How Do You Give Immunocal To Animals
Oxid Med Cell Longev. 2017; 2017: 3103272.
A Cystine-Rich Whey Supplement (Immunocal®) Provides Neuroprotection from Diverse Oxidative Stress-Inducing Agents In Vitro past Preserving Cellular Glutathione
Aimee North. Winter
1Department of Biological Sciences, Academy of Denver, 2199 Southward. Academy Blvd., Denver, CO 80208, USA
Erika K. Ross
iSection of Biological Sciences, University of Denver, 2199 S. University Blvd., Denver, CO 80208, U.s.
Vamsi Daliparthi
1Department of Biological Sciences, University of Denver, 2199 Due south. University Blvd., Denver, CO 80208, USA
Whitney A. Sumner
iDepartment of Biological Sciences, Academy of Denver, 2199 S. Academy Blvd., Denver, CO 80208, The states
Danielle M. Kirchhof
iSection of Biological Sciences, University of Denver, 2199 S. University Blvd., Denver, CO 80208, Usa
Evan Manning
1Department of Biological Sciences, Academy of Denver, 2199 South. Academy Blvd., Denver, CO 80208, U.s.a.
Heather 1000. Wilkins
1Department of Biological Sciences, University of Denver, 2199 Southward. Academy Blvd., Denver, CO 80208, USA
Daniel A. Linseman
oneDepartment of Biological Sciences, University of Denver, 2199 South. University Blvd., Denver, CO 80208, Us
2Knoebel Establish for Good for you Aging, University of Denver, 2155 Due east. Wesley Ave., Denver, CO 80208, USA
Received 2017 Feb xv; Accepted 2017 Jul 13.
Abstract
Oxidative stress is a main mechanism underlying the pathophysiology of neurodegeneration. Therefore, nutritional enhancement of endogenous antioxidant defenses may represent a viable treatment option. Nosotros investigated the neuroprotective properties of a unique whey protein supplement (Immunocal®) that provides an essential forerunner (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 and so afterward treated with agents known to induce oxidative stress. Immunocal protected CGNs against neurotoxicity induced by the Bcl-2 inhibitor, HA14-ane, the nitric oxide donor, sodium nitroprusside, CuCl2, and AlCl3. Immunocal also significantly reduced NSC34 cell death due to either HiiOtwo 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 betoken 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 past these diseases.
ane. Introduction
Oxidative stress and mitochondrial dysfunction are major factors underlying the pathophysiology of several neurodegenerative disorders including Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis (ALS) [ane–four]. For instance, complex I deficiency and the consequent increase in mitochondrial reactive oxygen species (ROS) play a critical office in the decease of dopaminergic neurons in Parkinson'due south disease [5, half-dozen]. In models of Alzheimer's illness, evidence of mitochondrial dysfunction and oxidative stress precedes the deposition of characteristic amyloid beta-plaques during disease progression [7, eight]. In the case of ALS, mutant forms of copper-zinc superoxide dismutase (SOD1), which are collectively responsible for approximately 20% of cases of familial ALS, accumulate at mitochondria and trigger a shift in the redox country of these organelles [9]. The above findings strongly indicate that oxidative stress, specially at the level of the mitochondria, plays a key part in the neuronal expiry that underlies a various group of neurodegenerative diseases.
Glutathione (GSH) is an endogenous tripeptide antioxidant that plays a key role in preventing oxidative stress, thereby preserving mitochondrial office and averting cellular apoptosis [10]. In many neurodegenerative disorders, GSH levels take been shown to be significantly depleted in patients suffering from these diseases, resulting in a diminished capacity to cope with increases in cellular ROS [11–13]. Indeed, decreases in GSH are often observed to precede other hallmarks of disease pathology, such every bit complex I deficiency and loss of dopaminergic neurons in Parkinson's illness [xiv]. Intriguingly, in vitro studies on GSH depletion have demonstrated that decreases in total cellular GSH levels tin recapitulate affliction pathology. For instance, in a dopaminergic PC12 cell line, deficiencies in GSH synthesis that led to an overall decrease in cellular GSH resulted in complex I inhibition, increased indices of oxidative stress, and deficits in mitochondrial respiration, every bit seen in cases of Parkinsonism [15]. Similarly, NSC34 motor neuron-like cells stably expressing the human G93A mutant form of SOD1 displayed a significant and selective depletion of mitochondrial GSH content in comparison to parental cells, reminiscent of some forms of familial ALS [16]. GSH depletion in vitro has also been shown to sensitize neurons to oxidative stress and mitochondrial dysfunction, leading to subsequent increases in ROS and apoptotic cell decease. This was clearly demonstrated by a study in which main cortical neurons treated with subtoxic levels of the GSH-depleting agent, buthionine sulfoximine (BSO), underwent apoptosis in the presence of trace amounts of extracellular copper [17]. Similarly, TAR DNA-bounden protein-43 (TDP-43) forms cytoplasmic inclusions, which are a hallmark pathology observed in sporadic ALS patients, in cultured neurons subjected to GSH depletion [18]. Collectively, these studies demonstrate a critical office for GSH depletion in disease progression and pathology in multiple neurodegenerative affliction states.
