BDNF stimulation of protein synthesis in cortical neurons requires the map kinase-interacting kinase MNK1

Although the MAP kinase-interacting kinases (MNKs) have been known for >15 years, their roles in the regulation of protein synthesis have remained obscure. Here, we explore the involvement of the MNKs in brain-derived neurotrophic factor (BDNF)-stimulated protein synthesis in cortical neurons from mice. Using a combination of pharmacological and genetic approaches, we show that BDNF-induced upregulation of protein synthesis requires MEK/ERK signaling and the downstream kinase, MNK1, which phosphorylates eukaryotic initiation factor (eIF) 4E. Translation initiation is mediated by the interaction of eIF4E with the m7GTP cap of mRNA and with eIF4G. The latter interaction is inhibited by the interactions of eIF4E with partner proteins, such as CYFIP1, which acts as a translational repressor. We find that BDNF induces the release of CYFIP1 from eIF4E, and that this depends on MNK1. Finally, using a novel combination of BONCAT and SILAC, we identify a subset of proteins whose synthesis is upregulated by BDNF signaling via MNK1 in neurons. Interestingly, this subset of MNK1-sensitive proteins is enriched for functions involved in neurotransmission and synaptic plasticity. Additionally, we find significant overlap between our subset of proteins whose synthesis is regulated by MNK1 and those encoded by known FMRP-binding mRNAs. Together, our data implicate MNK1 as a key component of BDNF-mediated translational regulation in neurons

Hemisphere asymmetry of response to pharmacologic treatment in an Alzheimer’s disease mouse model

The aim of this study was to examine hemisphere asymmetry of response to pharmacologic treatment in an Alzheimer’s disease mouse model using cilostazol as a chemical stimulus. Eight-month-old mice were assigned to vehicle or cilostazol treatment for three months and hemispheres were analyzed using quantitative proteomics. Bioinformatics interpretation showed that following treatment, aggregation of blood platelets significantly decreased in the right hemisphere whereas neurodegeneration significantly decreased and synaptic transmission increased in the left hemisphere only. Our study provides novel evidence on cerebral laterality of pharmacologic activity, with important implications in deciphering regional pharmacodynamic effects of existing drugs thus uncovering novel hemisphere-specific therapeutic targets

Quantitative proteomic profiling of white matter in cases of cerebral amyloid angiopathy reveals upregulation of extracellular matrix proteins and clusterin

Aims: Cerebral amyloid angiopathy (CAA) is the accumulation of amyloid beta (Aβ) in the walls of cerebral arterioles, arteries and capillaries. Changes in the white matter in CAA are observed as hyperintensities and dilated perivascular spaces on MRI suggesting impairment of fluid drainage but the pathophysiology behind these changes is poorly understood. We tested the hypothesis that proteins associated with clearance of Aβ peptides are upregulated in the white matter in cases of CAA.Methods: In this study, we compare the quantitative proteomic profile of white matter from post-mortem brains of patients with CAA and age-matched controls in order to gain insight into the cellular processes and key molecules involved in the pathophysiology of CAA.Results: Our proteomic analysis resulted in the profiling of 3,734 proteins (peptide FDR p<0.05). Of these, 189 were differentially expressed in CAA vs. control. Bioinformatics analysis of these proteins showed significant enrichment of proteins related to cell adhesion | cell-matrix interaction, mitochondrial dysfunction and hypoxia. Upregulated proteins in CAA included EMILIN2, COL4A2, TLN1, CLU, HSPG2. Downregulated proteins included DSP, IDE, HBG1.Conclusions: The present study reports an in-depth quantitative proteomic profiling of white matter from patients with CAA, highlighting extracellular matrix proteins and clusterin as key molecules in the pathophysiology of white matter changes in cases of CAA

LONP1 and mtHSP70 cooperate to promote mitochondrial protein folding

Most mitochondrial precursor polypeptides are imported from the cytosol into the mitochondrion, where they must efficiently undergo folding. Mitochondrial precursors are imported as unfolded polypeptides. For proteins of the mitochondrial matrix and inner membrane, two separate chaperone systems, HSP60 and mitochondrial HSP70 (mtHSP70), facilitate protein folding. We show that LONP1, an AAA+ protease of the mitochondrial matrix, works with the mtHSP70 chaperone system to promote mitochondrial protein folding. Inhibition of LONP1 results in aggregation of a protein subset similar to that caused by knockdown of DNAJA3, a co-chaperone of mtHSP70. LONP1 is required for DNAJA3 and mtHSP70 solubility, and its ATPase, but not its protease activity, is required for this function. In vitro, LONP1 shows an intrinsic chaperone-like activity and collaborates with mtHSP70 to stabilize a folding intermediate of OXA1L. Our results identify LONP1 as a critical factor in the mtHSP70 folding pathway and demonstrate its proposed chaperone activity

Increased plasma CD14 levels 1 year postpartum in women with pre-eclampsia during pregnancy: a case–control plasma proteomics study

