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.

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

Reports from neural cell cultures and experimental animal studies provide evidence of age- and disease-related changes in retrograde transport of spent or misfolded proteins destined for degradation or recycling. However, few studies address these issues in human brain from those who either age without dementia and overt neuropathology, or succumb to Alzheimer's; especially as such propensity may be influenced by APOE genotype. We studied the expression and distribution of the dynein subunit dynactin-P50, the β amyloid precursor protein (βAPP), and hyperphosphorylated tau (P-tau) in tissues and tissue sections of brains from non-demented, neuropathology-free patients and from Alzheimer patients, with either APOE ε3,3 or APOE ε4,4. We found that advanced age in patients without dementia or neuropathological change was associated with coordinated increases in dynactin-P50 and βAPP in neurons in pyramidal layers of the hippocampus. In contrast, in Alzheimer's, βAPP and dynactin were significantly reduced. Furthermore, the dynactin-P50 and βAPP that was present was located primarily in dystrophic neurites in Aβ plaques. Tissues from Alzheimer patients with APOE ε3,3 had less P-tau, more βAPP, dynactin-P50, and synaptophysin than did tissues from Alzheimer patients carrying APOE ε4,4. It is logical to conclude, then, that as neurons age successfully, there is coordination between retrograde delivery and maintenance and repair, as well as between retrograde delivery and degradation and/or recycling of spent proteins. The buildup of proteins slated for repair, synaptic viability, transport, and re-cycling in neuron soma and dystrophic neurites suggest a loss of this coordination in Alzheimer neurons. Inheritance of APOE ε3,3 rather than APOE ε4,4, is associated with neuronal resilience, suggestive of better repair capabilities, more synapses, more efficient transport, and less hyperphosphorylation of tau. We conclude that even in disease the ε3 allele is neuroprotective.

BackgroundNeuroinflammation, typified by elevated levels of interleukin-1 (IL-1) α and β, and deficits in proteostasis, characterized by accumulation of polyubiquitinated proteins and other aggregates, are associated with neurodegenerative disease independently and through interactions of the two phenomena. We investigated the influence of IL-1β on ubiquitination via its impact on activation of the E3 ligase parkin by either phosphorylated ubiquitin (P-Ub) or NEDD8.MethodsImmunohistochemistry and Proximity Ligation Assay were used to assess colocalization of parkin with P-tau or NEDD8 in hippocampus from Alzheimer patients (AD) and controls. IL-1β effects on PINK1, P-Ub, parkin, P-parkin, and GSK3β—as well as phosphorylation of parkin by GSK3β—were assessed in cell cultures by western immunoblot, using two inhibitors and siRNA knockdown to suppress GSK3β. Computer modeling characterized the binding and the effects of P-Ub and NEDD8 on parkin. IL-1α, IL-1β, and parkin gene expression was assessed by RT-PCR in brains of 2- and 17-month-old PD-APP mice and wild-type littermates.ResultsIL-1α, IL-1β, and parkin mRNA levels were higher in PD-APP mice compared with wild-type littermates, and IL-1α-laden glia surrounded parkin- and P-tau-laden neurons in human AD. Such neurons showed a nuclear-to-cytoplasmic translocation of NEDD8 that was mimicked in IL-1β-treated primary neuronal cultures. These cultures also showed higher parkin levels and GSK3β-induced parkin phosphorylation; PINK1 levels were suppressed. In silico simulation predicted that binding of either P-Ub or NEDD8 at a singular position on parkin opens the UBL domain, exposing Ser65 for parkin activation.ConclusionsThe promotion of parkin- and NEDD8-mediated ubiquitination by IL-1β is consistent with an acute neuroprotective role. However, accumulations of P-tau and P-Ub and other elements of proteostasis, such as translocated NEDD8, in AD and in response to IL-1β suggest either over-stimulation or a proteostatic failure that may result from chronic IL-1β elevation, easily envisioned considering its early induction in Down’s syndrome and mild cognitive impairment. The findings further link autophagy and neuroinflammation, two important aspects of AD pathogenesis, which have previously been only loosely related.