PMC

(A) Western blot of the phosphorylated forms of AMPK, mTOR and AKT (Thr803 and Ser473), which showed increased activation in SDS cells. Genetic correction with the SBDS gene restored normal phosphorylation levels of these proteins. (B) Densitometric analysis of the WB signals. The data are expressed as relative optical densities.

ATP/AMP ratio (A) in SDS lymphoblast and lymphocytes was comparable to the wt cells after leucine treatment as well as ATP and AMP levels (B) respiration rate (C), complex IV activity (D), extracellular lactate concentration (E) MDA lipid peroxidation (F) and [Ca²⁺]_i (G).

(A) A clonogenic assay on the SDS MNCs showed increased erythroid colonies after treatment with leucine (closed circles); however, no myeloid colony induced effect was observed (open circles). (B) The AMPK, mTOR and AKT (Thr803 and Ser473) phosphorylation levels in SDS cells was reduced after leucine treatment and were similar to wt cell levels and was representative of respiratory and energetic stress recovery after leucine treatment. (C). Densitometric analysis of the WB signals. The data are expressed as relative optical densities.

(A) Extracellular lactate production is a marker for the glycolysis pathway. Lactate production was higher in the SDS lymphocyte and lymphoblast medium, suggesting an increase in the glycolysis pathway to sustain the energy request. The data are the medians of at least 3 different experiments. ^#P < 0.005. (B) Malondialdehyde (MDA), a lipid peroxidation product, was utilized as a marker of oxidative stress. The MDA level was significantly increased in SDS cells compared with wt cells. The data are the medians of at least 3 different experiments. *p < 0.001. Legend: LB (lymphoblasts), LYC (lymphocytes), SDS (Shwachman-Diamond syndrome), wt (wild type), SDS-corr (Shwachman-Diamond syndrome cells transfected with the SBDS gene), SDS MOCK (Shwachman-Diamond syndrome cells transfected with empty vector).

(A) The scheme represents possible interconnections among the several aspects considered in this study (see Discussion). (B) The scheme focuses attention on the biochemical pathways involved in the energetic status of the cell. In particular, it is possible to hypothesize that the impairment of Complex IV may be a reason for the decreased energy production and increased oxidative stress. The loss of ATP synthesis may activate the AMPK pathway, inducing an increase in the glycolysis pathway to support the cell energy demands. Moreover, the mTOR pathway also seemed to be activated, probably to improve the energy production in the mitochondria. However, in the SDS cell, these reactions led to a vicious circle, which could increase the ATP/AMP ratio impairment.

(A) The AMP/ATP ratio was calculated using data from the table in (B) (*P < 0.001). (B) The ATP and AMP contents measured in lymphoblasts (LB) and lymphocytes (LYC) of wt, SDS and their isogenically corrected counterparts (SDS-corr) cells. The AMP and ATP assays routinely employed cell homogenates from 100 to 200,000 cells. The data are expressed as means ± SD of at least 3 different experiments. Panels (C–E) show the oxygen consumption in wt, SDS and SDS-corr cells measured with an amperometric electrode, respectively. Panel (F) shows the number of samples examined and the oxygen consumption values (μM O2/min/mg) at the level of complex I (pyruvate/malate) or complex II (succinate) of oxidative phosphorylation. The data are expressed as means ± SD of at least 3 different experiments. Two-hundred and fifty to 500,000 cells were routinely analysed for each oxymetric titration. (G) The complex IV (cytochrome c oxidase) activity was measured following oxidation of ascorbate-reduced cytochrome C at 550 nm. The data were obtained from the medians of at least 3 different experiments. *p < 0.001. (H) Western blots of COX5A and COX2, which are complex IV components of nuclear and mitochondrial DNA origin, respectively. SDS and wt lymphoblast and lymphocyte extracts showed comparable levels of both proteins, as demonstrated by the densitometric analysis reported in the table (the data are reported as the relative optical density normalized to the actin signal). Legend: LB (lymphoblasts), LYC (lymphocytes), SDS (Shwachman-Diamond syndrome), wt (wild type), SDS-corr (Shwachman-Diamond syndrome transfected with SBDS gene), SDS-mock (Shwachman-Diamond syndrome transfected with empty vector).