Development and characterization of phospho-ubiquitin antibodies to monitor PINK1-PRKN signaling in cells and tissue

The selective removal of dysfunctional mitochondria, a process termed mitophagy, is critical for cellular health and impairments have been linked to aging, Parkinson disease, and other neurodegenerative conditions. A central mitophagy pathway is orchestrated by the ubiquitin (Ub) kinase PINK1 together with the E3 Ub ligase PRKN/Parkin. The decoration of damaged mitochondrial domains with phosphorylated Ub (p-S65-Ub) mediates their elimination though the autophagy system. As such p-S65-Ub has emerged as a highly specific and quantitative marker of mitochondrial damage with significant disease relevance. Existing p-S65-Ub antibodies have been successfully employed as research tools in a range of applications including western blot, immunocytochemistry, immunohistochemistry, and enzyme-linked immunosorbent assay. However, physiological levels of p-S65-Ub in the absence of exogenous stress are very low, therefore difficult to detect and require reliable and ultrasensitive methods. Here we generated and characterized a collection of novel recombinant, rabbit monoclonal p-S65-Ub antibodies with high specificity and affinity in certain applications that allow the field to better understand the molecular mechanisms and disease relevance of PINK1-PRKN signaling. These antibodies may also serve as novel diagnostic or prognostic tools to monitor mitochondrial damage in various clinical and pathological specimens. Abbreviations: AD: Alzheimer disease; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ELISA: enzyme-linked immunosorbent assay; HEK293E cell: human embryonic kidney E cell; ICC: immunocytochemistry; IHC: immunohistochemistry: KO: knockout; LoB: limit of blank; LoD: limit of detection; LoQ: limit of quantification; MEF: mouse embryonic fibroblast; MSD: Meso Scale Discovery; n.s.: non-significant; nonTg: non-transgenic; PBMC: peripheral blood mononuclear cell; PD: Parkinson disease; p-S65-PRKN: phosphorylated PRKN at serine 65; p-S65-Ub: phosphorylated Ub at serine 65; Ub: ubiquitin; WT: wild-type.</p

Role of Helix 1 and the β-sheet in prion protein aggregation and its amyloid structure

Prionerkrankungen werden durch die Aggregation des nativen α-helikalen Prionproteins PrPC in seine pathologische Isoform PrPSc verursacht. In derzeitigen PrPSc-Strukturmodellen wird vermutet, dass die Helix 1 bevorzugt in ein β-Faltblatt bei der Aggregation des Prionproteins umfaltet. Diese Annahme wird durch die NMR-Struktur von nativem PrPC gestützt, da im Kontrast zur isolierten Helix 1, die Helices 2 und 3 nur über einen kurzen Loop miteinander verbunden sind und zusätzlich durch eine intrahelikale Disulfidbrücke stabilisiert werden. Allerdings ist Helix 1 sehr hydrophil und besitzt eine starke Tendenz zur Ausbildung helikaler Strukturen. Diese Beobachtungen führten zur Untersuchung der Rolle der Helix 1 bei der Aggregation des Prionproteins in humPrP23-159 einschliesslich Helix 1 (144-156) verglichen mit einer C-terminal trunkierten Isoform humPrP23-144, die beide mit einem Subtyp der humanen Prionerkrankungen, des Gerstmann-Sträussler-Scheinker-Syndroms, assoziiert sind. Unerwartet aggregierte humPrP23-159 signifikant schneller als humPrP23-144, was den aggregationsbeschleunigenden Einfluss der Helix 1 klar belegt. Interessanterweise ist die Helix 1 allerdings nicht resistent gegenüber Proteinase K-Verdau in fibrillärem humPrP23-159, was andeutet, dass Helix 1 nicht in ein β-Faltblatt umgewandelt wird. Dies wird bestätigt durch quantitative FTIR-Spektroskopie, da nahezu kein Unterschied im β-Faltblattgehalt zwischen fibrillärem humPrP23-159 und humPrP23-144 detektiert werden konnte. Zusätzlich konnte ein stark aggregationsbeschleunigender Effekt des β-Stranges S2 bei der Aggregation von humPrP23-173 im Vergleich zu humPrP23-159 aufgezeigt werden. Zusamenfassend erscheint es sehr evident, dass im Gegensatz zu früheren Annahmen Helix 1 bei der Konversion von PrPC in PrPSc nicht in ein β-Faltblatt umgefaltet wird

PDGF is Required for Remyelination-Promoting IgM Stimulation of Oligodendrocyte Progenitor Cell Proliferation

