Particle-based nanosensors offer a tool for determining the pH in the endosomal-lysosomal system of living cells. Measurements providing absolute values of pH have so far been restricted by the limited sensitivity range of nanosensors, calibration challenges and the complexity of image analysis. This protocol describes the design and application of a polyacrylamide-based nanosensor (∼60 nm) that covalently incorporates two pH-sensitive fluorophores, fluorescein (FS) and Oregon Green (OG), to broaden the sensitivity range of the sensor (pH 3.1-7.0), and uses the pH-insensitive fluorophore rhodamine as a reference fluorophore. The nanosensors are spontaneously taken up via endocytosis and directed to the lysosomes where dynamic changes in pH can be measured with live-cell confocal microscopy. The most important focus areas of the protocol are the choice of pH-sensitive fluorophores, the design of calibration buffers, the determination of the effective range and especially the description of how to critically evaluate results. The entire procedure typically takes 2-3 weeks.

Future improvements of non-viral vectors for siRNA delivery require better understanding of intracellular processing and vector interactions with target cells. Here, we have compared the siRNA delivery properties of a lipid derivative of bPEI 1.8kDa (DOPE-PEI) with branched polyethyleneimine (bPEI) with average molecular weights of 1.8kDa (bPEI 1.8kDa) and 25 kDa (bPEI 25kDa). We find mechanistic differences between the DOPE-PEI conjugate and bPEI regarding siRNA condensation and intracellular processing. bPEI 1.8kDa and bPEI 25kDa have similar properties with respect to condensation capability, but are very different regarding siRNA decondensation, cellular internalization and induction of reporter gene knockdown. Lipid conjugation of bPEI 1.8kDa improves the siRNA delivery properties, but with markedly different 2 formulation requirements and mechanisms of action compared to conventional PEIs. Interestingly, strong knockdown using bPEI 25kDa is dependent on the presence of a free vector fraction which does not increase siRNA uptake. Finally, we have investigated the effect on lysosomal pH induced by these vectors to elucidate the differences in the proton sponge effect between lipid conjugated PEI and conventional PEI: Neither DOPE-PEI nor bPEI 25kDa affected lysosomal pH as a function of time, underlining that the possible proton sponge effect is not associated with changes in lysosomal pH.

No abstract

Increasing evidence points to defects in autophagy as a common denominator in most neurodegenerative conditions. Progressive functional decline in the autophagy-lysosomal pathway (ALP) occurs with age, and the consequent impairment in protein processing capacity has been associated with a higher risk of neurodegeneration. Defects in cathepsin D (CD) processing and α-synuclein degradation causing its accumulation in lysosomes are particularly relevant for the development of Parkinson's disease (PD). However, the mechanism by which alterations in CD maturation and α-synuclein degradation leads to autophagy defects in PD neurons is still uncertain. Here we demonstrate that MPR300 shuttling between endosomes and the trans Golgi network is altered in α-synuclein overexpressing neurons. Consequently, CD is not correctly trafficked to lysosomes and cannot be processed to generate its mature active form, leading to a reduced CD-mediated α-synuclein degradation and α-synuclein accumulation in neurons. MPR300 is downregulated in brain from α-synuclein overexpressing animal models and in PD patients with early diagnosis. These data indicate MPR300 as crucial player in the autophagy-lysosomal dysfunctions reported in PD and pinpoint MRP300 as a potential biomarker for PD.