Caloric Restriction Suppresses Microglial Activation and Prevents Neuroapoptosis Following Cortical Injury in Rats

Traumatic brain injury (TBI) is a widespread cause of death and a major source of adult disability. Subsequent pathological events occurring in the brain after TBI, referred to as secondary injury, continue to damage surrounding tissue resulting in substantial neuronal loss. One of the hallmarks of the secondary injury process is microglial activation resulting in increased cytokine production. Notwithstanding that recent studies demonstrated that caloric restriction (CR) lasting several months prior to an acute TBI exhibits neuroprotective properties, understanding how exactly CR influences secondary injury is still unclear. The goal of the present study was to examine whether CR (50% of daily food intake for 3 months) alleviates the effects of secondary injury on neuronal loss following cortical stab injury (CSI). To this end, we examined the effects of CR on the microglial activation, tumor necrosis factor-α (TNF-α) and caspase-3 expression in the ipsilateral (injured) cortex of the adult rats during the recovery period (from 2 to 28 days) after injury. Our results demonstrate that CR prior to CSI suppresses microglial activation, induction of TNF-α and caspase-3, as well as neurodegeneration following injury. These results indicate that CR strongly attenuates the effects of secondary injury, thus suggesting that CR may increase the successful outcome following TBI

Cathepsin D genetic depletion in MEF cells does not cause cholesterol nor lysosomal proteins accumulation.

<p>(A) Confocal microscopy of CtsD KO MEFs. Cholesterol (filipin staining, white) and NPC1 (green). (B) Western blot analysis of CtsD KO MEFs using LC3, LAMP1 and NPC1 antibody. α-Tubulin was used as a loading control. (C) Quantification of Western blot results of the 3 independent experiments was performed by ImageJ. Student t-test was used for statistical analysis. Error bars present the mean ± standard deviation (** p < 0.01).</p

Cathepsin B/L genetic depletion in MEF cells causes accumulation of cholesterol and lysosomal proteins.

<p>(A) Confocal microscopy of CtsB KO, CtsL KO and CtsB/L double KO MEFs. Cholesterol (filipin staining, white) and NPC1 (green). (B) Western blot analysis of CtsB KO, CtsL KO and CtsB/L double KO MEFs using LC3, LAMP1 and NPC1 antibody. α-Tubulin was used as a loading control. (C) Quantification of Western blot results of the 3 independent experiments was performed by ImageJ. Student t-test was used for statistical analysis. Error bars present the mean ± standard deviation (** p < 0.01, *** p < 0.001).</p

Cathepsin B/L inhibition causes NPC disease-like cholesterol accumulation in CHO cells.

<p>(A) Confocal microscopy of CHOwt cells treated with different inhibitors. Cholesterol (filipin staining, white) and NPC1 (green). (B) Western blot of CHOwt cells treated with different inhibitors using ABCA1, NPC1 and NPC2 antibody. β-Actin was used as a loading control. (C) Quantification of Western blot results of the 3 independent experiments was performed by ImageJ. Student t-test was used for statistical analysis. Error bars present the mean ± standard deviation (** p < 0.01, *** p < 0.001).</p

Inhibition of cathepsin B/L and not cathepsin D causes lysosomal dysfunction.

<p>(A) EGFR degradation assay. Western blot analysis of EGFR in SH-SY5Y cells treated with different inhibitors at different time points. α-Tubulin was used as a loading control. (B) Quantification of Western blot results of the 3 independent experiments was performed by ImageJ. Student t-test was used for statistical analysis. Error bars present the mean ± standard deviation (* p < 0.05, ** p < 0.01). (C) Confocal microscopy of CHOwt cells treated with different inhibitors and CHO <i>NPC1</i>-null cells. LysoTracker (red) and Hoechst (blue). (D) Western blot of CHOwt cells treated with different inhibitors and CHO <i>NPC1</i>-null cells using LC3 antibody. β-Actin was used as a loading control. (E) Quantification of Western blot results of the 3 independent experiments was performed by ImageJ. Student t-test was used for statistical analysis. Error bars present the mean ± standard deviation (* p < 0.05, ** p < 0.01).</p

Cathepsin B/L inhibition causes NPC disease-like cholesterol accumulation in SH-SY5Y cells.

<p>(A) Confocal microscopy of SH-SY5Y control and PADK treated cells. Cholesterol (filipin staining, white) and NPC1 (green). (B) Western blot of SH-SY5Y control and PADK treated cells using LC3, ABCA1 and NPC1 antibody. α-Tubulin was used as a loading control. (C) Quantification of Western blot results of the 3 independent experiments was performed by ImageJ. Student t-test was used for statistical analysis. Error bars present the mean ± standard deviation (*** p < 0.001). (D) RT-PCR Expression of the cholesterol egress genes in the control, PADK, U18666A- and Leu/NH₄Cl-treated SH-SY5Y cells, normalized to β-actin and quantified by 2−ΔΔCt method using control sample as calibrator. ATP-Binding Cassette sub-family A member 1 (ABCA1) and Niemann-Pick C1 protein (NPC1) mRNA levels presented as a fold change. The error bars present the mean ± SEM (* p < 0.05).</p

Neuroapoptosis is present in the perilesioned area of AL animals.

<p>Fluoro-Jade B and Hoechst staining of brain sections from AL animals 2, 7, 14, 28 days after injury. Numerous Fluoro-Jade B/Hoechst positive cells (merged) were observed in the AL group on the 2^nd day following injury (arrows); these cells were not detected at later time points. Images are representative of brain sections at the site of the lesion (n = 3 animals per experimental group); c – physiological control; 2, 7, 14, 28 – days following injury; blood vessels (arrowheads); magnification 40×.</p

CR treatment suppresses neuroapoptosis in the perilesioned area.

<p>Fluoro-Jade B and Hoechst staining of brain sections from CR animals 2, 7, 14, 28 – days after injury. Fluoro-Jade B/Hoechst positive cells were not detected in the CR group at any time point. Images are representative of brain sections at the site of the lesion (n = 3 animals per experimental group); c – physiological control; 2, 7, 14, 28 – days after the injury; blood vessels (arrowheads); magnification 40×.</p