Ubiquitin Accumulation in Autophagy-Deficient Mice is Dependent on the Nrf2-Mediated Stress Response Pathway: A Potential Role for Protein Aggregation in Autophagic Substrate Selection

Genetic ablation of autophagy in mice leads to liver and brain degeneration accompanied by the appearance of ubiquitin (Ub) inclusions, which has been considered to support the hypothesis that ubiquitination serves as a cis-acting signal for selective autophagy. We show that tissue-specific disruption of the essential autophagy genes Atg5 and Atg7 leads to the accumulation of all detectable Ub–Ub topologies, arguing against the hypothesis that any particular Ub linkage serves as a specific autophagy signal. The increase in Ub conjugates in Atg7$^{−/−}$ liver and brain is completely suppressed by simultaneous knockout of either p62 or Nrf2. We exploit a novel assay for selective autophagy in cell culture, which shows that inactivation of Atg5 leads to the selective accumulation of aggregation-prone proteins, and this does not correlate with an increase in substrate ubiquitination. We propose that protein oligomerization drives autophagic substrate selection and that the accumulation of poly-Ub chains in autophagy-deficient circumstances is an indirect consequence of activation of Nrf2-dependent stress response pathways

Systems-based analyses of brain regions functionally impacted in Parkinson's disease reveals underlying causal mechanisms.

Detailed analysis of disease-affected tissue provides insight into molecular mechanisms contributing to pathogenesis. Substantia nigra, striatum, and cortex are functionally connected with increasing degrees of alpha-synuclein pathology in Parkinson's disease. We undertook functional and causal pathway analysis of gene expression and proteomic alterations in these three regions, and the data revealed pathways that correlated with disease progression. In addition, microarray and RNAseq experiments revealed previously unidentified causal changes related to oligodendrocyte function and synaptic vesicle release, and these and other changes were reflected across all brain regions. Importantly, subsets of these changes were replicated in Parkinson's disease blood; suggesting peripheral tissue may provide important avenues for understanding and measuring disease status and progression. Proteomic assessment revealed alterations in mitochondria and vesicular transport proteins that preceded gene expression changes indicating defects in translation and/or protein turnover. Our combined approach of proteomics, RNAseq and microarray analyses provides a comprehensive view of the molecular changes that accompany functional loss and alpha-synuclein pathology in Parkinson's disease, and may be instrumental to understand, diagnose and follow Parkinson's disease progression

Overlap between pathways significantly enriched with differentially expressed genes identified by microarray and RNA-sequencing.

<p>(<b>a</b>) Overlap of pathways enriched with upregulated genes together with the five most significant overlapping pathways. (<b>b</b>) Overlap of pathways enriched with downregulated genes together with the five most significant overlapping pathways.</p

Tracking peripheral biomarkers identified from the causal mapping of PD brain.

<p>(<b>a</b>) High confidence biomarkers consistently identified for PD cortex, striatum, and substantia nigra (S. nigra) using microarray analysis. Upregulated biomarkers are shown on the left together with fold changes in the three brain regions, downregulated biomarkers on the right. (<b>b</b>) Functional biomarker panel that combines DEGs, high confidence biomarkers shown in (a) and genes from the causal map. (<b>c</b>) Assessment of biomarkers in brain (top panels)(substantia nigra, SN) and blood (bottom panels) from age-matched control and PD patients using QuantiGene technology from Panomics. * <i>p</i>≤0.05 and ** <i>p</i>≤0.01 as determined using a two-tailed unpaired t-test with Welch's correction.</p

Identification of pathways dysregulated in PD-brain regions, and the overlap of differentially expressed genes (DEGs) between the PD-affected brain regions.

<p>The 10 key pathways most significantly enriched for DEGs in substantia nigra (<b>a</b>), striatum (<b>b</b>) and cortex (<b>c</b>) of PD brains compared to control as measured by microarray. Enrichments for upregulated genes are shown on the left, for downregulated genes on the right. The numbers of DEGs populating each pathway are denoted in the right columns (#DEGs). (<b>d</b>) Overlap between DEGs in PD cortex and striatum as measured by microarrays. The overlap of upregulated genes is shown on the left, the overlap of downregulated genes on the right. (<b>e</b>) Overlap between DEGs in striatum and substantia nigra (S.nigra) as measured by microarrays. The overlap of upregulated genes is shown on the left, the overlap of downregulated genes on the right. (<b>f</b>) Overlap between DEGs in PD cortex and substantia nigra (S. nigra) as measured by microarrays. The overlap of upregulated genes is shown on the left, the overlap of downregulated genes on the right. (<b>g</b>) Overlap between DEGs in PD cortex, striatum and substantia nigra (S. nigra) as measured by microarrays. The overlap of upregulated genes is shown on the left, the overlap of downregulated genes on the right.</p