Given the prominent relationship between GSH depletion and neurodegeneration, it is not surprising that many studies have been undertaken to determine the neuroprotective effects of bolstering GSH levels through various handling paradigms. Such treatments include administration of the GSH precursor, N-acetylcysteine (NAC), and GSH-monoethylester (GSH-MEE), a prison cell permeable form of GSH, and consecration of the transcription factor, nuclear factor erythroid 2-related factor-ii (Nrf2), which is involved in transcriptional regulation of γ-glutamyl-cysteine ligase, the charge per unit-limiting enzyme necessary for GSH synthesis [19]. Studies with NAC are extensive and indicate that NAC treatment offers a number of benefits beyond numerous disease models. For case, NAC demonstrated a significant protective capacity in a rotenone (circuitous I inhibition) rat model of Parkinson's disease past decreasing ROS generation, sustaining normal GSH levels, and ultimately preventing dopaminergic cell death [20]. In the G93A mutant SOD1 mouse model of familial ALS, NAC delayed the onset of disease-associated motor deficits and significantly extended survival, possibly due to its power to elevate GSH levels in these animals [21]. Lastly, SAMP8 senescence-accelerated mice, which display many of the pathological features of Alzheimer's illness, demonstrated an increased cognitive performance with NAC handling as compared to vehicle-treated controls [22]. Another study utilizing GSH-MEE in an MPTP rat model of Parkinson's illness demonstrated that GSH-MEE supplementation is capable of raising GSH levels in the brain when centrally delivered, and this increase in GSH corresponded to partial preservation of striatal dopamine levels [23]. Studies such as this have led to recent clinical trials testing the safety and tolerability of intranasal delivery of GSH to patients with PD [24]. Finally, Nrf2 induction or overexpression has shown similar promise in animal models of Parkinson's, ALS, and Alzheimer'southward disease. In the MPTP mouse model of Parkinson's disease, overexpression of Nrf2 in astrocytes attenuated the development of a Parkinsonian phenotype [25]. Likewise, astrocytic overexpression of Nrf2 in a mouse model of ALS both delayed onset and increased survival, as did treatment with chemical Nrf2 inducers [26, 27]. Comparatively, lentiviral Nrf2 overexpression caused significant improvements in observed learning deficits in a mouse model of Alzheimer's illness, accompanied past decreased amyloid plaque brunt [28]. Cumulatively, these data indicate that treatments aimed at increasing GSH levels in the brain may be a feasible option for treatment and prevention of neurodegenerative illness.
However, while existing treatment strategies take shown some hope in this capacity, the efficacy of such treatments is significantly limited by the relatively low stability and bioavailability of compounds such as GSH-MEE and NAC [23, 29]. Moreover, GSH-MEE requires direct injection into the brain for pregnant effects to be observed, further limiting its efficacy for treatment in human patients [23]. In the electric current study, nosotros investigated the neuroprotective potential of a nondenatured whey protein supplement, Immunocal, in vitro in several models of oxidative stress. Immunocal has previously been shown to substantially increment blood or lymphocyte GSH levels in patients with HIV infection or cystic fibrosis, respectively, owing to its loftier concentration of nondenatured whey proteins containing the cysteine forerunner, cystine (encounter Table 1 for composition) [30–32]. Cystine is resistant to trypsin proteolysis and able to travel through the bloodstream to the target cell where it is then readily reduced to ii cysteine molecules which tin serve as essential precursors for de novo GSH synthesis. In this manner, the stability of Immunocal lends itself to increased bioavailability, such that information technology tin act as a cysteine commitment arrangement. This is significant, as cysteine is spontaneously catabolized in the GI tract and bloodstream, and its supplementation alone tin produce toxicity [33]. Additionally, because of its superior stability, the effects of Immunocal are non dependent upon an invasive administration system as is needed for GSH-MEE and have been observed with standard oral dosing regimens. These unique characteristics spurred us to examine the neuroprotective potential of Immunocal.
Table i
Immunocal constituents by mass per one packet of supplement (one packet of Immunocal contains approximately 10 grand of protein supplement (1 serving) in fine powder form and 40 calories per serving).
| Component | Supplement content | Per centum of total supplement |
|---|---|---|
| Whey proteins (β-lactoglobulin, immunoglobulin, serum albumin, α-lactalbumin, and lactoferrin) | 8.eight–nine.2 yard | 88–92% |
| Fat | ~0.05 g | <0.five% |
| Lactose | ~0.xv g | <ane.5% |
| Minerals (Ca, Na) | ~0.30 g | <3.0% |
| Moisture | 0.5 g | ~5% |
two. Materials and Methods
2.1. Materials
Immunocal was provided past Immunotec Inc. (Quebec, Canada; Table ane). 2-Amino-6-bromo-α-cyano-three-(ethoxycarbonyl)-4H-1-benzopyran-4-acetic acid ethyl ester (HA14-1) and sodium nitroprusside (SNP) were obtained from Calbiochem (San Diego, CA). DL-buthionine-sulfoximine (BSO), iv, half-dozen-diamidino-ii-phenylindole (DAPI), Hoechst dye 33258, and a monoclonal antibody against β-tubulin (clone AA2; used at a dilution of i : 250) were from Sigma Aldrich Co. LLC (St Louis, MO). FITC-conjugated secondary antibodies were from Jackson Immunoresearch Laboratories (Westward Grove, PA).
