A Cystine-Rich Whey Supplement (Immunocal®) Provides Neuroprotection from Diverse Oxidative Stress-Inducing Agents In Vitro by Preserving Cellular Glutathione

Abstract

Oxidative stress is a principal mechanism underlying the pathophysiology of neurodegeneration. Therefore, nutritional enhancement of endogenous antioxidant defenses may represent a viable treatment option. We investigated the neuroprotective properties of a unique whey protein supplement (Immunocal®) that provides an essential precursor (cystine) for synthesis of the endogenous antioxidant, glutathione (GSH). Primary cultures of rat cerebellar granule neurons (CGNs), NSC34 motor neuronal cells, or HT22 hippocampal cells were preincubated in medium containing Immunocal and then subsequently treated with agents known to induce oxidative stress. Immunocal protected CGNs against neurotoxicity induced by the Bcl-2 inhibitor, HA14-1, the nitric oxide donor, sodium nitroprusside, CuCl2, and AlCl3. Immunocal also significantly reduced NSC34 cell death due to either H2O2 or glutamate and mitigated toxicity in HT22 cells overexpressing β-amyloid1-42. The neuroprotective effects of Immunocal were blocked by inhibition of γ-glutamyl-cysteine ligase, demonstrating dependence on de novo GSH synthesis. These findings indicate that sustaining GSH with Immunocal significantly protects neurons against diverse inducers of oxidative stress. Thus, Immunocal is a nutritional supplement worthy of testing in preclinical animal models of neurodegeneration and in future clinical trials of patients afflicted by these diseases.

Figures

Figure 1

Cells treated with Immunocal display healthy neuronal morphology. Cells were left untreated (a) or treated with Immunocal alone (b) and assessed for overall health and appearance. Left-hand panels are representative images of cell nuclei stained with DAPI. Right-hand panels depict the same fields as viewed under brightfield to assess the state of neuronal processes and soma. Con: control; ICAL: Immunocal. Scale bar, 10 μm.

Figure 2

Immunocal preserves cellular GSH and prevents apoptosis in CGNs exposed to the Bcl-2 inhibitor, HA14-1. (a) Representative images of CGNs left untreated (control), treated with HA14-1 (15 μM), or preincubated for 24 h with Immunocal before HA14-1 treatment for further 24 h. Panels from left to right, DAPI (nuclei), β-tubulin, merged images showing β-tubulin (green), and DAPI (blue). Scale bar, 10 μm. (b) Quantification of apoptosis for 4 independent experiments performed as in (a) except some cultures were preincubated with 200 μM BSO as well. Apoptotic cells were those with condensed or fragmented nuclei. Results are shown as mean ± SEM, n = 4. ∗∗∗ indicates p < 0.001 compared to control, ††† indicates p < 0.001 compared to HA14-1, ‡‡‡ indicates p < 0.001 compared to ICAL + HA14-1. (c) CGNs were treated exactly as described in (b). Total cellular GSH was measured as described in Materials and Methods. Data shown represent the percent of control cellular GSH concentration, mean ± SEM, n = 4. ∗∗∗ indicates p < 0.001 compared to control, † indicates p < 0.05 compared to HA14-1, and ‡‡ indicates p < 0.01 compared to ICAL + HA14-1. Significant differences were determined by one-way ANOVA with a post hoc Tukey's test. Con: control; ICAL: Immunocal; BSO: buthionine sulfoximine.

Figure 3

Immunocal decreases CuCl2-induced apoptosis and lipid peroxidation in CGNs. (a) Representative images of CGNs left untreated (control), treated with CuCl2 (50 μM), or preincubated with Immunocal for 24 h before CuCl2 treatment for further 24 h. Immunofluorescence shows β-tubulin (green) and DAPI (blue). Scale bar, 10 μm. (b) Quantification of apoptosis for 4 independent experiments performed as in (a). Results are shown as mean ± SEM, n = 4. ∗∗ indicates p < 0.01 compared to control and †† indicates p < 0.01 compared to CuCl2. (c) Cellular lipid peroxidation (malondialdehyde (MDA)) was measured as described in Materials and Methods. Results are shown as mean ± SEM, n = 5. ∗∗ indicates p < 0.01 compared to control, ††† indicates p < 0.001 compared to CuCl2. Con: control; ICAL: Immunocal.

