p62, Ref(2)P and ubiquitinated proteins are conserved markers of neuronal aging, aggregate formation and progressive autophagic defects

Abstract

Suppression of macroautophagy, due to mutations or through processes linked to aging, results in the accumulation of cytoplasmic substrates that are normally eliminated by the pathway. This is a significant problem in long-lived cells like neurons, where pathway defects can result in the accumulation of aggregates containing ubiquitinated proteins. The p62/Ref(2)P family of proteins is involved in the autophagic clearance of cytoplasmic protein bodies or sequestosomes. These unique structures are closely associated with protein inclusions containing ubiquitin as well as key components of the autophagy pathway. In this study we show that detergent fractionation followed by western blot analysis of insoluble ubiquitinated proteins (IUP), mammalian p62 and its Drosophila homologue, Ref(2)P can be used to quantitatively assess the activity level of aggregate clearance (aggrephagy) in complex tissues. Using this technique we show that genetic or age-dependent changes that modify the long-term enhancement or suppression of aggrephagy can be identified. Moreover, using the Drosophila model system this method can be used to establish autophagy-dependent protein clearance profiles that are occurring under a wide range of physiological conditions including developmental, fasting and altered metabolic pathways. This technique can also be used to examine proteopathies that are associated with human disorders such as frontotemporal dementia, Huntington and Alzheimer disease. Our findings indicate that measuring IUP profiles together with an assessment of p62/Ref(2)P proteins can be used as a screening or diagnostic tool to characterize genetic and age-dependent factors that alter the long-term function of autophagy and the clearance of protein aggregates occurring within complex tissues and cells.

Figure 1

Accumulation of IUP, Ref(2)P and cytoplasmic inclusions associated with neuronal aging and autophagy defects. (A) Wild-type flies were aged between 1 day and 4 weeks (1 D, 1 W, 2 W, 4 W) before heads were collected and serially detergent extracts prepared for western blot analysis. Neural extracts were also prepared from 6-week-old flies with enhanced Atg8a expression (Atg8a⁺, APPL-GAL4/UAS-Atg8a) or from 1-week-old Atg8a^−/− mutant flies (Atg8a¹/Atg8a²). Immunoblots were probed with the Ref(2)P and actin antibodies and the fold increase in SDS soluble Ref(2)P levels compared to 1-day controls. (B) Matching IUP profiles. (C) qRT-PCR analysis was used to examine the expression profiles of the Ref(2)P and Atg8a gene from multiple head RNA extractions were taken from wild-type heads at 1 (n = 5), 2 (n = 2) and 4 weeks (n = 3) of age. Atg8a and ref(2)P mRNA values were normalized to individual actin•5c profiles. 1-week values were set at 1- and 2- and 4-week readings averaged and standard deviations calculated. Both Ref(2)P and Atg8a mRNA levels show a similar age-dependent decline. (D) Western blot analysis of neural extracts prepared from 1 day-, 1 week- and 2-week-old bchs^−/− mutant flies, shows Ref(2)P accumulation profiles in both the Triton-X and SDS protein fractions.

Figure 2

TEM analysis of bchs mutant neurons. (A) TEM image of 2-week-old adult bchs^−/− mutant flies (bchs³/bchs⁶) shows three nerve cells within a single image that contain inclusion bodies (IB) located near the nucleus (N). These perinuclear structures show hallmark features of aggresomes and morphology of other subcellular organelles are abnormal. (B) Enlargement shows the indentation of the nucleus by the inclusion body (black arrows) and the formation of vacuoles (white arrows). (C) In a different neuron the inclusion body is surrounded by multiple membrane layers (white arrows) and is adjacent to two large double membrane organelles (black arrows). (D) Multiple abnormal structures are detected in a different neuronal cell body that include multilamellar bodies (MLB) and electron dense structures that may represent a population of autophagic vesicles or inclusion body-like structures.