2.2. Jail cell Culture and Treatment
Rat cerebellar granule neurons (CGNs) were isolated every bit previously described from 7-day-old Sprague-Dawley rat pups of both sexes [34]. CGNs were seeded on 35 mm bore plastic dishes coated with poly-L-lysine at an boilerplate density of 2.0 × 106 cells/mL in basal modified Eagle's medium containing 10% fetal bovine serum, 25 mM KCl, 2 mM Fifty-glutamine, and penicillin (100 units/mL)/streptomycin (100μg/mL). Cytosine arabinoside (xμM) was added to the culture medium 24 h after plating. Experiments were performed after half dozen days in culture. In general, cells were pretreated with Immunocal at a concentration of iii.3%, w/five (unless otherwise noted) in serum-free medium for 24 h prior to treatment with the specified insult (i.east., SNP, HA14-1, etc.) for an boosted 24 h.
NSC34 cells were maintained in DMEM with high glucose containing 10% fetal bovine serum, 2 mM L-glutamine, and penicillin (100 units/mL)/streptomycin (100μm/mL). NSC34 cells were preincubated with Immunocal for 24 h prior to exposure to H2O2 or glutamate. For glutamate experiments, NSC34 cells were differentiated by withdrawing serum for vii days prior to experimentation.
For transient transfection, HT22 mouse hippocampal cells were seeded in vi-well plates at an estimate confluency of one.0 × 10half-dozen cells/mL and then cultured for 24 h in DMEM with low glucose containing ten% fetal bovine serum, ii mM L-glutamine, and penicillin (100 units/mL)/streptomycin (100μgrand/mL). Cells were transfected using lipofection (vμg Dna/mL, 5μL lipofectamine/mL) in OptiMEM medium for 4 h with either empty pIRES 2DsRed-Express2 bicistronic vector (Clontech, Mountain View, CA) or vector containing the sequence for amyloid-beta ane-42 (Aβ 1-42). Following transfection, OptiMEM medium was replaced with DMEM culture medium, and cells were treated with Immunocal for 24 h. Percent apoptosis was then adamant for only transfected (DSRed-positive) cells based on nuclear morphology.
two.3. Cell Viability, Lipid Peroxidation, and Cellular GSH Assay
All assays were performed according to commercially available manufacturer's instructions. GSH/GSSG analysis kit was purchased from Oxford Biomedical Research (Oxford, MI). MTT jail cell viability assay was from BioAssay Systems (Hayward, CA). Malondialdehyde (MDA) lipid peroxidation assay was obtained from OXIS Research Inc. (Foster City, CA).
2.4. Immunofluorescence Microscopy
After handling, cells were fixed in 4% paraformaldehyde for 1 h, washed one time in PBS, and then permeabilized and blocked in 0.2% Triton X-100 and 5% bovine serum albumin (BSA) in PBS. Primary antibody (monoclonal antibody against β-tubulin; clone AA2; used at a dilution of i : 250; Sigma Aldrich Co. LLC, St Louis, MO) was diluted in two% BSA and 0.ii% Triton X-100 in PBS, and cells were incubated with primary antibodies for 24 h at 4°C. They were then washed 5 times in PBS and then incubated for ane h in FITC-conjugated secondary antibody diluted in 2% BSA and 0.2% Triton-X 100 in PBS with DAPI. The cells were washed 5 times with PBS before the addition of antiquench (0.one% p-phenylenediamine in PBS). Images were captured using a Zeiss Axiovert 200 One thousand epifluorescence microscope equipped with Zeiss Axiovision software.
2.5. Statistical Assay
Each experiment was washed in duplicate and repeated a minimum of three times; information are reported as mean ± SEM. Statistical significance was analyzed with a ane-style analysis of variance (ANOVA) followed by post hoc Tukey'south test.
3. Results
3.1. Immunocal Preserves Cellular GSH and Prevents Apoptosis in CGNs Exposed to the Bcl-2 Inhibitor, HA14-1
Initially, master CGNs were incubated with iii.3% (w/v) Immunocal for 24 h to appraise any potential toxicity that this supplement might induce. Immunocal is composed of 5 primary cystine- and glutamylcysteine-containing proteins, β-lactoglobulin, immunoglobulin, α-lactalbumin, serum albumin, and lactoferrin (Table 2) [35, 36]. Based upon the relative percentages for each of these four proteins within the whey poly peptide fraction and the number of cystine or glutamylcysteine residues contained inside each poly peptide, nosotros calculated the estimate concentration of each of these GSH precursors with which CGNs were treated (Table 3). In general, a three.iii% solution of Immunocal in civilization medium contains 85.iii mM cystine and xxx mM glutamylcysteine, both of which have the potential to act as GSH precursors; however, it should be noted that since both precursors are contained inside much larger proteins information technology is unlikely that all cystine and glutamylcysteine molecules are freely available to exist utilized in GSH synthesis. Thus, the values calculated in Table iii for these precursors should be considered as concentrations that could potentially be achieved rather than accented concentrations.
