The Receptor-interacting Serine/Threonine Protein Kinase 1 (RIPK1) Regulates Progranulin Levels - PubMed (original) (raw)

The Receptor-interacting Serine/Threonine Protein Kinase 1 (RIPK1) Regulates Progranulin Levels

Amanda R Mason et al. J Biol Chem. 2017.

Abstract

Progranulin (PGRN), a secreted growth factor, is a key regulator of inflammation and is genetically linked to two common and devastating neurodegenerative diseases. Haploinsufficiency mutations in GRN, the gene encoding PGRN, cause frontotemporal dementia (FTD), and a GRN SNP confers significantly increased risk for Alzheimer's disease (AD). Because cellular and animal data indicate that increasing PGRN can reverse phenotypes of both FTD and AD, modulating PGRN level has been proposed as a therapeutic strategy for both diseases. However, little is known about the regulation of PGRN levels. In this study, we performed an siRNA-based screen of the kinome to identify genetic regulators of PGRN levels in a rodent cell-based model system. We found that knocking down receptor-interacting serine/threonine protein kinase 1 (Ripk1) increased both intracellular and extracellular PGRN protein levels by increasing the translation rate of PGRN without affecting mRNA levels. We observed this effect in Neuro2a cells, wild-type primary mouse neurons, and _Grn_-haploinsufficient primary neurons from an FTD mouse model. We found that the effect of RIPK1 on PGRN is independent of the kinase activity of RIPK1 and occurs through a novel signaling pathway. These data suggest that targeting RIPK1 may be a therapeutic strategy in both AD and FTD.

Keywords: Alzheimer disease; Ripk1; frontotemporal dementia; neurobiology; neurodegeneration; neurodegenerative disease; neurological disease; progranulin.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

FIGURE 1.

A screen of the kinome identifies Ripk1 as a genetic regulator of PGRN level. A, results of primary screen. The Ripk1 result is highlighted with an arrowhead. B, results of secondary screen. The Ripk1 result is highlighted with an arrowhead. C, secondary screen result for Ripk1 siRNA. NT, nontargeting. n = 3 samples/condition. **, p < 0.01; ****, p < 0.0001 versus nontargeting siRNA by ANOVA. Error bars, S.D.

FIGURE 2.

Ripk1 siRNAs increase PGRN by ELISA and Western blotting. A and B, PGRN ELISA on Neuro2a cells transfected with nontargeting (NT) siRNA, siRNA against Grn, or eight different siRNAs against Ripk1. n = 6–12 replicates/condition. Results are representative of at least three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 versus nontargeting siRNA by ANOVA. C, Western blotting on concentrated medium from Neuro2a cells transfected with nontargeting or Ripk1 siRNA. Native gel blotted with ELISA capture antibody. Results are representative of two independent experiments. F, Western blotting on concentrated medium from Neuro2a cells transfected with nontargeting or Ripk1 siRNA. Reducing gel was blotted with anti-PGRN (linker 1) antibody. Results are representative of two independent experiments. D and E, quantification of gels shown in C and F by densitometry. **, p < 0.01; ***, p < 0.001 compared with nontargeting control by t test. Error bars, S.D.

FIGURE 3.

Ripk1 knockdown does not increase cell number or global secretion. A and B, cell number determined by CellTiterGlo assay in Neuro2a cells transfected with nontargeting siRNA (NT), Grn siRNA, or eight different Ripk1 siRNAs. n = 6–12 replicates/condition. Results are representative of at least three independent experiments. C, collagen IV level secreted by Neuro2a cells transfected with nontargeting, Grn, or Ripk1 siRNA and assessed by collagen IV ELISA (antibodies-online.com). n = 6 replicates/condition. Results are representative of two independent experiments. *, p < 0.05; **, p < 0.01; ****, p < 0.0001 compared with nontargeting control by ANOVA. Error bars, S.D.

FIGURE 4.

