RIPK1 mediates a disease-associated microglial PNAS PLUS response in Alzheimer’s disease Dimitry Ofengeima,1, Sonia Mazzitellia,1, Yasushi Itoa, Judy Park DeWitta, Lauren Mifflina, Chengyu Zoua, Sudeshna Dasb,c, Xian Adiconisd, Hongbo Chena, Hong Zhua, Michelle A. Kellihere, Joshua Z. Levind, and Junying Yuana,2 aDepartment of Cell Biology, Harvard Medical School, Boston, MA 02115; bMassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Cambridge, MA 02139; cDepartment of Neurology, Harvard Medical School, Boston, MA 02115; dBroad Institute, Cambridge, MA 02142; and eDepartment of Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605 Contributed by Junying Yuan, August 15, 2017 (sent for review August 11, 2017; reviewed by J. Marie Hardwick and David Rubinsztein) Dysfunction of microglia is known to play an important role in identified disease-associated microglia (DAM) present in spatial Alzheimer’s disease (AD). Here, we investigated the role of RIPK1 proximity to Aβ plaques found in both postmortem human AD in microglia mediating the pathogenesis of AD. RIPK1 is highly brain samples and in an AD mouse model (12). Cellular markers expressed by microglial cells in human AD brains. Using the amyloid of DAM include Cst7, which encodes cystatin F, an endosomal/ precursor protein (APP)/presenilin 1 (PS1) transgenic mouse model, lysosomal cathepsin inhibitor (13). However, the functional role of we found that inhibition of RIPK1, using both pharmacological and DAM has not been well-characterized, nor do we know the sig- genetic means, reduced amyloid burden, the levels of inflammatory nificance of Cst7 expression in this microglial population. cytokines, and memory deficits. Furthermore, inhibition of RIPK1 RIPK1, a death-domain containing Ser/Thr kinase, has an promoted microglial degradation of Aβ in vitro. We characterized established role in mediating the deleterious mechanisms down- the transcriptional profiles of adult microglia from APP/PS1 mice and stream of type I TNFα receptor (TNFR1) (14). Mice carrying the identified a role for RIPK1 in regulating the microglial expression of RIPK1 D138N or K45A kinase-dead knock-in mutation develop CH25H and Cst7, a marker for disease-associated microglia (DAM), normally but are resistant to a TNFα-induced systemic inflamma- which encodes an endosomal/lysosomal cathepsin inhibitor named tory response (15, 16). We have developed a highly specific and Cystatin F. We present evidence that RIPK1-mediated induction of CNS-permeable inhibitor of RIPK1 kinase, Necrostatin-1 (Nec-1s, Cst7 leads to an impairment in the lysosomal pathway. These data R-7-Cl-O-Nec-1) (Kd = 3.1 nM) (17–20). Inhibition of RIPK1 by NEUROSCIENCE suggest that RIPK1 may mediate a critical checkpoint in the transition Nec-1s phenocopied the genetic inactivation of RIPK1 by D138N to the DAM state. Together, our study highlights a non-cell death or K45A mutations in protection against TNFα-induced systemic mechanism by which the activation of RIPK1 mediates the induction inflammatory response and animal models of neurodegeneration of a DAM phenotype, including an inflammatory response and a re- (15, 16, 21, 22). While RIPK1 is involved in mediating the acti- duction in phagocytic activity, and connects RIPK1-mediated transcrip- vation of RIPK3 and MLKL to promote necroptosis, RIPK1 has tion in microglia to the etiology of AD. Our results support that also been implicated in regulating inflammation, independent of RIPK1 is an important therapeutic target for the treatment of AD. cell death (14, 20, 23). However, it remains unclear how the acti- vation of RIPK1 kinase might be involved in mediating neuro- RIPK1 | microglia | Alzheimer’s disease | RIP1 | inflammation inflammation in AD (21, 22, 24). Here, we investigated the role of RIPK1 in AD. We found that lzheimer’s disease (AD) is a devastating neurodegenerative RIPK1 is highly expressed by microglial cells in human AD brains. Adisorder and the leading cause of age-related dementia. About To test the involvement of RIPK1 in AD, we examined the impact 5.5 million Americans and 1 in 10 people worldwide over the age of of RIPK1 in the amyloid precursor protein (APP)/presenilin 1 65 are currently living with AD. Chronic brain inflammation, characterized by an increased number of microglia and elevated Significance levels of proinflammatory cytokines, is a hallmark of AD (1). Re- cent genome-wide association studies (GWASs) have identified Dysfunction of microglia plays a fundamental role in the patho- specific alleles of multiple genes involved in regulating innate im- genesis of Alzheimer’s disease (AD), the most common form of munity and inflammation as risk factors for late onset AD (LOAD), dementia. However, there is a lack of knowledge about targets including CH25H, TREM2, complement receptor 1 (CR1), clus- that can be safely manipulated for modulating microglia for the terin, CD33, the MS4A6-MS4A4 cluster, ABCA7, CD2AP, EPHA1, treatment of AD. The presence of a unique subtype of disease- HLA-DRB5–DRB1, INPP5D,andMEF2C (2–7). In addition, in- associated microglia (DAM) has recently been implicated in creased levels of TNFα are found in the cerebral spinal fluid (CSF) mediating pathogenesis of AD. However, the mechanism that of patients with mild cognitive impairment (MCI) at risk to develop promotes the development of DAM is unclear, nor is it known AD, suggesting that CNS inflammation is an early event during how DAM may modulate the progression of AD. This study the pathogenesis of AD (8). It remains unclear, however, how the demonstrates that RIPK1-dependent transcription promotes DAM LOAD risk factors mediate neuroinflammation and onset of the and lysosomal defects to mediate the accumulation of amyloid disease. Furthermore, while neuroinflammation has been recog- plaques in AD. Thus, targeting RIPK1 may provide an important nized as an important therapeutic target for the treatment of therapeutic strategy for the treatment of AD. AD (9), there is a lack of understanding how neuroinflammatory Author contributions: D.O., S.M., J.Z.L., and J.Y. designed research; D.O., S.M., Y.I., J.P.D., mechanisms can be specifically and safely modulated. L.M., C.Z., X.A., H.C., and H.Z. performed research; M.A.K. contributed new reagents/analytic Microglia, the macrophage-like cells of the central nervous tools; D.O., S.M., S.D., J.Z.L., and J.Y. analyzed data; and D.O. and J.Y. wrote the paper. system, can uptake extracellular amyloid beta (Aβ)species,which Reviewers: J.M.H., Johns Hopkins University; and D.R., University of Cambridge. are then degraded via the autophagic/lysosomal system (10, 11). Conflict of interest statement: Denali Therapeutics has licensed the Necrostatin program An altered inflammatory milieu is known to impair the ability of (including Nec-1s) from Harvard Medical School. J.Y. is a consultant of Denali Therapeutics. microglia to properly internalize and degrade amyloid beta (Aβ); 1D.O. and S.M. contributed equally to this work. however, the molecular mechanism by which inflammation im- 2To whom correspondence should be addressed. Email: [email protected]. β pairs the ability of microglia to mediate the clearance of A is not This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. well-understood. Recently, a single-cell RNA-sequencing study 1073/pnas.1714175114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1714175114 PNAS Early Edition | 1of10 Downloaded by guest on September 28, 2021 (PS1) transgenic mouse model. We found that, compared with imals (Fig. 2 A and B and Fig. S2). Furthermore, we character- control APP/PS1 animals, plaque burden, and insoluble Aβ levels ized the levels of soluble and insoluble Aβ species in vehicle- were strongly reduced in RIPK1D138N kinase-dead knock-in;APP/ treated and Nec-1s–treated mice and found that the treatment PS1 double mutant mice, and by the treatment of APP/PS1 mice of Nec-1s reduced the levels of both Aβ species (Fig. 2C). Our with the RIPK1 inhibitor Nec-1s. Furthermore, inhibition of data suggest that administration of Nec-1s mediates a histolog- RIPK1 attenuated the behavioral deficits observed in the APP/ ical and biochemical reduction of amyloid burden in the APP/ PS1 mice. Moreover, in both RIPK1D138N;APP/PS1 mice and PS1 mice. APP/PS1 mice dosed with Nec-1s, there was a significant re- To assess if pharmacological inhibition of RIPK1 can also res- duction in amyloid plaque-associated microglia and the levels of cue behavioral deficits in the APP/PS1 mice, we examined WT proinflammatory cytokines. We demonstrate that the kinase ac- and APP/PS1 mice at 5 mo of age in an open field test for the total tivity of RIPK1 mediates the transcriptional up-regulation of Cst7, distance traveled in a new environment. Consistent with the effect which encodes an endosomal/lysosomal cathepsin inhibitor named of expressing APP/PS1 in promoting hyperactivity of mice (29, 30), Cystatin F (25), and CH25H in microglia. We present evidence APP/PS1 mice showed significant increased spontaneous loco- that RIPK1-dependent induction of Cst7 leads to inhibition of motor activity, illustrated by a significantly greater distance trav- cathepsin activity and impairment in the lysosomal pathway. eled in the testing chamber during the first hour of the open field Consistently, inhibition of RIPK1 could promote the degradation test compared with WT mice (Fig. 2 D and E). After confirming of Aβ by microglia in vitro. Together, our study provides a mech- these data, APP/PS1 mice were then divided into groups so their anism by which activation of the RIPK1-mediated transcriptional baseline activity levels did not differ between genotype-matched response promotes a DAM state, which in turn impairs the groups that received vehicle or Nec-1s. APP/PS1 mice and WT microglial phagocytic capacity. Thus, we propose that RIPK1 is a controls were tested again after dosing with vehicle or Nec-1s for critical mediator in microglial response to the extracellular envi- 30 d (n = 14 mice per genotype and treatment) in the open field ronment to promote pathogenesis of AD.