Table 2
Cystine [(Cys)ii] and glutamylcysteine [Glu-(Cys)2] content of Immunocal whey proteins.
| Whey protein | Molecular mass (kDa) | Percent of protein fraction | Cystine (Cys)2 per molecule | Glu-(Cys)2 per molecule |
|---|---|---|---|---|
| β-Lactoglobulin | 18,400 | 56.three% | 2 | 0 |
| Immunoglobulin | 166,000 | nine.2% | iv | 0 |
| α-Lactalbumin | 14,200 | 22.8% | 4 | 0 |
| Serum albumin | 66,000 | 11.1% | 17 | six |
| Lactoferrin | 77,000 | 0.7% | 17 | 4 |
Table 3
Cystine [(Cys)2] and glutamylcysteine [Glu-(Cys)ii] content of Immunocal in preincubation civilisation medium (3.three%, westward/five final concentration).
| Whey poly peptide | Total molecules per mL | Full number of (Cys)2 per mL | Total number of Glu-(Cys)ii per mL |
|---|---|---|---|
| β-Lactoglobulin | 5.44 × x14 | 1.09 × ten15 | 0 |
| Immunoglobulin | 9.91 × x12 | 3.96 × 1013 | 0 |
| α-Lactalbumin | 2.91 × 10xiv | 1.17 × x15 | 0 |
| Serum albumin | 3.01 × 10xviii | 5.xi × 1019 | 1.80 × 10xix |
| Lactoferrin | 1.63 × 1016 | 2.76 × 1017 | 6.50 × 1016 |
| Last concentration | — | 85.3 mM | xxx.0 mM |
Following Immunocal treatment, cells were fixed and stained with DAPI to clarify nuclear morphology. Cells treated with Immunocal alone displayed nuclear morphology comparable to that of untreated control cells (Figure ane). Moreover, observation under brightfield demonstrated that cells treated with Immunocal maintained a healthy neuronal morphology with intact processes and large somas, comparable to cells that were non supplemented with Immunocal (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 country of neuronal processes and soma. Con: control; ICAL: Immunocal. Scale bar, 10μm.
Having established that Immunocal displayed no overt toxicity to CGNs, cells were next treated with Immunocal and so exposed to the Bcl-2 homology-3 domain (BH3) mimetic, HA14-ane. We have previously shown this Bcl-2 inhibitor to induce GSH-sensitive mitochondrial oxidative stress and intrinsic apoptosis in CGNs [37, 38]. HA14-1 induced marked nuclear condensation and microtubule disruption (Effigy 2(a)) indicative of apoptosis (Figure 2(b)), while also causing significant depletion of GSH (Figure 2(c)). Immunocal significantly protected CGNs from apoptosis induced by HA14-1 and significantly preserved GSH levels. To ostend that the mechanism of protection was dependent, at to the lowest degree in part, on enhanced GSH synthesis, CGNs were cotreated with Immunocal and the γ-glutamyl-cysteine ligase inhibitor, BSO, which prevents GSH synthesis [39]. Coincubation with Immunocal and BSO for 24 h before HA14-1 treatment completely prevented whatsoever protective effect that Immunocal solitary displayed confronting the Bcl-2 inhibitor (Effigy 2(b)). Moreover, the capacity of Immunocal to preserve cellular GSH levels upon HA14-one exposure was eliminated by BSO cotreatment (Figure ii(c)).
Immunocal preserves cellular GSH and prevents apoptosis in CGNs exposed to the Bcl-2 inhibitor, HA14-1. (a) Representative images of CGNs left untreated (command), treated with HA14-1 (fifteenμK), or preincubated for 24 h with Immunocal before HA14-ane treatment for further 24 h. Panels from left to right, DAPI (nuclei), β-tubulin, merged images showing β-tubulin (dark-green), and DAPI (blueish). Scale bar, 10μm. (b) Quantification of apoptosis for 4 independent experiments performed as in (a) except some cultures were preincubated with 200μM BSO as well. Apoptotic cells were those with condensed or fragmented nuclei. Results are shown every bit hateful ± SEM, n = 4. ∗∗∗ indicates p < 0.001 compared to command, ††† indicates p < 0.001 compared to HA14-i, ‡‡‡ indicates p < 0.001 compared to ICAL + HA14-1. (c) CGNs were treated exactly every bit described in (b). Total cellular GSH was measured as described in Materials and Methods. Information shown represent the percent of control cellular GSH concentration, mean ± SEM, due north = iv. ∗∗∗ indicates p < 0.001 compared to control, † indicates p < 0.05 compared to HA14-1, and ‡‡ indicates p < 0.01 compared to ICAL + HA14-i. Pregnant differences were determined by i-style ANOVA with a post hoc Tukey's test. Con: command; ICAL: Immunocal; BSO: buthionine sulfoximine.