Ripk1 knockdown increases PGRN in microglial-like cells and rescues PGRN level in FTD model neurons. A, PGRN ELISA on microglial-like BV-2 cells transfected with nontargeting (NT) siRNA, siRNA against Grn, or four different siRNAs against Ripk1. n = 6–12 replicates/condition. Results are representative of two independent experiments. Note that because Grn and Ripk1 siRNAs were toxic to BV-2 cells, all values are normalized to cell number. ****, p < 0.0001 compared with nontargeting control by ANOVA. B, PGRN level in mouse cortical neurons transduced with nontargeting, Grn, or Ripk1 shRNA lentivirus, assessed by ELISA. n = 6 replicates/condition. Results are representative of at least three independent experiments. Lentivirus was added on day in vitro (DIV) 2, and qPCR or ELISA samples were collected DIV 7. *, p < 0.05; ****, p < 0.0001 compared with +/+ nontargeting control by ANOVA. #, p < 0.05; ####, p < 0.0001 compared with +/− nontargeting control by ANOVA. Error bars, S.D.

FIGURE 5.

Ripk1 knockdown increases PGRN level by increasing PGRN translation without affecting Grn transcription or secretion. A, qPCR for Grn on Neuro2a cells transfected with nontargeting (NT), Grn, or Ripk1 siRNA. n = 4 replicates/condition. Results are representative of at least three independent experiments. B, PGRN level in Neuro2a cells transfected with nontargeting, Grn, or Ripk1 siRNA, assessed by ELISA. The two graphs represent the cell lysate (right) and medium (left) of the same samples. n = 6–12 replicates/condition. Results are representative of at least three independent experiments. C, autoradiography for [35S]PGRN in Neuro2a cells transfected with nontargeting or Ripk1 siRNA and incubated with [35S]methionine (top, PGRN band is indicated by an asterisk) and quantification of autoradiogram (bottom). Representative of at least three independent experiments. D, total protein synthesis, as measured by GFP fluorescence (in arbitrary units, a.u.) using the OPP assay, in Neuro2a cells transfected with nontargeting or Ripk1 siRNA. n = 149–192 cells/condition. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 compared with nontargeting control by ANOVA; *, p < 0.05 for comparison of slopes by linear regression. Error bars, S.D.

FIGURE 6.

The effect of Ripk1 knockdown on PGRN is kinase-independent and proceeds through a novel signaling pathway. A, PGRN ELISA on Neuro2a cells treated with DMSO or Nec1 (0.1, 0.4, 1.6, or 6.4 μ

). Results are representative of three independent experiments. B, PGRN ELISA on brain lysates from wild-type (+/+) or Ripk1 D138N/D138N kinase-dead knock-in mice. n = 3 animals/condition. C, ELISA for PGRN in Neuro2a cells transfected with nontargeting, Grn, Ripk1, or Nf_κ_b1 siRNA. n = 6–12 samples/condition. Results are representative of three independent experiments. D, ELISA for PGRN in Neuro2a cells transfected with nontargeting, Grn, Ripk1, or Casp8 siRNA. n = 6–12 samples/condition. Results are representative of three independent experiments. E, ELISA for PGRN in Neuro2a cells transfected with nontargeting, Grn, Ripk1, or Ripk3 siRNA. n = 6–12 samples/condition. Results are representative of three independent experiments. F, ELISA for PGRN in Neuro2a cells transfected with nontargeting or Pkr siRNA. n = 2–8 samples/condition. For C–E, results were normalized to cell number (from CellTiterGlo assay) because siRNAs to Nf_κ_b1, Casp8, and Ripk3 caused a significant change in cell number. *, p < 0.05; **, p < 0.01; ****, p < 0.0001 compared with nontargeting control by ANOVA. Error bars, S.D.

References

1. Knopman D. S., and Roberts R. O. (2011) Estimating the number of persons with frontotemporal lobar degeneration in the US population. J. Mol. Neurosci. 45, 330–335 -PMC -PubMed
1. Onyike C. U., and Diehl-Schmid J. (2013) The epidemiology of frontotemporal dementia. Int. Rev. Psychiatry 25, 130–137 -PMC -PubMed
1. World Health Organization and Alzheimer's Disease International (2012) Dementia: A Public Health Priority, World Health Organization, Geneva
1. Petkau T. L., and Leavitt B. R. (2014) Progranulin in neurodegenerative disease. Trends Neurosci. 37, 388–398 -PubMed
1. Jian J., Konopka J., and Liu C. (2013) Insights into the role of progranulin in immunity, infection, and inflammation. J. Leukoc. Biol. 93, 199–208 -PMC -PubMed

The Receptor-interacting Serine/Threonine Protein Kinase 1 (RIPK1) Regulates Progranulin Levels - PubMed (original) (raw)