Point mutations of the p150 subunit of dynactin (DCTN1) gene in ALS.

The authors report mutation screening of the p150 subunit of dynactin (DCTN1) and the cytoplasmic dynein heavy chain (DNCHC1) genes in 250 patients with ALS and 150 unrelated control subjects. Heterozygous missense mutations of the DCTN1 gene were detected in one apparently sporadic case of ALS (T1249I), one individual with familial ALS (M571T), two patients with familial ALS, and two unaffected relatives in the same kindred (R785W). The allelic variants of the DCTN1 gene may represent a previously unknown genomic risk factor for ALS.

Late compartments of amyloid precursor protein transport in SY5Y cells are involved in beta-amyloid secretion.

Amyloid plaques, composed mainly of the 39-43 amino acid betaA4 peptide, are a characteristic feature of Alzheimer's disease. Generation of betaA4 by proteolytic processing of the amyloid precursor protein (APP) is thought to occur in a pathway that includes the activity of two as yet unknown proteases, with beta-secretase cleaving at the N terminus and gamma-secretase releasing the C terminus of betaA4. Inhibition studies and the finding that cell surface APP can serve as a direct precursor of betaA4 suggest that the endosomal/lysosomal compartment is involved in the proteolysis of APP into betaA4. In this study we targeted APP695 chimeric proteins directly into the endosomal/lysosomal compartment. This decreased the amount of released betaA4, while the generation of the betaA4 N terminus continued. APP695 proteins were constructed also, which carried sorting signals responsible for recycling between the trans-Golgi network (TGN) and the cell surface. These proteins were processed into secreted betaA4 at even higher levels than wild-type APP695. Moreover, retention of APP695 proteins in the endoplasmic reticulum led to neither betaA4 secretion nor to processing by beta-secretase in human SH-SY5Y neuroblastoma cells. These data suggest that a beta-cleavage activity resides in a late endosomal compartment and that a gamma-cleavage occurs in early endosomes, resulting in the generation of betaA4 peptides with the majority ending at residue 40.

Human amyloid precursor-like protein 1--cDNA cloning, ectopic expression in COS-7 cells and identification of soluble forms in the cerebrospinal fluid.

Amyloid precursor-like protein 1 (APLP1) represents an integral membrane type 1 protein of unknown function which was originally cloned from a mouse cDNA library on the basis of sequence similarity with the Alzheimer's amyloid precursor protein (APP). Here we report on the molecular cloning and expression of the human APLP1 (hAPLP1). hAPLP1 consists of 650 amino acids, displays 89% identity on the amino acid level to its mouse homologue and has a calculated molecular mass of 72 kDa. hAPLP1 synthesized in a cell-free system displays an apparent molecular mass of approximately 80 kDa in SDS-containing gels and becomes N-glycosylated when the in vitro translation is performed in the presence of microsomes. The hAPLP1 cDNA was also expressed ectopically in COS-7 cells and the protein expression was analyzed by immunoprecipitation and western blotting. We have demonstrated that hAPLP1 represents a novel glycoprotein which carries both N- and O-linked glycans. Moreover, hAPLP1 undergoes limited proteolysis which results in the secretion of the carboxy-terminal truncated molecule into the cells conditioned medium. Examination of cells transfected with hAPLP1 cDNA by confocal laser microscopy reveals an intense perinuclear and Golgi staining, a pattern resembling the subcellular distribution of APP. Using a novel hAPLP1-specific antiserum, we identified soluble hAPLP1 in the human cerebrospinal fluid, which suggests that secretion of hAPLP1 from brain cells also takes place in vivo.