The Rab5 activator RME-6 is required for amyloid precursor protein endocytosis depending on the YTSI motif.

Endocytosis of the amyloid precursor protein (APP) is critical for generation of ß-amyloid, aggregating in Alzheimer's disease. APP endocytosis depending on the intracellular NPTY motif is well investigated, whereas involvement of the YTSI (also termed BaSS) motif remains controversial. Here, we show that APP lacking the YTSI motif (ΔYTSI) displays reduced localization to early endosomes and decreased internalization rates, similar to APP ΔNPTY. Additionally, we show that the YTSI-binding protein, PAT1a interacts with the Rab5 activator RME-6, as shown by several independent assays. Interestingly, knockdown of RME-6 decreased APP endocytosis, whereas overexpression increased the same. Similarly, APP ΔNPTY endocytosis was affected by PAT1a and RME-6 overexpression, whereas APP ΔYTSI internalization remained unchanged. Moreover, we could show that RME-6 mediated increase of APP endocytosis can be diminished upon knocking down PAT1a. Together, our data identify RME-6 as a novel player in APP endocytosis, involving the YTSI-binding protein PAT1a.

beta-amyloid precursor protein can be transported independent of any sorting signal to the axonal and dendritic compartment.

In neurons, amyloid precursor protein (APP) is localized to the dendritic and axonal compartment. Changes in subcellular localization affect secretase cleavage of APP, altering the generation of Abeta, and presumably also its pathogenic features. It was reported that APP is sorted initially to the axon and transcytosed subsequently to the somatodendritic compartment. This may be carried out by a recessive dendritic sorting signal in the cytoplasmic C-terminus, possibly the tyrosine based basolateral sorting signal (BaSS), and an axonal sorting motif within the extracellular juxtamembraneous domain. We investigated whether the C- or N-terminal domain of APP contains an independent dendritic or axonal sorting signal. We generated different APP deletion mutants, and produced chimeric proteins of APP and a non-related Type I transmembrane protein. Quantitative immunocytochemical analyses of transfected primary neurons showed that similar amounts of all APP mutants, lacking either the N- or C-terminus, were transported to the axonal and dendritic compartment. Investigations of the chimeric proteins showed that neither the N- nor the C-terminus of APP functions as independent sorting signal, whereas another tyrosine based dendritic sorting signal was sufficient to prevent axonal entry of APP. This data shows that, under steady state conditions, Heterologously expressed APP is transported equally to axons and dendrites irrespective of any putative sorting signal in its N- or C-terminus. This shows that APP can enter the axon in absence of the initial axonal sorting motif, indicating the existence of an alternative pathway allowing axonal entry of APP.

PAT1a modulates intracellular transport and processing of amyloid precursor protein (APP), APLP1, and APLP2.

Understanding the intracellular transport of the beta-amyloid precursor protein (APP) is a major key to elucidate the regulation of APP processing and thus beta-amyloid peptide generation in Alzheimer disease pathogenesis. APP and its two paralogues, APLP1 and APLP2 (APLPs), are processed in a very similar manner by the same protease activities. A putative candidate involved in APP transport is protein interacting with APP tail 1 (PAT1), which was reported to interact with the APP intracellular domain. We show that PAT1a, which is 99.0% identical to PAT1, binds to APP, APLP1, and APLP2 in vivo and describe their co-localization in trans-Golgi network vesicles or endosomes in primary neurons. We further demonstrate a direct interaction of PAT1a with the basolateral sorting signal of APP/APLPs. Moreover, we provide evidence for a direct role of PAT1a in APP/APLP transport as overexpression or RNA interference-mediated knockdown of PAT1a modulates APP/APLPs levels at the cell surface. Finally, we show that PAT1a promotes APP/APLPs processing, resulting in increased secretion of beta-amyloid peptide. Taken together, our data establish PAT1a as a functional link between APP/APLPs transport and their processing.

Development of an 18S rRNA gene-targeted PCR-based diagnostic for the blue crab parasite Hematodinium sp..

The 18S rRNA gene from Hematodinum sp., a parasitic dinoflagellate that infects blue crabs, was amplified, cloned, and sequenced. The sequence showed a high similarity (95% at the nucleotide level) to sequences obtained from other dinoflagellate species, including both free-living and symbiotic species. Sequence similarity was much lower when compared with parasites of other marine invertebrates with similar life histories and with the 18S rRNA gene from the blue crab. Based on comparison of sequence alignments between Hematodinium, other dinoflagellate species, protozoan pathogens of oysters, and blue crab 18S rRNA gene sequences, 2 sets of PCR primers that specifically amplified fragments of the Hematodinium 18S rRNA gene were developed and tested. One of these primer sets (Hemat-F-1487 and Hemat-R-1654) amplified a 187 bp fragment that could be used routinely as a diagnostic test for the presence of Hematodinium in hemolymph from blue crabs. This fragment was consistently amplified from genomic DNA extracted from hemolymph of Hematodinium infected blue crabs. Comparison between the PCR technique and standard histological examination indicated that the PCR technique was reliable and provided 1000 times more sensitivity than the histological methods. The sensitivity of the PCR diagnostic was estimated to be one parasite cell among 300,000 crab hemocytes. Preliminary studies using the PCR diagnostic technique suggest that Hematodinium sp. is absent in crabs collected from waters with low salinity (5 to 10 ppt), but common in crabs from higher salinity environments in estuarine waters from southeastern Georgia (USA).