (b), a subunit of the core proteasome particle (20S), and with the anti-Rpt6 antibody (c), an ATPase subunit of the 19S regulatory particle. Proteasomal 26S (two caps and one cap) and 20S forms are indicated by on the left.

Fig. 2

Setup for making a glycerol gradient. For explanation, see Subheading 3.2, steps 1–8.

Fig. 3

Sedimentation velocity of proteasomes in SK-N-SH cells. Cells were treated for 24 h with DMSO (control, vehicle) or epoxomicin (25 nM). Total lysates (2 mg protein/sample) were fractionated by glycerol density gradient centrifugation (10–40% glycerol corresponding to fractions 13 to 1). (a) Aliquots (50 μl) of each fraction obtained from control (black squares)- and epoxomicin (white circles)-treated cells were assayed for chymotrypsin-like activity with Suc-LLVY-AMC. (b) Immunoblot analyses of each fraction probed with antibodies that react with the proteasome (α4, core particle; Rpt6, 19S regulatory particle). Proteins were precipitated with acetone from 450 μl of each fraction. The fractions were obtained from control- and epoxomicin-treated cells.

Fig. 4

Proteasome activities in SK-N-SH cells. Cells were treated for 24 h with DMSO (control, vehicle) or epoxomicin (25 nM). Proteasome activities were measured in cleared supernatants obtained from total cell homogenates (50 μg of protein/sample). Peptidase activities were assayed colorimetrically after a 24-h incubation at 37°C. The chymotrypsin-like activity was measured with Suc-LLVY-AMC, the trypsin-like activity with Z-GGR-βNA, and the caspase-like activity with Z-LLE-βNA.

Fig. 5

Proteasome subunit levels in rat E18 primary cortical neuronal cultures. Aliquots of the supernatant (cleared lysate) and pellet fractions obtained from samples prepared for the “in-gel” assay (see Fig. 1) were run on SDS-PAGE (10% gel) followed by immunobloting with the same antibodies listed in Fig. 1 as well as with anti-β-actin. 40 μg of protein were loaded per lane.

Fig. 6

Accumulation of ubiquitinated proteins in rat E18 primary cortical neuronal cultures. Cells were treated for 24 h with DMSO [control, vehicle (−)] or with 20 μM PGJ2 (+). Total cell extracts were subjected to western blot analysis (8% gel) to detect ubiquitinated proteins (40 μg of protein/lane). Equal protein loading was demonstrated by probing the immunoblots with the anti-β-actin antibody.

Fig. 7

Filter trap assay to measure ubiquitin-protein aggregates in rat E18 primary cortical neuronal cultures. (a) In a preliminary test, waterproof drawing ink (100 μl per well) was applied to the nitrocellulose membrane to assess flow through each well. If vacuum is properly equilibrated, dye dots corresponding to each well should appear round and neat on the nitrocellulose membrane. (b) Influence of membrane composition and porosity on detection of ubiquitin-protein aggregates. Cells were treated for 16 h with DMSO [control, vehicle (0)] or with 15 μM or 20 μM PGJ2. Cell extracts were subjected to the filter trap assay to detect ubiquitin-protein aggregates (100 μg of protein/sample). Nitrocellulose-yielded optimal assay sensitivity relative to polyvinylidene fluoride (PVDF) or cellulose acetate.

Fig. 8

Immunofluorescence detection of ubiquitin-protein aggregates in rat E18 primary cortical neuronal cultures. Ubiquitinated proteins and βIII-tubulin immunofluorescence staining of cortical cultures treated with DMSO (top 4 panels) or 15 μM PGJ2 for 16 h (bottom 4 panels). Nuclei are stained with DAPI. Large arrows point to protein aggregates and small arrows to dystrophic neurites. Scale bar = 10 μm.