Metabolic and Stress Response Changes Precede Disease Onset in the Spinal Cord of Mutant SOD1 ALS Mice

Many Amyotrophic Lateral Sclerosis (ALS) patients experience hypermetabolism, or an increase in measured vs. calculated metabolic rate. The cause of hypermetabolism and the effects on neuronal metabolism in ALS are currently unknown, but the efficacy of dietary interventions shows promise for metabolism as an ALS therapeutic target. The goal of this study is to measure changes in metabolic pathways as a function of disease progression in spinal cords of the SOD1G93A mouse model of ALS. We conducted a comprehensive assessment of protein expression for metabolic pathways, antioxidants, chaperones, and proteases in lumbar spinal cord from male SOD1G93A mice at pre-onset, onset, and end-stages of the disease using targeted proteomic analysis. These results reveal that protein content of metabolic proteins including proteins involved in glycolysis, β-oxidation, and mitochondrial metabolism is altered in SOD1G93A mouse spinal cord well before disease onset. The changes in mitochondrial metabolism proteins are associated with decreased maximal respiration and glycolytic flux in SOD1G93A dermal fibroblasts and increased hydrogen peroxide and lipid hydroperoxide production in mitochondria from sciatic nerve and gastrocnemius muscle fibers at end stage of disease. Consistent with redox dysregulation, expression of the glutathione antioxidant system is decreased, and peroxiredoxins and catalase expression are increased. In addition, stress response proteases and chaperones, including those involved in the mitochondrial unfolded protein response (UPRmt), are induced before disease onset. In summary, we report that metabolic and stress response changes occur in SOD1G93A lumbar spinal cord before motor symptom onset, and are primarily caused by SOD1G93A expression and do not vary greatly as a function of disease course

FOXP3 Expression Is Upregulated in CD4+T Cells in Progressive HIV-1 Infection and Is a Marker of Disease Severity

Understanding the role of different classes of T cells during HIV infection is critical to determining which responses correlate with protective immunity. To date, it is unclear whether alterations in regulatory T cell (Treg) function are contributory to progression of HIV infection.FOXP3 expression was measured by both qRT-PCR and by flow cytometry in HIV-infected individuals and uninfected controls together with expression of CD25, GITR and CTLA-4. Cultured peripheral blood mononuclear cells were stimulated with anti-CD3 and cell proliferation was assessed by CFSE dilution.HIV infected individuals had significantly higher frequencies of CD4(+)FOXP3(+) T cells (median of 8.11%; range 1.33%-26.27%) than healthy controls (median 3.72%; range 1.3-7.5%; P = 0.002), despite having lower absolute counts of CD4(+)FOXP3(+) T cells. There was a significant positive correlation between the frequency of CD4(+)FOXP3(+) T cells and viral load (rho = 0.593 P = 0.003) and a significant negative correlation with CD4 count (rho = -0.423 P = 0.044). 48% of our patients had CD4 counts below 200 cells/microl and these patients showed a marked elevation of FOXP3 percentage (median 10% range 4.07%-26.27%). Assessing the mechanism of increased FOXP3 frequency, we found that the high FOXP3 levels noted in HIV infected individuals dropped rapidly in unstimulated culture conditions but could be restimulated by T cell receptor stimulation. This suggests that the high FOXP3 expression in HIV infected patients is likely due to FOXP3 upregulation by individual CD4(+) T cells following antigenic or other stimulation.FOXP3 expression in the CD4(+) T cell population is a marker of severity of HIV infection and a potential prognostic marker of disease progression

A Quantitative Proteomic Profile of the Nrf2-Mediated Antioxidant Response of Macrophages to Oxidized LDL Determined by Multiplexed Selected Reaction Monitoring

<div><p>The loading of macrophages with oxidized low density lipoprotein (LDL) is a key part of the initiation and progression of atherosclerosis. Oxidized LDL contains a wide ranging set of toxic species, yet the molecular events that allow macrophages to withstand loading with these toxic species are not completely characterized. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a master regulator of the cellular stress response. However, the specific parts of the Nrf2-dependent stress response are diverse, with both tissue- and treatment-dependent components. The goal of these experiments was to develop and use a quantitative proteomic approach to characterize the Nrf2-dependent response in macrophages to oxidized LDL. Cultured mouse macrophages, the J774 macrophage-like cell line, were treated with a combination of oxidized LDL, the Nrf2-stabilizing reagent tert- butylhydroquinone (tBHQ), and/or Nrf2 siRNA. Protein expression was determined using a quantitative proteomics assay based on selected reaction monitoring. The assay was multiplexed to monitor a set of 28 antioxidant and stress response proteins, 6 housekeeping proteins, and 1 non-endogenous standard protein. The results have two components. The first component is the validation of the multiplexed, quantitative proteomics assay. The assay is shown to be fundamentally quantitative, precise, and accurate. The second component is the characterization of the Nrf2-mediated stress response. Treatment with tBHQ and/or Nrf2 siRNA gave statistically significant changes in the expression of a subset of 11 proteins. Treatment with oxidized LDL gave statistically significant increases in the expression of 7 of those 11 proteins plus one additional protein. All of the oxLDL-mediated increases were attenuated by Nrf2 siRNA. These results reveal a specific, multifaceted response of the foam cells to the incoming toxic oxidized LDL.</p> </div

Short run gel electrophoresis used for sample preparation and in-gel digestion.

