Blood RNA biomarkers in prodromal PARK4 and rapid eye movement sleep behavior disorder show role of complexin 1 loss for risk of Parkinson's disease.

Parkinson's disease (PD) is a frequent neurodegenerative process in old age. Accumulation and aggregation of the lipid-binding SNARE complex component α-synuclein (SNCA) underlies this vulnerability and defines stages of disease progression. Determinants of SNCA levels and mechanisms of SNCA neurotoxicity have been intensely investigated. In view of the physiological roles of SNCA in blood to modulate vesicle release, we studied blood samples from a new large pedigree with SNCA gene duplication (PARK4 mutation) to identify effects of SNCA gain of function as potential disease biomarkers. Downregulation of complexin 1 (CPLX1) mRNA was correlated with genotype, but the expression of other Parkinson's disease genes was not. In global RNA-seq profiling of blood from presymptomatic PARK4 indviduals, bioinformatics detected significant upregulations for platelet activation, hemostasis, lipoproteins, endocytosis, lysosome, cytokine, Toll-like receptor signaling and extracellular pathways. In PARK4 platelets, stimulus-triggered degranulation was impaired. Strong SPP1, GZMH and PLTP mRNA upregulations were validated in PARK4. When analysing individuals with rapid eye movement sleep behavior disorder, the most specific known prodromal stage of general PD, only blood CPLX1 levels were altered. Validation experiments confirmed an inverse mutual regulation of SNCA and CPLX1 mRNA levels. In the 3'-UTR of the CPLX1 gene we identified a single nucleotide polymorphism that is significantly associated with PD risk. In summary, our data define CPLX1 as a PD risk factor and provide functional insights into the role and regulation of blood SNCA levels. The new blood biomarkers of PARK4 in this Turkish family might become useful for PD prediction.

Effect of crowding agents, signal peptide, and chaperone SecB on the folding and aggregation of E. coli maltose binding protein.

SecB, a soluble cytosolic chaperone component of the Sec export pathway, binds to newly synthesized precursor proteins and prevents their premature aggregation and folding and subsequently targets them to the translocation machinery on the membrane. PreMBP, the precursor form of maltose binding protein, has a 26-residue signal sequence attached to the N-terminus of MBP and is a physiological substrate of SecB. We examine the effect of macromolecular crowding and SecB on the stability and refolding of denatured preMBP and MBP. PreMBP was less stable than MBP (DeltaT(m )= 7 +/- 0.5 K) in both crowded and uncrowded solutions. Crowding did not cause any substantial changes in the thermal stability of MBP (DeltaT(m )= 1 +/- 0.4 K) or preMBP (DeltaT(m )= 0 +/- 0.6 K), as observed in spectroscopically monitored thermal unfolding experiments. However, both MBP and preMBP were prone to aggregation while refolding under crowded conditions. In contrast to MBP aggregates, which were amorphous, preMBP aggregates form amyloid fibrils. Under uncrowded conditions, a molar excess of SecB was able to completely prevent aggregation and promote disaggregation of preformed aggregates of MBP. When a complex of the denatured protein and SecB was preformed, SecB could completely prevent aggregation and promote folding of MBP and preMBP even in crowded solution. Thus, in addition to maintaining substrates in an unfolded, export-competent conformation, SecB also suppresses the aggregation of its substrates in the crowded intracellular environment. SecB is also able to promote passive disaggregation of macroscopic aggregates of MBP in the absence of an energy source such as ATP or additional cofactors. These experiments also demonstrate that signal peptide can greatly influence protein stability and aggregation propensity.

Fatty alcohols or fatty acids as niosomal hybrid carrier: effect on vesicle size, encapsulation efficiency and in vitro dye release.

Niosomal hybrid mixtures are prepared with bilayer stabilizer cholesterol from non-ionic surfactants span 20 (HLB value 8.6), span 60 (HLB 4.7) and span 85 (HLB 1.8) in presence of dicetyl phosphate (DCP) where fatty acids or fatty alcohols (C14, C16 and C18) are used as carrier. Hybrid mixtures upon hydration with aqueous phosphate buffer (pH 7.4) spontaneously produce vesicular phase which can encapsulate 5(6)-carboxyfluorescein (CF). Effect of fatty alcohols and fatty acids on the vesicle size has been studied by dynamic light scattering (DLS), freeze-fracture scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Release rates of CF from vesicular suspensions are found to be dependent on carriers as well as surfactants used. Fatty acid coated hybrids form large multilamellar vesicles (LMV) (size range 10-15 microm) where as fatty alcohol coated hybrid systems form small multilamellar vesicles (SMV) with an average diameter of 400 nm, in all cases vesicles are polydisperse (PI approximately 0.9) in nature.