Impaired SorLA maturation and trafficking as a new mechanism for SORL1 missense variants in Alzheimer disease

The SorLA protein, encoded by the SORL1 gene, is a major player in Alzheimer disease (AD) pathophysiology. Functional and genetic studies demonstrated that SorLA deficiency results in increased production of Aβ peptides, and thus a higher risk of AD. A large number of SORL1 missense variants have been identified in AD patients, but their functional consequences remain largely undefined. Here, we identified a new pathophysiological mechanism, by which rare SORL1 missense variants identified in AD patients result in altered maturation and trafficking of SorLA protein. An initial screening, based on the overexpression of 71 SorLA variants in HEK293 cells, revealed that 15 of them (S114R, R332W, G543E, S564G, S577P, R654W, R729W, D806N, Y934C, D1535N, D1545E, P1654L, Y1816C, W1862C, P1914S) induced a maturation and trafficking-deficient phenotype. Three of these variations (R332W, S577P, and R654W) and two maturation-competent variations (S124R and N371T) were further studied in details in CRISPR/Cas9-modified hiPSCs. When expressed at endogenous levels, the R332W, S577P, and R654W SorLA variants also showed a maturation defective profile. We further demonstrated that these variants were largely retained in the endoplasmic reticulum, resulting in a reduction in the delivery of SorLA mature protein to the plasma membrane and to the endosomal system. Importantly, expression of the R332W and R654W variants in hiPSCs were associated with a clear increase of Aβ secretion, demonstrating a loss-of-function effect of these SorLA variants regarding this ultimate readout, and a direct link with AD pathophysiology. Finally, structural analysis of the impact of missense variations on SorLA protein structure indicated that impaired cellular trafficking of SorLA protein could be due to subtle variations of the protein structure resulting from changes in the interatomic interactions.

THE EXPRESSION OF THE MAIN AND ALTERNATIVE TRANSCRIPT (SORL1-Δ2) OF THE SORL1 GENE IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS AFFECTED BY THE CHORNOBYL ACCIDENT

Objective: to study clinical-hematological data and expression of the main and alternative transcripts of SORL1 gene in chronic lymphocytic leukemia (CLL) patients affected by the Chornobyl catastrophe. Methods. Analysis was performed in the main group of 34 CLL patients irradiated due to the Chornobyl NPP accident (30 clean-up workers, and 4 evacuees) and in the control group of 27 non-irradiated CLL patients. Groups of patients were comparable by age, sex, stage of disease, mutational status of IGHV genes. Expression of the main and alternative transcripts of SORL1 gene was evaluated by Quantitative Real-time polymerase chain reaction (PCR). The IGHV gene mutational status, TP53 and SF3B1 mutations were studied by PCR followed by direct sequencing. Data were analyzed with the SPSS software package, version 20.0. Results. Relative expression level of the main transcript of SORL1 gene was low (mean 1.71 ± 0.55, median 0.57), did not correlate with the IGHV gene mutational status, TP53 and SF3B1 mutations, stage of disease. The expression of B transcript was not detected, F transcript was expressed at a very low level in 9 patients. The average relative expression level of SORL1-Δ2 transcript was 14.1 ± 6.04 (median 3.48; range 0.01–90.51). The expression of SORL1-Δ2 transcript above the median was more frequent among patients on C stage (p = 0.001), and in patients with unmutated IGHV genes was associated with an extremely negative course of CLL (median of overall survival 9 months vs 61 months at low expression). Relative expression levels of the main and alternative transcripts of SORL1 gene in patients of the main and the control groups did not differ. Conclusions. Our preliminary data suggest that increased expression of SORL1-Δ2 transcript in CLL patients with unmutated IGHV genes can be considered as a negative prognostic marker. Key words: chronic lymphocytic leukemia, SORL1, SORL1-Δ2, Chornobyl NPP accident.