Regulators of proteostasis are translationally repressed in fibroblasts from patients with sporadic and LRRK2-G2019S Parkinson's disease.

Deficits in protein synthesis are associated with Parkinson's disease (PD). However, it is not known which proteins are affected or if there are synthesis differences between patients with sporadic and Leucine-Rich Repeat Kinase 2 (LRRK2) G2019S PD, the most common monogenic form. Here we used bio-orthogonal non-canonical amino acid tagging for global analysis of newly translated proteins in fibroblasts from sporadic and LRKK2-G2019S patients. Quantitative proteomic analysis revealed that several nascent proteins were reduced in PD samples compared to healthy without any significant change in mRNA levels. Using targeted proteomics, we validated which of these proteins remained dysregulated at the static proteome level and found that regulators of endo-lysosomal sorting, mRNA processing and components of the translation machinery remained low. These proteins included autophagy-related protein 9A (ATG9A) and translational stability regulator YTH N6-ethyladenosine RNA binding protein 3 (YTHDF3). Notably, 77% of the affected proteins in sporadic patients were also repressed in LRRK2-G2019S patients (False discovery rate (FDR) < 0.05) in both sporadic and LRRK2-G2019S samples. This analysis of nascent proteomes from PD patient skin cells reveals that regulators of proteostasis are repressed in both sporadic and LRRK2-G2019S PD.

Long-term osteogenic differentiation of human bone marrow stromal cells in simulated microgravity: novel proteins sighted.

Microgravity-induced bone loss is a major concern for space travelers. Ground-based microgravity simulators are crucial to study the effect of microgravity exposure on biological systems and to address the limitations posed by restricted access to real space. In this work, for the first time, we adopt a multidisciplinary approach to characterize the morphological, biochemical, and molecular changes underlying the response of human bone marrow stromal cells to long-term simulated microgravity exposure during osteogenic differentiation. Our results show that osteogenic differentiation is reduced while energy metabolism is promoted. We found novel proteins were dysregulated under simulated microgravity, including CSC1-like protein, involved in the mechanotransduction of pressure signals, and PTPN11, SLC44A1 and MME which are involved in osteoblast differentiation pathways and which may become the focus of future translational projects. The investigation of cell proteome highlighted how simulated microgravity affects a relatively low number of proteins compared to time and/or osteogenic factors and has allowed us to reconstruct a hypothetical pipeline for cell response to simulated microgravity. Further investigation focused on the application of nanomaterials may help to increase understanding of how to treat or minimize the effects of microgravity.

PhosPiR: an automated phosphoproteomic pipeline in R.

Large-scale phosphoproteome profiling using mass spectrometry (MS) provides functional insight that is crucial for disease biology and drug discovery. However, extracting biological understanding from these data is an arduous task requiring multiple analysis platforms that are not adapted for automated high-dimensional data analysis. Here, we introduce an integrated pipeline that combines several R packages to extract high-level biological understanding from large-scale phosphoproteomic data by seamless integration with existing databases and knowledge resources. In a single run, PhosPiR provides data clean-up, fast data overview, multiple statistical testing, differential expression analysis, phosphosite annotation and translation across species, multilevel enrichment analyses, proteome-wide kinase activity and substrate mapping and network hub analysis. Data output includes graphical formats such as heatmap, box-, volcano- and circos-plots. This resource is designed to assist proteome-wide data mining of pathophysiological mechanism without a need for programming knowledge.

Protein synthesis is suppressed in sporadic and familial Parkinson's disease by LRRK2.

Gain of function LRRK2-G2019S is the most frequent mutation found in familial and sporadic Parkinson's disease. It is expected therefore that understanding the cellular function of LRRK2 will provide insight on the pathological mechanism not only of inherited Parkinson's, but also of sporadic Parkinson's, the more common form. Here, we show that constitutive LRRK2 activity controls nascent protein synthesis in rodent neurons. Specifically, pharmacological inhibition of LRRK2, Lrrk2 knockdown or Lrrk2 knockout, all lead to increased translation. In the rotenone model for sporadic Parkinson's, LRRK2 activity increases, dopaminergic neuron translation decreases, and the neurites atrophy. All are prevented by LRRK2 inhibitors. Moreover, in striatum and substantia nigra of rotenone treated rats, phosphorylation changes are observed on eIF2α-S52(↑), eIF2s2-S2(↓), and eEF2-T57(↑) in directions that signify protein synthesis arrest. Significantly, translation is reduced by 40% in fibroblasts from Parkinson's patients (G2019S and sporadic cases alike) and this is reversed upon LRRK2 inhibitor treatment. In cells from multiple system atrophy patients, translation is unchanged suggesting that repression of translation is specific to Parkinson's disease. These findings indicate that repression of translation is a proximal function of LRRK2 in Parkinson's pathology.