Figure 4

Immunocal preserves CGN viability and protects from apoptosis after exposure to SNP. (a) Representative images of CGNs left untreated (control), treated with SNP (100 μM), or preincubated with Immunocal for 24 h before SNP treatment for further 24 h. Immunofluorescence shows β-tubulin (green) and DAPI (blue). Scale bar, 10 μm. (b) Quantification of apoptosis for 5 independent experiments performed as in (a). Results are shown as mean ± SEM, n = 5. (c) MTT cell viability was measured as described in Materials and Methods. Results are shown as mean ± SEM, n = 3. For (b) and (c), ∗∗∗ indicates p < 0.001 compared to control, and ††† indicates p < 0.001 compared to SNP. Con: control; ICAL: Immunocal.

Figure 5

Immunocal protects CGNs from AlCl3-induced toxicity. (a) Representative images of CGNs left untreated (control), treated with AlCl3 (10 μM), or preincubated with Immunocal for 24 h before AlCl3 treatment for further 48 h. Panels from left to right, DAPI (nuclei), β-tubulin, and merged image showing β-tubulin (green), and DAPI (blue). Scale bar, 10 μm. (b) CGN apoptosis was quantified for 4 independent experiments as described in (a). Results are shown as mean ± SEM, n = 4. ∗∗∗ indicates p < 0.001 compared to control, and †† indicates p < 0.01 compared to AlCl3. Con: control; ICAL: Immunocal.

Figure 6

Immunocal protects NSC34 cells from H2O2 and glutamate/glycine-induced excitotoxicity. (a) Cell survival was quantified with MTT cell viability assay for 5 independent experiments in undifferentiated NSC34 left untreated (control), treated with H2O2 (250 μM), or preincubated with Immunocal for 24 h before H2O2 treatment for further 24 h. Results are shown as mean ± SEM, n = 5. ∗∗ indicates p < 0.01 compared to control, † indicates p < 0.05 compared to H2O2, and †† indicates p < 0.01 compared to H2O2. (b) Representative images showing morphological differences between undifferentiated (wildtype (WT)) and differentiated (DIFF) NSC34 cells, β-tubulin (green), and DAPI (blue). Scale bar, 10 μm. (c) Cell survival was quantified for 5 independent experiments with an MTT cell viability assay in differentiated NSC34 cells left untreated (control), treated with glutamate/glycine (1 mM/100 μM), or preincubated with Immunocal for 24 h before glutamate/glycine treatment for further 24 h. ∗ indicates p < 0.05 compared to control, and † indicates p < 0.05 compared to glutamate/glycine. Con: control; ICAL: Immunocal; GG: glutamate/glycine.

Figure 7

Immunocal protects HT22 cells from toxicity induced by overexpression of Aβ1-42. (a) Representative images of HT22 cells transfected with either empty vector (IRES) or Aβ1-42. Top panels display colored images showing successful transfection of the cells, and bottom panels display decolorized images of cell nuclei to visualize nuclear condensation. Arrows indicate transfected cells. (b) Quantification of apoptosis for 4 independent experiments performed as in (a). Results are shown as mean ± SEM, n = 4. ∗∗∗ indicates p < 0.001 compared to control, and ††† indicates p < 0.001 compared to cells transfected with Aβ1-42 without Immunocal preincubation. Aβ: amyloid-beta; ICAL: Immunocal.

Figure 8

Proposed neuroprotective mechanism of Immunocal. Immunocal provides the essential GSH precursor, cystine, which is transported into cerebellar granule neurons via the system xc− antiporter (Sxc−). Upon entry into the cell, cystine is rapidly hydrolyzed to form two cysteine molecules, which are then utilized in the de novo synthesis of GSH by γ-glutamylcysteine ligase (γ-GCL) and glutathione synthase (GSS). Newly synthesized glutathione inhibits oxidation caused by a variety of insults, thereby preventing mitochondrial oxidative stress (MOS) and subsequent induction of apoptosis.