Aging Cell. 2015 Feb;14(1):35-48. doi: 10.1111/acel.12296.

Proteins in aggregates functionally impact multiple neurodegenerative disease models by forming proteasome-blocking complexes.

Ayyadevara S, Balasubramaniam M, Gao Y, Yu LR, Alla R, Shmookler Reis R.

McClellan Veterans Medical Center, Central Arkansas Veterans Healthcare Service, Little Rock, AR, 72205, USA; Department of Geriatrics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.

 

Abstract

Age-dependent neurodegenerative diseases progressively form aggregates containing both shared components (e.g., TDP-43, phosphorylated tau) and proteins specific to each disease. We investigated whether diverse neuropathies might have additional aggregation-prone proteins in common, discoverable by proteomics. Caenorhabditis elegans expressing unc-54p/Q40::YFP, a model of polyglutamine array diseases such as Huntington’s, accrues aggregates in muscle 2-6 days posthatch. These foci, isolated on antibody-coupled magnetic beads, were characterized by high-resolution mass spectrometry. Three Q40::YFP-associated proteins were inferred to promote aggregation and cytotoxicity, traits reduced or delayed by their RNA interference knockdown. These RNAi treatments also retarded aggregation/cytotoxicity in Alzheimer’s disease models, nematodes with muscle or pan-neuronal Aβ1-42 expression and behavioral phenotypes. The most abundant aggregated proteins are glutamine/asparagine-rich, favoring hydrophobic interactions with other random-coil domains. A particularly potent modulator of aggregation, CRAM-1/HYPK, contributed < 1% of protein aggregate peptides, yet its knockdown reduced Q40::YFP aggregates 72-86% (P < 10(-6) ). In worms expressing Aβ1-42 , knockdown of cram-1 reduced β-amyloid 60% (P < 0.002) and slowed age-dependent paralysis > 30% (P < 10(-6)). In wild-type worms, cram-1 knockdown reduced aggregation and extended lifespan, but impaired early reproduction. Protection against seeded aggregates requires proteasome function, implying that normal CRAM-1 levels promote aggregation by interfering with proteasomal degradation of misfolded proteins. Molecular dynamic modeling predicts spontaneous and stable interactions of CRAM-1 (or human orthologs) with ubiquitin, and we verified that CRAM-1 reduces degradation of a tagged-ubiquitin reporter. We propose that CRAM-1 exemplifies a class of primitive chaperones that are initially protective and highly beneficial for early reproduction, but ultimately impair aggregate clearance and limit longevity.

KEYWORDS: (protein) aggregation; Alzheimer (disease); C. elegans; Huntington (disease); neurodegeneration; proteasome

PMID: 25510159

 

SUPPLEMENTS:

We began with a C. elegans model of Huntington Disease, which expresses a tract of 40 glutamines (Q40) fused in-frame to yellow fluorescent protein (YFP), in body wall muscle. Figure 1 shows Q40::YFP aggregates formed in adult muscle; like Huntington Disease aggregates, they can be either cytoplasmic (orange arrows) or nuclear (yellow arrows).

rr fig1Figure 1. Nuclear and cytoplasmic localization of aggregates in a model of Huntington Disease. C. elegans adults (strain AM141, expressing Q40:: YFP in muscle) at day 5 post-hatch were stained with DAPI (shown here as green, identifying nuclei), and visualized for both DAPI and YFP (shown here as orange). Nuclear aggregates have both DAPI and YFP fluorescence, and thus appear gold/yellow.

 

Aggregates, isolated from adults of the same AM141 strain, were purified by YFP immunoaffinity and their insolubility in a reducing solution containing a strong detergent (Figure 2). Constituent proteins were then identified and roughly quantified by high-resolution mass spectrometry.

 

rr fig2Figure 2. Isolation of Q40::YFP aggregates from adult worms. (A) Schematic of the procedure used. (B) Fluorescence microscopy fields containing magnetic beads (top) or the unbound fraction (bottom). Aggregates (top) are similar in size to those seen in worms; back­ground fluorescence is attributed to Q40::YFP monomers and oligomers.

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Figure 3.  The number of aggregates seen in day-5 adults is reduced by RNAi knock¬down of proteins identified in the aggregates.

 

rr fig4Figure 4. Rescue by cram-1 knockdown, of paralysis in strain CL4176, is blocked by treatment with the proteasome inhibitor MG132, but enhanced by oleuropein, a proteasome activator. Chi-squared significance vs. FV controls: *p<0.005;  **p<5E–5;  ***p<5E–24

 

We found that aggregate number and size increased with adult age, in both Q40::YFP-expressing worms and wild-type worms, consistent with previous reports. When RNA interference was used to knock down several genes encoding proteins that were identified in aggregates, however, the number of Q40::YFP-containing aggregates in expressing worms was significantly reduced (Figure 3). These genes encode CRAM-1, an uncharacterized protein with some homology to HYPK, human Huntingtin-interacting protein K; PQN-22 and PQN-53, “prion-like” proteins with Gln/Asn-rich domains; ATX-2, orthologous to human ataxin-2; and UNC-108, a neuronal Rab GTPase. CRAM-1 is exceptional, in that cram-1 knockdown confers protection far exceeding its protein abundance in aggregates.

Surprisingly, knockdown of cram-1 (and to a lesser extent, of PQN genes) also rescued traits such as paralysis and loss of chemotaxis, associated with aggregation in Alzheimer models ― worms with induced or age-dependent expression of human Aβ peptide in muscle or neurons.

Sparing of aggregation after cram-1 knockdown, in each model assessed, was entirely dependent on expression and function of proteasomes, the primary means for disposing of misfolded proteins. Thus, if protea­somes were inhibited with MG132, or by RNAi to a proteasomal catalytic subunit, all benefit of cram-1 knock­­down was lost (e.g., as shown in Figure 4).

By molecular modeling, we predicted that CRAM-1 would bind directly and strongly to ubiquitin on proteins tagged for degradation, and thus block their normal targeting to either protea­somes or auto­phagosomes. This prediction was supported in vivo by use of a ubiquitin:: mCherry reporter (see full paper). In situ immunofluorescence (Figure 5) indicates that cram-1 knockdown frees proteasomes from apparent entrapment within Q40::YFP aggregates.

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Figure 5. Dual fluorescence images of C. elegans strain AM141, expressing Q40:: YFP in body-wall muscle, showing YFP aggregation (green) and proteasomal catalytic subunits (red) co-localizing in control worms (left panels), but not in worms exposed to RNAi that targets cram-1 (right panels). Upper images show YFP fluorescence, lower ones show fluor-tagged secondary antibody to proteasomes, and merged images are centered.

 

Why, then, would a protein evolve to block normal clearance of misfolded proteins and their aggregates? We show that cram-1 knockdown has no effect on the rate of development, but strikingly reduces early reproduction. CRAM-1 thus exhibits antagonistic pleiotropy, since its beneficial effect on the maximal rate of population expansion would drive natural selection, overwhelming any deleterious consequences later in life. [Please see the full paper for much more extensive experimental and molecular-modeling data, as well as discussion of human orthologs, implications for neurodegenerative diseases, and evolutionary aspects.]

 

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