Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic ...

Perelman School of Medicine at the University of Pennsylvania

The contribution of inflammation to neurodegenerative diseases is increasingly recognized, but the role of inflammation in sporadic amyotrophic lateral sclerosis (sALS) is not well understood and no animal model is available. We used enzyme-linked immunosorbent assays (ELISAs) to measure the cytokine interleukin-17A (IL-17A) in the serum of ALS patients (n = 32; 28 sporadic ALS (sALS) and 4 familial ALS (fALS)) and control subjects (n = 14; 10 healthy subjects and 4 with autoimmune disorders). IL-17A serum concentrations were 5767 ± 2700 pg/ml (mean ± SEM) in sALS patients and 937 ± 927 pg/ml in fALS patients in comparison to 7 ± 2 pg/ml in control subjects without autoimmune disorders (p = 0.008 ALS patients vs. control subjects by Mann-Whitney test). Sixty-four percent of patients and no control subjects had IL-17A serum concentrations > 50 pg/ml (p = 0.003 ALS patients vs. healthy subjects by Fisher's exact test). The spinal cords of sALS (n = 8), but not control subjects (n = 4), were infiltrated by interleukin-1β- (IL-1β-), and tumor necrosis factor-α-positive macrophages (co-localizing with neurons), IL-17A-positive CD8 cells, and IL-17A-positive mast cells. Mononuclear cells treated with aggregated forms of wild type superoxide dismutase-1 (SOD-1) showed induction of the cytokines IL-1β, interleukin-6 (IL-6), and interleukin-23 (IL-23) that may be responsible for induction of IL-17A. In a microarray analysis of 28,869 genes, stimulation of peripheral blood mononuclear cells by mutant superoxide dismutase-1 induced four-fold higher transcripts of interleukin-1α (IL-1α), IL-6, CCL20, matrix metallopeptidase 1, and tissue factor pathway inhibitor 2 in mononuclear cells of patients as compared to controls, whereas the anti-inflammatory cytokine interleukin-10 (IL-10) was increased in mononuclear cells of control subjects. Aggregated wild type SOD-1 in sALS neurons could induce in mononuclear cells the cytokines inducing chronic inflammation in sALS spinal cord, in particular IL-6 and IL-17A, damaging neurons. Immune modulation of chronic inflammation may be a new approach to sALS.

IL-17A is increased in the serum and in spinal cord CD8 and mast cells of ALS patients.

Practical biomarkers of Alzheimer's disease (AD) prognosis are lacking. Correspondingly, no drugs are known to decrease disease progression, although vitamin D3 has positive effects on cognition in vivo and 1α, 25-dihydroxyvitamin D3 (1,25 D3) on amyloid-β 1-42 (Aβ) phagocytosis in vitro. We have examined in a pilot study a new biomarker in peripheral blood mononuclear cells, the transcription of mRNA of β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase (MGAT3), the essential gene for Aβ phagocytosis. The transcription of MGAT3 stimulated by Aβ distinguishes macrophages into Type 0 (very low MGAT3 transcription), Type I (low MGAT3 transcription up regulated by bisdemethoxycurcumin), and Type II (high MGAT3 transcription down regulated by bisdemethoxycurcumin). In this pilot study of 20 AD patients and 20 control subjects, 45% patients, but only 10% control subjects, were Type 0 (p-value = 0.009). Type 0 AD patients had worse 2-year prognosis regarding loss of independence than Type I and Type II patients (p-value = 0.013). Phagocytosis of Aβ in Type I and II patients was shown to be dependent on 1,25 D3 using a specific inhibitor of the 1,25 D3-VDR activated nuclear receptor transcription factor. In a Type II patient, recovery from cognitive dysfunction related to surgical anesthesia was preceded by an improvement in phagocytosis of Aβ. The results of this pilot study suggest that the MGAT3 Type biomarker may characterize subgroups of AD patients with different disease progression. In vitro results suggest that vitamin D3 supplementation might be beneficial in both Type I and II patients, whereas curcuminoids only in Type I. These results must be investigated in a large prospective study.

