👤 Niccolò Parenti

High cholesterol levels are a risk factor for the development of Alzheimer's disease. Experiments investigating the influence of cholesterol on the proteolytic processing of the amyloid precursor protein (APP) by the β-secretase Bace1 and on their proximity in cells have led to conflicting results. By using a fluorescence bioassay coupled with flow cytometry we found a direct correlation between the increase in membrane cholesterol amount and the degree of APP shedding in living human neuroblastoma cells. Analogue results were obtained for cells overexpressing an APP mutant that cannot be processed by α-secretase, highlighting the major influence of cholesterol enrichment on the cleavage of APP carried out by Bace1. By contrast, the cholesterol content was not correlated with changes in membrane dynamics of APP and Bace1 analyzed with single molecule tracking, indicating that the effect of cholesterol enrichment on APP processing by Bace1 is uncoupled from changes in their lateral diffusion. Show less

Batten disease, one of the most devastating types of neurodegenerative lysosomal storage disorders, is caused by mutations in CLN3. Here, we show that CLN3 is a vesicular trafficking hub connecting the Golgi and lysosome compartments. Proteomic analysis reveals that CLN3 interacts with several endo-lysosomal trafficking proteins, including the cation-independent mannose 6 phosphate receptor (CI-M6PR), which coordinates the targeting of lysosomal enzymes to lysosomes. CLN3 depletion results in mis-trafficking of CI-M6PR, mis-sorting of lysosomal enzymes, and defective autophagic lysosomal reformation. Conversely, CLN3 overexpression promotes the formation of multiple lysosomal tubules, which are autophagy and CI-M6PR-dependent, generating newly formed proto-lysosomes. Together, our findings reveal that CLN3 functions as a link between the M6P-dependent trafficking of lysosomal enzymes and lysosomal reformation pathway, explaining the global impairment of lysosomal function in Batten disease. Show less

The development of neuronal apoptosis depends on an intrinsic transcriptional program. By DNA microarray technology, we have previously implicated a number of genes in different paradigms of neuronal apoptosis. In the present study, we investigated the spatiotemporal pattern of expression of two of these genes, gastric inhibitory polypeptide (Gip) and its receptor (Gipr) in the rat central nervous system. The levels of their transcripts were measured with real-time quantitative polymerase chain reaction and in situ-hybridization. Widespread expression of Gip and Gipr was found in adult rat brain, whereas during postnatal cerebellum development, they were highly expressed in the external and internal granule layer, and in Purkinje cells. To investigate the possible biological function of Gip we examined its effects in vitro. Addition of Gip to cultured cerebellar granule neurons reduced the extent of apoptotic death induced by switching the growing medium from 25 to 5 mM K+. This neurotrophic effect was mimicked by that of PACAP38 and IGF1. We conclude that Gip acts as an endogenous neurotrophic factor and supports neuronal survival. Show less