SummaryNeurodegenerative diseases are distinguished by characteristic protein aggregates initiated by disease‐specific ‘seed’ proteins; however, roles of other co‐aggregated proteins remain largely unexplored. Compact hippocampal aggregates were purified from Alzheimer's and control‐subject pools using magnetic‐bead immunoaffinity pulldowns. Their components were fractionated by electrophoretic mobility and analyzed by high‐resolution proteomics. Although total detergent‐insoluble aggregates from Alzheimer's and controls had similar protein content, within the fractions isolated by tau or Aβ1–42 pulldown, the protein constituents of Alzheimer‐derived aggregates were more abundant, diverse, and post‐translationally modified than those from controls. Tau‐ and Aβ‐containing aggregates were distinguished by multiple components, and yet shared >90% of their protein constituents, implying similar accretion mechanisms. Alzheimer‐specific protein enrichment in tau‐containing aggregates was corroborated for individuals by three analyses. Five proteins inferred to co‐aggregate with tau were confirmed by precise in situ methods, including proximity ligation amplification that requires co‐localization within 40 nm. Nematode orthologs of 21 proteins, which showed Alzheimer‐specific enrichment in tau‐containing aggregates, were assessed for aggregation‐promoting roles in C. elegans by RNA‐interference ‘knockdown’. Fifteen knockdowns (71%) rescued paralysis of worms expressing muscle Aβ, and 12 (57%) rescued chemotaxis disrupted by neuronal Aβ expression. Proteins identified in compact human aggregates, bound by antibody to total tau, were thus shown to play causal roles in aggregation based on nematode models triggered by Aβ1–42. These observations imply shared mechanisms driving both types of aggregation, and/or aggregate‐mediated cross‐talk between tau and Aβ. Knowledge of protein components that promote protein accrual in diverse aggregate types implicates common mechanisms and identifies novel targets for drug intervention.

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.

ApoE4-CLEAR interactions may account for suppressed autophagy in APOE ɛ4/ɛ4 carriers and, in this way, contribute to earlier AD onset.