iii.two. Immunocal Protects CGNs from CuCl2-Induced Oxidative Damage and Decreases Cellular Lipid Peroxidation
To further investigate the neuroprotective potential of Immunocal in master neurons, we used copper chloride (CuCl2) equally a model of oxidative stress. Copper overload is associated with free radical-induced lipid peroxidation and disruption of mitochondrial complex activeness [twoscore, 41]. Immunofluorescence analysis of the microtubule network revealed robust protection from this transition metal in CGNs pretreated with Immunocal (Figure three(a)). Quantification of apoptotic cells revealed that at that place was a significant reduction in CGN apoptosis with Immunocal pretreatment compared to CGNs treated with CuCl2 alone (Figure 3(b)). The antioxidant effect of Immunocal was confirmed with a lipid peroxidation analysis which revealed a pregnant decrease in malondialdehyde content in CGNs pretreated with Immunocal (Figure 3(c)).
Immunocal decreases CuClii-induced apoptosis and lipid peroxidation in CGNs. (a) Representative images of CGNs left untreated (control), treated with CuClii (lμM), or preincubated with Immunocal for 24 h before CuCl2 treatment for farther 24 h. Immunofluorescence shows β-tubulin (dark-green) and DAPI (blue). Scale bar, tenμm. (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 command and †† indicates p < 0.01 compared to CuCltwo. (c) Cellular lipid peroxidation (malondialdehyde (MDA)) was measured equally 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.
3.3. Immunocal Protects CGNs Exposed to Sodium Nitroprusside- (SNP-) Generated Nitric Oxide Species and from AlCl3-Induced Neurotoxicity
SNP is a nitric oxide donor that causes dissipation of the mitochondrial membrane potential and enhanced generation of mitochondrial ROS in cortical neurons and CGNs [42, 43]. Every bit expected, nitric oxide species generated by SNP caused overt apoptotic cell death in CGNs which was significantly mitigated past pretreatment with Immunocal (Figure 4(a)). Apoptotic cell counts confirmed that there was significant neuroprotection in CGNs pretreated with Immunocal, decreasing apoptosis by approximately 80% (Figure 4(b)). An MTT cell viability assay demonstrated similar results and showed that mitochondrial viability was too significantly preserved in Immunocal-pretreated cells, compared to CGNs treated with SNP solitary (Effigy four(c)).
Immunocal preserves CGN viability and protects from apoptosis afterward exposure to SNP. (a) Representative images of CGNs left untreated (control), treated with SNP (100μM), or preincubated with Immunocal for 24 h earlier SNP treatment for further 24 h. Immunofluorescence shows β-tubulin (green) and DAPI (blue). Scale bar, xμ1000. (b) Quantification of apoptosis for five independent experiments performed as in (a). Results are shown as mean ± SEM, due north = v. (c) MTT jail cell viability was measured equally described in Materials and Methods. Results are shown as mean ± SEM, due north = 3. For (b) and (c), ∗∗∗ indicates p < 0.001 compared to control, and ††† indicates p < 0.001 compared to SNP. Con: control; ICAL: Immunocal.
Aluminum is a neurotoxic metallic that impairs mitochondrial construction and office in neural cells exposed in vitro and in vivo [44, 45]. Aluminum chloride- (AlCliii-) induced toxicity in CGNs was characterized by nuclear condensation and marked disruption of the microtubule network; these furnishings were markedly decreased in CGNs pretreated with Immunocal (Figure 5(a)). To confirm that this protection was due to cysteine supplementation, and non metallic chelation, we removed the Immunocal later on the pretreatment period and washed the CGNs with serum-costless media before treating with AlCl3. Under these conditions, we still observed a significant reduction in apoptosis compared to CGNs treated with AlCl3 alone (Effigy five(b)).
Immunocal protects CGNs from AlCl3-induced toxicity. (a) Representative images of CGNs left untreated (control), treated with AlCl3 (tenμThou), or preincubated with Immunocal for 24 h before AlCl3 handling for further 48 h. Panels from left to correct, DAPI (nuclei), β-tubulin, and merged image showing β-tubulin (light-green), and DAPI (blue). Scale bar, 10μm. (b) CGN apoptosis was quantified for 4 contained experiments equally described in (a). Results are shown as mean ± SEM, n = 4. ∗∗∗ indicates p < 0.001 compared to command, and †† indicates p < 0.01 compared to AlCliii. Con: control; ICAL: Immunocal.