<p>A 12.5% polyacrylamide gel was used to immobilize the samples and facilitate a highly reproducible in-gel digestion. Each lane contains 20 µg total protein. The gel was run at 150 V for 15 min to give a length of approximately 1.5 cm. After fixing, the gel was stained for 5 min. The staining pattern shows equal loading of each sample. Each lane was then cut as a single sample for processing via a typical in-gel digestion protocol. The treatment groups in this gel are: 1, control; 2 −+oxLDL; 3+Nrf2 siRNA; 4+both. Each treatment was n = 5 to give a set of 20 samples making two gels necessary.</p

Summary of proteins measured in the antioxidant protein assay.

<p>Summary of proteins measured in the antioxidant protein assay.</p

Identification of proteins induced in J774 cells treated with tert-butyl hydroquinone (tBHQ) and oxidized LDL (oxLDL).

<p>A total of 28 proteins were monitored in the assay and data are presented for the proteins that were detected. These data utilize the raw SRM signal intensity for each protein in the LC-tandem MS analyses. The SRM intensity values for the treated samples were then normalized to respective comparison group. The corresponding percent change relative to the control group is shown. A) Treatment with tBHQ (25 µM for 24h) was used to identify a subset of Nrf2-target proteins. After the six housekeeping proteins, the proteins targeted in the assay are presented in an order determined by the magnitude of the tBHQ effect. B) Treatment with Nrf2 siRNA (15 nmol/mL for 48 h) followed by tBHQ (25 µM for 24 h) was then used to verify the Nrf2-dependence. C) Treatment with oxLDL (50 µg/mL for 24 h) was compared to this signature. For each treatment, the proteins with statistically significant changes in expression are shown as a red bar (mean ± SEM, n = 5, p<0.05, Student’s t-test). Proteins with unchanged expression are shown as a blue bar.</p

Equal content of the housekeeping proteins validated by a variety of approaches.

<p>A) Statistical analysis of the response in the selected reaction monitoring assay. Data for five of the housekeeping proteins (Hspd1, Calr, Rsp8, Ldha, and Ncl) and the internal standard (Lyz) plotted as the raw signal strength for all samples (mean ± SEM, m = 5). No statistically significant change was seen across the four treatment groups for any protein by either a Students t-test or a one-way analysis of variance. B) Enzyme activity analysis for lactate dehydrogenase in all samples. No statistically significant changes were seen across the four treatment groups by either a Students t-test or a one-way analysis of variance. C) Western blot analysis of Hspd1 showing equal content in all treatment groups. Representative samples from a control, oxidized LDL-treated (+oxLDL), Nrf2 siRNA-treated (+Nrf2 siRNA), and treated with both Nrf2 siRNA and oxidized LDL (+both).</p

Combined effects of oxLDL- and Nrf2 siRNA-treatment on protein expression.

<p>Data are shown for effects of Nrf2 siRNA pretreatment on the 8 proteins identified as upregulated by oxLDL. For each protein, results of the SRM assay are shown for control, +oxLDL (50 µg/mL, 24 h), +both. The +both group was pretreated with Nrf2 siRNA (15 nmol/mL) for 48 h prior to the oxLDL treatement. Statistically significant differences are designated with an * (p<0.05, n = 5, ANOVA with a Tukey multiple comparisons test).</p

A demonstration of accuracy for the selected reaction monitoring result for catalase.

<p>The changing expression of catalase (Cat) in J774 cells treated with tBHQ, Cat esiRNA, or a combination of both was tested by several methods. A) A summary of the SRM results. The SRM signal intensities were determined for all samples in each group and are plotted as the mean ± SEM, n = 5. B) Analysis of the catalase expression by Western blot analysis with densitometry shown in (C). Two representative samples from each group were selected for analysis. The densitometry values are plotted as the average for the two determinations. D) Catalase enzyme activity assay. For A) and D) ANOVA with a Tukey multiple comparisons test was used and statistically significant differences are noted (p<0.05, n = 5).</p

Possible ion suppression tested through a range of sample loading amounts.

<p>Increasing amounts of total protein equivalents ranging from 0.25 µg to 1.25 µg were injected. A) Responses for glucose-6-phsophate dehydrogenase (G6pd), catalase (Cat), thioredoxin reductase (Txnrd1), and glutathione reductase (GSR) plotted using the raw abundance data from the SRM experiment (mean ± SEM, n = 5). B) The average response for all proteins monitored in this experiment. For this representation, the SRM responses for each protein were normalized to the average values for the respective 0.25 µg injection. This normalization gave an expected slope of 4.0 through the linear range.</p