Figure 3

Defects in autophgy-lysosomal trafficking promotes Ref(2)P accumulation in the adult nervous system. Ref(2)P immunoblot were prepared from total protein extracts taken from 1-week-old control, autophagy and lysosomal trafficking mutant flies (eight heads per genotype). (A) Autophagy mutations that produced viable adults include Atg8a (Atg8a¹/Atg8a²), Atg7 (Atg7^EY10058, Atg7^d06990) and Atg3 (Atg3^EY08386). (B) Viable adult mutations in lysosomal trafficking genes were also examined. At 1 week Deep orange mutants (Dor¹) show elevated neuronal levels of Ref(2)P, while other lysosomal trafficking mutants (orange¹, orange⁴⁹, ruby¹, carmine¹, carnation¹ and spinster^EY10097) are not significantly different from that of age-matched controls (Canton-S, CS). Quantification of Ref(2)P levels are illustrated in Supplemental Figure 2A and B.

Figure 4

Metabolic Changes and Altered IUP and Ref(2)P Neural Profiles. (A) Early and mid 3^rd instar larvae (LE3, LM3) were maintained under fed (F) or starvation (S) conditions, as were 2-day-old adult Drosophila. Larval fat tissues and adult heads were collected after 3 hours or 12 hours of fasting, respectively. Immunoblots were prepared from the Triton-X and SDS fractions and probed with ubiquitin, Ref(2)P and actin antibodies. (B) SDS head extract immunoblots were prepared from 1-day-old, 1-, 3- or 4-week-old wild-type, Ref(2)P^−/− mutants (Ref(2)P^c/e) and heterozygous^−/+ and homozygous chico^−/− mutant flies (chico^1/+, chico^1/2). Both IUP and Ref(2)P profiles show similar age and genotype-dependent accumulation patterns with the exception of Ref(2)P null flies. (C) IUP, Ref(2)P and actin values for each age and genotype were quantified using ImageJ. The corrected IUP and Ref(2)P values for 1-day-old wild-type flies were set at one, other values adjusted and graphed using these values.

Figure 5

p62 profiles from human Alzheimer disease neural tissues. (A) Mid-frontal cortical tissue samples were taken from AD patients or control subjects (age and gender matched), flash frozen in liquid nitrogen and stored at −80°C. Protein extracts (2% SDS) were examined for p62 and actin. Previous histological examinations were performed on adjacent tissue samples and plaque density score (PDS) for a particular individual were assigned. PDS, age (years) and gender (M = male or F = female) are indicated for each sample. (B) Densitometry values for the two major p62 bands (arrows) were corrected using actin and normalized to the average p62 levels detected in control samples (PDS = 0, N = 8). Individuals in the PDS1 group had p62 levels that were not substantially different than controls (n = 2, p > 0.05), while samples from individuals with higher PDS showed a significant accumulation of p62 (one way ANOVA with Bonferroni post-test, p < 0.01, PSD3 N = 2; PDS N = 4).

Figure 6

Mammalian cell culture proteopathies models and p62 profiles. (A) Atg5^+/+ (wild type) and Atg5^−/− mouse embryonic fibroblasts (MEFs) were transfected with Flag-HttQ65 (48 hrs) or were untreated before extraction in 1.0% Triton-X (soluble) and 2% SDS buffers (insoluble). Samples were immunoblotted with anti-Flag, anti-Atg5 and anti-p62 antibodies. Equal loading was verified using anti-α-Tubulin and Atg5 protein levels confirmed using anti-Atg5 antibodies. Clearance of SDS-soluble Flag-HttQ65 inclusions was further analyzed by filter-trap assays (upper part). (B) HeLa cells were transfected with control or Vps24 siRNAs for 5 days and then processed for p62 western blot analysis. Knockdown efficiencies and loading were verified using Vps24 and α-Tubulin levels. (C) HeLa cells were transfected for 48 hrs with myc-tagged constructs containing wild-type myc-CH MP2B or a mutant myc-CHMP2B^intron5 construct and mock-transfected cells were used as control. Cells were extracted, immunoblotted and probed with p62, myc and α-Tubulin antibodies. Cells expressing mutant CHMP2B show p62 accumulation, whereas control and cells expressing wild-type myc-CHMP2B have similar low levels of p62. Anti-myc and anti-α-Tubulin immunoblots were used as expression and loading controls. (D) HeLa cells were transfected for 48 hrs with myc-CHMP2B^intron5, fixed and processed for immunofluorescence analysis. Cells were labeled with antibodies against myc, ubiquitin and p62/SQSTM1 and imaged using confocal microscopy. Co-localization is indicated in white (right panel, scale bar = 10 µm).