3.iv. Immunocal Protects NSC34 Motor Neuron-Like Cells from HtwoO2 and Glutamate/Glycine-Induced Excitotoxicity
H2O2-mediated cell death is a archetype model of ROS toxicity in neuronal systems, as it generates free radicals that are implicated in neurodegeneration [46]. As expected, ROS generated by H2Oii caused an overt loss of viability in NSC34 cells, which was significantly mitigated by pretreatment with Immunocal. An MTT cell viability assay demonstrated that mitochondrial viability was preserved in Immunocal-pretreated cells in a dose-dependent manner, compared to NSC34 cells treated with HiiO2 alone (Effigy 6(a)). Incubation with Immunocal alone had no significant adverse issue on NSC34 cell viability assessed past MTT analysis (data not shown).
Immunocal protects NSC34 cells from HiiO2 and glutamate/glycine-induced excitotoxicity. (a) Cell survival was quantified with MTT prison cell viability assay for five independent experiments in undifferentiated NSC34 left untreated (command), treated with H2O2 (250μM), or preincubated with Immunocal for 24 h before H2Otwo handling 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 H2Otwo, and †† indicates p < 0.01 compared to HtwoOtwo. (b) Representative images showing morphological differences between undifferentiated (wildtype (WT)) and differentiated (DIFF) NSC34 cells, β-tubulin (green), and DAPI (blue). Calibration bar, 10μgrand. (c) Cell survival was quantified for 5 independent experiments with an MTT cell viability analysis in differentiated NSC34 cells left untreated (command), treated with glutamate/glycine (ane mM/100μM), or preincubated with Immunocal for 24 h earlier 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.
Glutamate excitotoxicity is thought to play a significant role in several forms of neurodegenerative disease, leading to neuronal harm and prison cell death through both apoptotic and nonapoptotic mechanisms. NSC34 motor neuron-like cells practise not typically express functional glutamate receptors, which are the chief mediators of excitotoxicity. However, if they are exposed to serum withdrawal for vii days, so they attain a semi-differentiated state and express functional N-methyl-D-aspartate (NMDA) receptors (Figure 6(b)). Later this, betoken cells become sensitive to glutamate excitotoxicity [47]. We observed that exposure to glutamate/glycine caused a significant loss of viability in NSC34 cells differentiated by serum withdrawal. An MTT cell viability assay demonstrated that mitochondrial viability was significantly preserved in Immunocal-pretreated cells in a dose-dependent mode, compared to NSC34 cells treated with glutamate/glycine lonely (Figure six(c)).
3.5. Immunocal Protects HT22 Mouse Hippocampal Cells from Toxicity Induced past Overexpression of Amyloid-Beta Peptide (Aβ 1-42)
Aβ 1-42 is the major constituent of senile plaques, which form in the brains of Alzheimer'south patients, leading to the hypothesis that increased production of this protein from aberrant processing of amyloid precursor protein is a major contributor to neuronal death and disease pathogenesis [48]. HT22 mouse hippocampal cells transfected with Aβ 1-42 displayed a marked increase in apoptosis compared to controls transfected with empty vector, indicated by the presence of condensed and fragmented nuclei (Figure 7(a)). Strikingly, this effect was entirely mitigated by handling with Immunocal, which preserved neuronal viability to an extent similar to that of empty vector controls (Figure 7(b)).
Immunocal protects HT22 cells from toxicity induced by overexpression of Aβ i-42. (a) Representative images of HT22 cells transfected with either empty vector (IRES) or Aβ ane-42. Top panels display colored images showing successful transfection of the cells, and bottom panels display decolorized images of prison cell nuclei to visualize nuclear condensation. Arrows betoken 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 command, and ††† indicates p < 0.001 compared to cells transfected with Aβ one-42 without Immunocal preincubation. Aβ: amyloid-beta; ICAL: Immunocal.
4. Word
Strategies aimed at scavenging ROS, including those that enhance the capacity of endogenous antioxidant defenses like GSH, are actively existence investigated equally therapeutic approaches for neurodegenerative diseases. In the present study, we assessed the neuroprotective potential of Immunocal, a cystine-rich whey protein supplement, against oxidative stress in vitro. This supplement contains high concentrations of proteins such as serum albumin, blastoff-lactalbumin, and lactoferrin, which possess a substantial number of cystine residues in the unique nondenatured preparation. In improver, the direct GSH forerunner, glutamylcysteine, is as well present in the serum albumin fraction of this supplement. Due to these unique features, Immunocal has been used as a cysteine delivery organization to boost GSH levels in individuals diagnosed with diseases for which oxidative stress is a prominent underlying gene [31, 32, 49]. Therefore, Immunocal may be an effective approach to elevate GSH in cases of neurodegeneration for which oxidative stress plays a meaning function. To this cease, we studied the potential of Immunocal to protect neurons in vitro from a diverse array of oxidative insults, which are not only known to cause oxidative damage and mitochondrial dysfunction but besides to imitate some pathogenic factors in neurodegeneration such as diminished Bcl-2 function, increased levels of nitric oxide, or metal ion toxicity (Figure eight).
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 grade two cysteine molecules, which are and 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 diversity of insults, thereby preventing mitochondrial oxidative stress (MOS) and subsequent consecration of apoptosis.
