Analysis of the hippocampal proteome in ME7 prion disease reveals a predominant astrocytic signature and highlights the brain-restricted production of clusterin in chronic neurodegeneration.
Bottom Line: The observed changes associated with reactive glia highlighted some specific proteins that dominate the proteome in late-stage disease.Four of the up-regulated proteins (GFAP, high affinity glutamate transporter (EAAT-2), apo-J (Clusterin), and peroxiredoxin-6) are selectively expressed in astrocytes, but astrocyte proliferation does not contribute to their up-regulation.This does not preclude an important role of Clusterin in late-stage disease, but it cautions against the assumption that brain levels provide a surrogate peripheral measure for the progression of brain degeneration.
Affiliation: From the Centre for Biological Sciences and.
Prion diseases are characterized by accumulation of misfolded protein, gliosis, synaptic dysfunction, and ultimately neuronal loss. This sequence, mirroring key features of Alzheimer disease, is modeled well in ME7 prion disease. We used iTRAQ(TM)/mass spectrometry to compare the hippocampal proteome in control and late-stage ME7 animals. The observed changes associated with reactive glia highlighted some specific proteins that dominate the proteome in late-stage disease. Four of the up-regulated proteins (GFAP, high affinity glutamate transporter (EAAT-2), apo-J (Clusterin), and peroxiredoxin-6) are selectively expressed in astrocytes, but astrocyte proliferation does not contribute to their up-regulation. The known functional role of these proteins suggests this response acts against protein misfolding, excitotoxicity, and neurotoxic reactive oxygen species. A recent convergence of genome-wide association studies and the peripheral measurement of circulating levels of acute phase proteins have focused attention on Clusterin as a modifier of late-stage Alzheimer disease and a biomarker for advanced neurodegeneration. Since ME7 animals allow independent measurement of acute phase proteins in the brain and circulation, we extended our investigation to address whether changes in the brain proteome are detectable in blood. We found no difference in the circulating levels of Clusterin in late-stage prion disease when animals will show behavioral decline, accumulation of misfolded protein, and dramatic synaptic and neuronal loss. This does not preclude an important role of Clusterin in late-stage disease, but it cautions against the assumption that brain levels provide a surrogate peripheral measure for the progression of brain degeneration.
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Mentions: We investigated progressive changes in the identified glial genes and their expressed proteins across disease. Five hippocampi from ME7 and NBH animals at 8, 13, and 21 weeks were homogenized as individual samples and divided in 2 aliquots from which either protein or total RNA was extracted. For quantitative Western blot analyses, we used a pool of SDS buffer-extracted 8-, 13-, and 21-week NBH hippocampal homogenates as control (NBH control), which was compared with 13- and 21-week ME7 animals and probed with antibodies against PrP, GFAP, Clusterin, EAAT-2, and Prdx6 (Fig. 6, A–J). The presence of an increasing level of PrPc/PrPSc in each sample was indicated by the increasing anti-PrP antibody immunoreactivity and the emergence of high order oligomers that resist dissociation by SDS at both the middle and late stages of the disease (Fig. 6, A and F, p < 0.01) (32). GFAP immunoreactivity increased with the disease (Fig. 6B), and quantification showed a significant increase relative to NBH control at the 13-week time point (Fig. 6B, p < 0.01) that further increased by 21 weeks (Fig. 6B, p < 0.01).