<div><h3>Background</h3><p>Promotion of remyelination is a major goal in treating demyelinating diseases such as <em>multiple sclerosis</em> (MS). The recombinant human monoclonal IgM, rHIgM22, targets myelin and oligodendrocytes (OLs) and promotes remyelination in animal models of MS. It is unclear whether rHIgM22-mediated stimulation of lesion repair is due to promotion of oligodendrocyte progenitor cell (OPC) proliferation and survival, OPC differentiation into myelinating OLs or protection of mature OLs. It is also unknown whether astrocytes or microglia play a functional role in IgM-mediated lesion repair.</p> <h3>Methods</h3><p>We assessed the effect of rHIgM22 on cell proliferation in mixed CNS glial and OPC cultures by tritiated-thymidine uptake and by double-label immunocytochemistry using the proliferation marker, Ki-67. Antibody-mediated signaling events, OPC differentiation and OPC survival were investigated and quantified by Western blots.</p> <h3>Results</h3><p>rHIgM22 stimulates OPC proliferation in mixed glial cultures but not in purified OPCs. There is no proliferative response in astrocytes or microglia. rHIgM22 activates PDGFαR in OPCs in mixed glial cultures. Blocking PDGFR-kinase inhibits rHIgM22-mediated OPC proliferation in mixed glia. We confirm in isolated OPCs that rHIgM22-mediated anti-apoptotic signaling and inhibition of OPC differentiation requires PDGF and FGF-2. We observed no IgM-mediated effect in mature OLs in the absence of PDGF and FGF-2.</p> <h3>Conclusion</h3><p>Stimulation of OPC proliferation by rHIgM22 depends on co-stimulatory astrocytic and/or microglial factors. We demonstrate that rHIgM22-mediated activation of PDGFαR is required for stimulation of OPC proliferation. We propose that rHIgM22 lowers the PDGF threshold required for OPC proliferation and protection, which can result in remyelination of CNS lesions.</p> </div

RHIgM22 reduces expression of OL differentiation markers in the presence of PDGF/FGF-2. A:

<p>OLs were cultured for 1–7 days on fibronectin with rHIgM22 (5 µg/ml) or the human isotype control IgM (IC) (5 µg/ml) in the absence or presence of growth factors PDGF-AA and FGF-2. Representative Western blots showing the levels of CNPase, MBP and β-actin as a loading control. <b>B:</b> Quantitative analysis of 3 independent experiments as described in A. Background is subtracted from each value and normalized against β-actin levels. Data are presented as mean ± S.D. (<i>n = </i>3). * <i>p</i><0.05 compared to controls. IC: isotype control IgM.</p

rHIgM22 Induced Thymidine Uptake in Primary Mixed Glial Cultures, but not in isolated OPCs.

<p><b>A-C Mixed glial cultures.</b> Mixed glial cells were maintained in serum-containing medium for 5 days prior to the addition of IgMs and/or growth factors PDGF and FGF-2 in serum free media for 48 h. <b>A</b>. Tritiated thymidine, IgMs or PDGF/FGF-2 were added at day 5 in a serum-free medium. The mitogens PDGFAA and FGF2 were included as positive controls and at the highest concentrations consistently increased tritium uptake 3-fold over medium alone. PDGFAA (20 ng/ml) and FGF2 (10 ng/ml) were added at serial 10-fold dilutions. rHIgM22 induced thymidine uptake by progenitor oligodendrocytes at both 20 µg/ml, 10 µg/ml and 1 µg/ml over medium alone or human isotype control IgM (p<0.001 by one way ANOVA). In contrast isotype control IgM, which does not promote remyelination <i>in vivo</i>, did not induce uptake of thymidine. Data points are mean and standard error of triplicate wells and represent one of three independent tritium uptake assays using 3 different cultures. <b>B.</b> Quantitative analysis of immunocytochemical data from mixed glial cultures treated with human isotype control IgM (IC) or rHIgM22 showing the extent of co-localization between glial markers Olig-1, Olig-2, GFAP, CD68 and proliferation marker Ki-67 in percent of cells positive for glial marker (e.g. Olig-1). <b>C.</b> BRDU-assay in mixed glial cultures after treatment with IgMs for 48 h (10 µg/ml each) or 24 h of PDGF/FGF-2. Representative images show the extent of BRDU-positive cells (anti-BRDU) relative to total cell numbers (DAPI) in isotype control (IC), rHIgM22 and PDGF/FGF-2 treated mixed glia plus quantitation of BRDU-positive cells per 15×field for all treatment groups. <b>D+E</b>. Mixed glial cultures were treated with rHIgM22 or control IgMs for 7 days in serum-free medium. D. Representative Western blots from one of three independent experiments show levels of proliferation marker Ki-67 in addition to loading control beta-actin. E. Quantitative analysis of Western blots from experiments shown in D. Data are presented as mean ± S.D. (<i>n = </i>3). * <i>p</i><0.05 compared to controls. <b>F.</b> Tritiated thymidine, human isotype control (IC), rHIgM22 or PDGF/FGF-2 were added at day 5 identical to under A. At the highest concentrations PDGF-AA and FGF-2 consistently increased tritium uptake 2-fold over both IgM treatments. rHIgM22 did not induce thymidine uptake by progenitor oligodendrocytes at all concentrations used compared to human isotype control IgM. Data points are mean and standard error of triplicate wells and represent one of three independent tritium uptake assays using 3 different cultures.</p

Secondary antibodies.

<p>Secondary antibodies.</p

Proliferating cell types upon IgM-treatment.

<p>Proliferating cell types upon IgM-treatment.</p

Primary antibodies.

<p>Primary antibodies.</p