GSH depletion is a widely studied phenomenon in cases of neurodegeneration. Although there are multiple mechanisms by which GSH may be depleted, one involves the downregulation of Bcl-2 expression or function. Increased expression of Bcl-2 leads to enhanced GSH synthesis and decreased GSH efflux from the prison cell [l, 51]. On the other paw, Bcl-ii knockdown leads to decreased levels of tissue GSH [52]. In the electric current study, we utilized the Bcl-two inhibitor, HA14-1, to mimic loss of Bcl-2 function and assess the neuroprotective potential of Immunocal. Nosotros have previously shown HA14-ane to subtract the cellular GSH pool with a propensity to bear on the mitochondrial GSH pool starting time and induce mitochondrial oxidative stress and intrinsic apoptosis in CGNs [37, 38]. Under these atmospheric condition, Immunocal displayed robust neuroprotection, indicating a capacity to counter the effects of mitochondrial GSH depletion and oxidative stress induced by loss of Bcl-ii function. Moreover, the protective event of Immunocal against Bcl-2 inhibition is dependent upon de novo GSH synthesis as coincubation of Immunocal with BSO blocked neuroprotection.
Another cistron implicated in the pathogenesis of several neurodegenerative diseases is copper toxicity. GSH is known to play a significant role in mitigating copper toxicity past facilitating the transport of copper to proteins that can safely store this toxic metal in the intracellular environment [53]. Depletion of GSH disrupts this important process and sensitizes neuronal cells to copper toxicity through copper-associated ROS generation, even when exposed to only trace amounts of copper [17, 54, 55]. Thus, copper toxicity may exist a process that is dependent on GSH depletion, and indeed, increased concentrations of copper and dysregulation of copper homeostasis are observed in several neurodegenerative diseases in which GSH status is reduced, including Alzheimer'southward disease and models of ALS [54, 56]. In our study, acme of GSH levels in cultured primary neurons with Immunocal proved to exist an constructive fashion to ameliorate the toxic effects of copper treatment past attenuating copper-induced lipid peroxidation, resulting in reduced cell expiry.
Neuroinflammation, in which microglia and astrocytes take on an inflammatory phenotype and secrete toxic factors such as cytokines and nitric oxide, is another major component of neurodegenerative disease [57, 58]. Induction of nitric oxide synthase (NOS) and subsequent production of nitric oxide is a well-established mechanism by which inflammatory cells trigger neuronal cell death [57]. Markers of nitrosative stress are prevalent in tissues from both Parkinson's and Alzheimer'due south disease patients, indicating a significant role for nitric oxide in affliction pathogenesis [59, lx]. Reactive nitrogen species (RNS) such as nitric oxide promote harm to mitochondrial components, leading to dissipation of mitochondrial membrane potential and further increases in ROS and RNS production [42, 43]. This feed forward cycle ultimately exacerbates inflammatory responses and somewhen results in neuronal decease. GSH is known to detoxify both ROS and RNS, making it an essential antioxidant and primal neuroprotective molecule. Consistent with this, preincubation with Immunocal significantly protected CGNs from toxicity induced by the nitric oxide donor SNP.
The neurotoxic furnishings of aluminum exposure are well documented, and recently, environmental aluminum and aluminum-containing vaccines have garnered attention as potential causes of neurodegeneration. In general, in vitro exposure of neural cells to aluminum has been shown to result in pronounced alterations in mitochondrial structure and office, leading to marked increases in ROS, reduction of mitochondrial enzyme activity, and prison cell death [45]. Aluminum also interferes with the activity of NADP-isocitrate at the mitochondria, decreasing the pool of NADPH that is available and necessary for the regeneration of GSH, and thereby decreasing GSH levels [61]. In vivo examination of aluminum neurotoxicity has demonstrated that healthy mice treated with aluminum hydroxide display significant motor deficits and develop pathological features similar to those observed in ALS [62]. These results are notable in that Veterans of the 1990-1991 Gulf War who received vaccines containing aluminum hydroxide adjuvant demonstrate a significant increase in the incidence of ALS, implicating aluminum toxicity every bit i potential ecology factor in some forms of sporadic ALS [62, 63]. Our experiments conspicuously demonstrate that Immunocal pretreatment is capable of significantly reducing the degree of neurotoxicity observed with aluminum in CGN cultures. We further confirmed that the protective effects of Immunocal were not due to metal chelation past removing Immunocal-containing media prior to the add-on of AlCl3.
To determine if the protective action of Immunocal observed in CGNs was reproducible in other neuronal cell types bearing relevance to neurodegenerative illness, we examined the chapters of this supplement to protect NSC34 motor neuron-like cells from oxidative stress and excitotoxicity. NSC34 cells are a hybrid cell line consisting of spinal cord motor neurons fused with mouse neuroblastoma cells [64]. Nosotros first analyzed the ability of Immunocal to protect NSC34 cells from H2Oii-induced oxidative stress. Immunocal pretreatment of NSC34 cells dose-dependently adulterate H2O2-induced cell death. Nosotros side by side examined the potential of Immunocal to ameliorate harm induced by excitotoxic insult in NSC34 cells, which were differentiated by prolonged serum withdrawal to induce the expression of NMDA receptors [47]. Excitotoxicity is known to play a prevalent role in the pathogenesis of multiple neurodegenerative diseases, including ALS, and is intimately linked with both oxidative and nitrosative stress [65]. Immunocal pretreatment of differentiated NSC34 motor neuron-like cells significantly reduced the injurious effects of glutamate excitotoxicity in a dose-dependent way.
