Impact of inflammatory preconditioning on murine microglial proteome response induced by focal ischemic brain injury.

Preconditioning with lipopolysaccharide (LPS) induces neuroprotection against subsequent cerebral ischemic injury, mainly involving innate immune pathways. Microglia are resident immune cells of the central nervous system (CNS) that respond early to danger signals through memory-like differential reprogramming. However, the cell-specific molecular mechanisms underlying preconditioning are not fully understood. To elucidate the distinct molecular mechanisms of preconditioning on microglia, we compared these cell-specific proteomic profiles in response to LPS preconditioning and without preconditioning and subsequent transient focal brain ischemia and reperfusion, - using an established mouse model of transient focal brain ischemia and reperfusion. A proteomic workflow, based on isolated microglia obtained from mouse brains by cell sorting and coupled to mass spectrometry for identification and quantification, was applied. Our data confirm that LPS preconditioning induces marked neuroprotection, as indicated by a significant reduction in brain infarct volume. The established brain cell separation method was suitable for obtaining an enriched microglial cell fraction for valid proteomic analysis. The results show a significant impact of LPS preconditioning on microglial proteome patterns by type I interferons, presumably driven by the interferon cluster regulator proteins signal transducer and activator of transcription1/2 (STAT1/2).

Impaired biogenesis of basic proteins impacts multiple hallmarks of the aging brain.

Aging and neurodegeneration entail diverse cellular and molecular hallmarks. Here, we studied the effects of aging on the transcriptome, translatome, and multiple layers of the proteome in the brain of a short-lived killifish. We reveal that aging causes widespread reduction of proteins enriched in basic amino acids that is independent of mRNA regulation, and it is not due to impaired proteasome activity. Instead, we identify a cascade of events where aberrant translation pausing leads to reduced ribosome availability resulting in proteome remodeling independently of transcriptional regulation. Our research uncovers a vulnerable point in the aging brain's biology - the biogenesis of basic DNA/RNA binding proteins. This vulnerability may represent a unifying principle that connects various aging hallmarks, encompassing genome integrity and the biosynthesis of macromolecules.

Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD.

Functional loss of TDP-43, an RNA-binding protein genetically and pathologically linked to ALS and FTD, leads to inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. However, the possibility of de novo protein synthesis from cryptic exon transcripts has not been explored. Here, we show that mRNA transcripts harboring cryptic exons generate de novo proteins both in TDP-43 deficient cellular models and in disease. Using coordinated transcriptomic and proteomic studies of TDP-43 depleted iPSC-derived neurons, we identified numerous peptides that mapped to cryptic exons. Cryptic exons identified in iPSC models were highly predictive of cryptic exons expressed in brains of patients with TDP-43 proteinopathy, including cryptic transcripts that generated de novo proteins. We discovered that inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Finally, we showed that these de novo peptides were present in CSF from patients with ALS. The demonstration of cryptic exon translation suggests new mechanisms for ALS pathophysiology downstream of TDP-43 dysfunction and may provide a strategy for novel biomarker development.

HAT cofactor TRRAP modulates microtubule dynamics via SP1 signaling to prevent neurodegeneration.

Brain homeostasis is regulated by the viability and functionality of neurons. HAT (histone acetyltransferase) and HDAC (histone deacetylase) inhibitors have been applied to treat neurological deficits in humans; yet, the epigenetic regulation in neurodegeneration remains elusive. Mutations of HAT cofactor TRRAP (transformation/transcription domain-associated protein) cause human neuropathies, including psychosis, intellectual disability, autism, and epilepsy, with unknown mechanism. Here we show that Trrap deletion in Purkinje neurons results in neurodegeneration of old mice. Integrated transcriptomics, epigenomics, and proteomics reveal that TRRAP via SP1 conducts a conserved transcriptomic program. TRRAP is required for SP1 binding at the promoter proximity of target genes, especially microtubule dynamics. The ectopic expression of Stathmin3/4 ameliorates defects of TRRAP-deficient neurons, indicating that the microtubule dynamics is particularly vulnerable to the action of SP1 activity. This study unravels a network linking three well-known, but up-to-date unconnected, signaling pathways, namely TRRAP, HAT, and SP1 with microtubule dynamics, in neuroprotection.

Reduced proteasome activity in the aging brain results in ribosome stoichiometry loss and aggregation.

A progressive loss of protein homeostasis is characteristic of aging and a driver of neurodegeneration. To investigate this process quantitatively, we characterized proteome dynamics during brain aging in the short-lived vertebrate Nothobranchius furzeri combining transcriptomics and proteomics. We detected a progressive reduction in the correlation between protein and mRNA, mainly due to post-transcriptional mechanisms that account for over 40% of the age-regulated proteins. These changes cause a progressive loss of stoichiometry in several protein complexes, including ribosomes, which show impaired assembly/disassembly and are enriched in protein aggregates in old brains. Mechanistically, we show that reduction of proteasome activity is an early event during brain aging and is sufficient to induce proteomic signatures of aging and loss of stoichiometry in vivo. Using longitudinal transcriptomic data, we show that the magnitude of early life decline in proteasome levels is a major risk factor for mortality. Our work defines causative events in the aging process that can be targeted to prevent loss of protein homeostasis and delay the onset of age-related neurodegeneration.