MGAT3 mRNA: a biomarker for prognosis and therapy of Alzheimer's disease by vitamin D and curcuminoids.

As immune defects in amyloid-β (Aβ) phagocytosis and degradation underlie Aβ deposition and inflammation in Alzheimer's disease (AD) brain, better understanding of the relation between Aβ phagocytosis and inflammation could lead to promising preventive strategies. We tested two immune modulators in peripheral blood mononuclear cells (PBMCs) of AD patients and controls: 1α,25(OH)2-vitamin D3 (1,25D3) and resolvin D1 (RvD1). Both 1,25D3 and RvD1 improved phagocytosis of FAM-Aβ by AD macrophages and inhibited fibrillar Aβ-induced apoptosis. The action of 1,25D3 depended on the nuclear vitamin D and the protein disulfide isomerase A3 receptors, whereas RvD1 required the chemokine receptor, GPR32. The activities of 1,25D3 and RvD1 commonly required intracellular calcium, MEK1/2, PKA, and PI3K signaling; however, the effect of RvD1 was more sensitive to pertussis toxin. In this case study, the AD patients: a) showed significant transcriptional up regulation of IL1RN, ITGB2, and NFκB; and b) revealed two distinct groups when compared to controls: group 1 decreased and group 2 increased transcription of TLRs, IL-1, IL1R1 and chemokines. In the PBMCs/macrophages of both groups, soluble Aβ (sAβ) increased the transcription/secretion of cytokines (e.g., IL1 and IL6) and chemokines (e.g., CCLs and CXCLs) and 1,25D3/RvD1 reversed most of the sAβ effects. However, they both further increased the expression of IL1 in the group 1, sβ-treated cells. We conclude that in vitro, 1,25D3 and RvD1 rebalance inflammation to promote Aβ phagocytosis, and suggest that low vitamin D3 and docosahexaenoic acid intake and/or poor anabolic production of 1,25D3/RvD1 in PBMCs could contribute to AD onset/pathology.

1α,25-dihydroxyvitamin D3 and resolvin D1 retune the balance between amyloid-β phagocytosis and inflammation in Alzheimer's disease patients.

Patients with sporadic amyotrophic lateral sclerosis (sALS) show inflammation in the spinal cord and peripheral blood. The inflammation is driven by stimulation of macrophages by aggregated superoxide dismutase 1 (SOD1) through caspase1, interleukin 1 (IL1), IL6 and chemokine signaling. Inflammatory gene activation is inhibited in vitro by tocilizumab, a humanized antibody to IL6 receptor (IL6R). Tocilizumab inhibits global interleukin-6 (IL6) signaling, a key mechanism in chronic rheumatoid disorders. Here we studied in vivo baseline inflammatory gene transcription in peripheral blood mononuclear cells (PBMCs) of 10 sALS patients, and the effects of tocilizumab (Actemra(R)) infusions. At baseline, one half of ALS subjects had strong inflammatory activation (Group 1) (8 genes up regulated >4-fold, P<0.05 vs. controls) and the other half (Group 2) had weak activation. All patients showed greater than four-fold up regulation of MMP1, CCL7, CCL13 and CCL24. Tocilizumab infusions in the Group 1 patients resulted in down regulation of inflammatory genes (in particular IL1β), whereas in the Group 2 patients in up regulation of inflammatory genes. Post-infusion serum and CSF concentrations of tocilizumab inhibited caspase1 activation in vitro. Three of 5 patients receiving tocilizumab infusions showed time-limited attenuation of clinical progression. In conclusion, inflammation of sALS patients at baseline is up- or down-regulated in comparison to controls, but is partially normalized by tocilizumab infusions.

Rachel Weitzman

Department of Biochemistry and Biophysics

Perelman School of Medicine at the University of Pennsylvania

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Rachel Weitzman

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry and Biophysics

Perelman School of Medicine at the University of Pennsylvania

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Department of Biochemistry and Biophysics