Lastly, we evaluated the ability of Immunocal to defend HT22 mouse hippocampal cells from toxicity induced past the overexpression of Aβ ane-42. Every bit previously discussed, Aβ 1-42 is the primary elective of senile plaques, one of the hallmarks of Alzheimer's affliction pathology. In improver, this protein is also known to accrue with amyloid forerunner protein at mitochondria, leading to significant mitochondrial dysfunction [48]. Indeed, Aβ 1-42 accumulation at the mitochondria has been shown to occur both in transgenic mouse models of the affliction and in the brains of Alzheimer's patients [66–68]. Our information point that pretreatment with Immunocal was able to preserve HT22 hippocampal cell viability to a significant degree, indicating that GSH supplementation may exist an constructive way to mitigate cell decease caused by Aβ 1-42-induced toxicity.
5. Conclusions
Immunocal was initially studied for awarding to clinical disorders of immune organisation deficiency and cancer every bit an arroyo to augment the bachelor GSH pool and increase cellular antioxidant and immune system defenses. More recently, Immunocal has been investigated as a potential handling for disorders involving the CNS. Oral administration of Immunocal for 45 days has been shown to elevate GSH levels in the brains of healthy, nontransgenic mice by up to 300% compared to casein-treated controls, demonstrating that this supplement is able to direct affect the antioxidant status of tissues in the CNS [69]. Furthermore, we recently demonstrated that oral administration of Immunocal in the G93A mutant SOD1 mouse model of ALS delayed illness onset and preserved grip strength to a meaning caste, in comparison to untreated transgenic mice [70]. These therapeutic effects correlated with preservation of both blood and spinal cord GSH levels in comparison to untreated transgenic controls, indicating that Immunocal is able to act straight on the CNS to preserve GSH status in the context of neurodegenerative disease. Based on the above studies and the data shown here, we propose that Immunocal might agree meaning potential as a novel therapeutic arroyo to eternalize GSH levels in neurodegenerative disorders for which the underlying pathology involves significant oxidative stress. In the future, it will be of involvement to farther assess the therapeutic do good of GSH precursor supplementation with Immunocal in additional preclinical animal models of neurodegeneration and ultimately in clinical trials of patients afflicted with neurodegenerative disorders.
Acknowledgments
This study was supported in role past funding from Immunotec Inc. (Quebec, Canada).
Abbreviations
| Aβ: | Amyloid beta |
| ALS: | Amyotrophic lateral sclerosis |
| BH3: | Bcl-ii homology domain-3 |
| BSA: | Bovine serum albumin |
| BSO: | Buthionine sulfoximine |
| CGN: | Cerebellar granule neuron |
| DAPI: | 4,6-Diamidino-ii-phenylindole |
| γ-GCL: | γ-Glutamyl-cysteine ligase |
| GSH: | Glutathione |
| GSH-MEE: | Glutathione monoethylester |
| GSS: | Glutathione synthase |
| HA14-1: | 2-Amino-6-bromo-α-cyano-3-(ethoxycarbonyl)-4H-i-benzopyran-4-acetic acid ethyl ester |
| ICAL: | Immunocal |
| NAC: | N-Acetyl cysteine |
| NMDA: | N-Methyl-D-aspartate |
| Nrf2: | Nuclear cistron erythroid 2-related gene-2 |
| ROS: | Reactive oxygen species |
| RNS: | Reactive nitrogen species |
| SOD1: | Copper-zinc superoxide dismutase |
| SNP: | Sodium nitroprusside |
| Sxc −: | Organization xc − |
| TDP-43: | TAR Deoxyribonucleic acid binding poly peptide-43. |
Disclosure
Significant portions of this work have previously been published every bit part of one of the coauthor's master'south thesis (Erika K. Ross, "Nutraceutical Antioxidants and Their Therapeutic Potential in Neurodegeneration" (2012). Electronic Theses and Dissertations. Paper 563). This work has previously been presented in poster form at the Society for Neuroscience Annual Coming together.
Conflicts of Involvement
The authors take received funding from Immunotec Inc. (Quebec, Canada) to support the research on the neuroprotective effects of Immunocal.
Authors' Contributions
Erika K. Ross, Vamsi Daliparthi, Aimee N. Winter, Whitney A. Sumner, Danielle Grand. Kirchhof, Evan Manning, and Heather 1000. Wilkins had substantial contributions to the formulation and design, acquisition, and analysis or estimation of data. Aimee N. Winter, Erika K. Ross, and Daniel A. Linseman are involved in drafting the article or revising it critically for important intellectual content. Daniel A. Linseman is responsible for the final approval of the version to exist published.
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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5574309/
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