Epidemiological data suggest that pre-eclampsia (PE) is associated with an increased risk of post-delivery metabolic dysregulation. The aim of the present case–control observational study was to examine the global plasma proteomic profile 1 year postpartum in women who developed PE during pregnancy (n = 5) compared to controls (n = 5), in order to identify a novel predictive marker linking PE with long-term metabolic imbalance. Key findings were verified with enzyme-linked immunosorbent assay (ELISA) in a separate cohort (n = 17 women with PE and n = 43 controls). One hundred and seventy-two proteins were differentially expressed in the PE vs. control groups. Gene ontology analysis showed that Inflammatory|Immune responses, Blood coagulation and Metabolism were significantly enriched terms. CD14, mapping to the inflammatory response protein network, was selected for verification based on bibliographic evidence. ELISA measurements showed CD14 to be significantly increased 1 year postpartum in women with PE during pregnancy compared to controls [PE group (median ± SD): 296.5 ± 113.6; control group (median ± SD): 128.9 ± 98.5; Mann–Whitney U test p = 0.0078]. Overall, the identified proteins could provide insight into the long-term disease risk among women with PE during pregnancy and highlight the need for their postpartum monitoring. CD14 could be examined in larger cohorts as a predictive marker of insulin resistance and type II diabetes mellitus among women with PE

Ursodeoxycholic acid inhibits uptake and vasoconstrictor effects of taurocholate in human placenta

Intrahepatic cholestasis of pregnancy (ICP) causes increased transfer of maternal bile acids to the fetus and an increased incidence of sudden fetal death. Treatment includes ursodeoxycholic acid (UDCA), but it is not clear if UDCA protects the fetus. This study explores the placental transport of the bile acid taurocholate (TC) by the organic anion–transporting polypeptide, (OATP)4A1, its effects on the placental proteome and vascular function, and how these are modified by UDCA. Various methodological approaches including placental villous fragments and Xenopus laevis oocytes were used to investigate UDCA transport. Placental perfusions and myography investigated the effect of TC on vasculature. The effects of acute TC exposure on placental tissue were investigated using quantitative proteomics. UDCA inhibited OATP4A1 activity in placental villous fragments and oocytes. TC induced vasoconstriction in placental and rat vasculature, which was attenuated by UDCA. Quantitative proteomic analysis of villous fragments showed direct effects of TC on multiple placental pathways, including oxidative stress and autophagy. The effects of TC on the placental proteome and vasculature demonstrate how bile acids may cause fetal distress in ICP. UDCA inhibition of OATP4A1 suggests it will protect the mother and fetus against the vascular effects of TC by inhibiting its cellular uptake. UDCA may protect the fetus in ICP by inhibiting OATP4A1-mediated bile acid transfer and TC-induced placental vasoconstriction. Understanding the physiologic mechanisms of UDCA may allow better therapeutic interventions to be designed specifically for the fetus in the future.—Lofthouse, E. M., Torrens, C., Manousopoulou, A., Nahar, M., Cleal, J. K., O’Kelly, I. M., Sengers, B. G., Garbis, S. D., Lewis, R. M. Ursodeoxycholic acid inhibits uptake and vasoconstrictor effects of taurocholate in human placenta

Quantitative non-canonical amino acid tagging based proteomics identifies distinct patterns of protein synthesis rapidly induced by hypertrophic agents in cardiomyocytes, revealing new aspects of metabolic remodeling

Cardiomyocytes undergo growth and remodeling in response to specific pathological or physiological conditions. Pathological myocardial growth is a risk factor for cardiac failure to which faster protein synthesis is a major driving element. We aimed to quantify the rapid effects of different pro-hypertrophic stimuli on the synthesis of specific proteins in ARVC and to determine whether such effects are due to alterations on mRNA abundance or the translation of specific mRNAs. Cardiomyocytes have very low rates of protein synthesis, posing a challenging problem in terms of studying changes in the synthesis of specific proteins, which also applies to other non-dividing primary cells. To address this, an optimized QuaNCAT LC/MS method was used to selectively quantify newly synthesized proteins in such cells. The study showed both classical (phenylephrine; PE) and more recent (insulin) pathological cardiac hypertrophic agents increased the synthesis of proteins involved in glycolysis, the Krebs cycle / beta-oxidation, and sarcomeric components. However, insulin increased synthesis of many metabolic enzymes to a greater extent than PE. Using a novel validation method, we confirmed that synthesis of selected candidates is indeed up-regulated by PE and insulin. Synthesis of all proteins studied was upregulated by signaling through mTORC1 without changes in their mRNA levels, showing the key importance of translational control in the rapid effects of hypertrophic stimuli. Expression of PKM2 was upregulated in rat hearts following TAC. This isoform possesses specific regulatory properties that may be involved in metabolic remodeling and as a novel candidate biomarker. Levels of translation factor eEF1 also increased during TAC, likely contributing to faster cell mass accumulation. Interestingly, PKM2 and eEF1 were not up-regulated in pregnancy or exercise induced CH, suggesting them as pathological CH specific markers. The study methods may be of utility to the examination of protein synthesis in primary cells