Junying Yuan, Ph.D - US grants

Programmed cell death is integral to the normal development and homeostasis of many tissues. Genes responsible for programmed cell death have been best defined in the nematode C. elegans, from which two genes required for cell death -- ced-3 and ced-4 -- have been cloned. This proposal is to characterize cell death genes in vertebrate species, using chicken interdigital cell death as a model. Four approaches will be taken to isolate genes that direct the normal physiological program of vertebrate cell death. First, based upon the significant degree to which many genes are evolutionarily conserved, vertebrate homologs of ced-3 and ced-4 will be sought. In preliminary experiments, ced-3 and ced-4 have been cloned from other nematode species, defining regions of these proteins that are conserved. Degenerate oligonucleotides based upon these conserved sequences will be used in PCR experiments to seek similar vertebrate genes. Second, monoclonal antibodies will be isolated that specifically recognize dying cells in chicken interdigital tissue. These antibodies will be used to characterize the process of programmed cell death and will be studied for possible usefulness in analyzing the cell death that occurs in aging and disease. Genes that encode proteins recognized by some of these antibodies will be cloned. One antibody that recognizes dying chicken interdigital cells has already been obtained. Third, genes that are activated during chicken interdigital cell death will be isolated, using the techniques of subtractive and differential screening of cDNA libraries and differential PCR. An activin receptor homolog and a zinc-finger protein have been identified in preliminary screens. Fourth, cell death genes identified in these various ways will be characterized molecularly and biochemically, and their patterns of expression will be determined. These genes will be expressed in cells in culture to see if they induce programmed cell death.

Junying Yuan IBN #9418785 The overall objective of this ambitious project is the molecular characterization of the vertebrate programmed cell death gene Ice-3, a homolog of the nematode C. elegans cell death gene ced-3 and mammalian cell death gene interleukin 1-beta converting enzyme (ICE). Naturally occurring cell death is an integral part of normal development and homeostasis. In the nematode C elegans, a genetic pathway of programmed cell death has been identified. Genetic mosaic analysis showed the ced-3 is most likely acting within dying cells to cause cell death. ICE is a vertebrate homolog of ced-3. Overexpression of ICE in mammalian fibroblasts causes cells to undergo programmed cell death. A family of vertebrate homologs of the C.elegans ced-3 gene has been identified by this laboratory. Ice- 3 is one of the most interesting homologs because it appears to encode both an activator and an inhibitor of cell death. The mRNA of Ice-3 is alternatively spliced into two different forms: Ice-3L, and Ice-3S. When expressed in Rat-1 cells, Ice-3L causes cells to die, while Ice-3S has the ability to prevent Rat-1 cell death induced by serum deprivation. A model is proposed in which ICE and ICE-3 act in parallel to control cell death. This model will be tested in a variety of experiments. Biochemical and enzymatic properties of the ICE-3 protein will be studied to reveal how ICE-3 acts to induce cell death. The developmental pattern of Ice-3 expression will be examined. Attempts will be made to isolate the activator, inhibitor and substrate of ICE-3 to identify upstream and downstream elements of Ice-3. Antibodies against ICE-3 will be generated and used to determine when and where the ICE-3 protein is made. ICE-3 protein made by E. coli or baculovirus will be used to study the enzymatic properties of ICE-3, with particular attention paid to the different potential roles of Ice-3L vs. Ice-3S. The yeast two hybrid system will also be used to isolate the genes that may encode proteins interacting with the ICE-3 protein. Through these studies, it is hoped to gain an understanding of the mechanism and function of Ice-3 in controlling programmed cell death. ***

DESCRIPTION: The investigators long-term objective is to understand the role of interleukin 1-b converting enzyme (ICE) family in normal and pathological conditions. The goal of this application is to characterize the mechanism and function of ICH-3, a member of the ICE family, in mediating apoptosis and inflammatory responses. Members of the mammalian ICE family are homologs of C. elegans cell death gene product ced-3. Increasing evidence suggests that the ICE protease family play important roles in controlling apoptosis. Ich-3-/- thymocytes are partially resistant to Fas induced apoptosis. Ich-3-/-embryonic fibroblast cells are resistant to granzyme B induced apoptosis. These results showed that ICH-3 may be a downstream component of Fas and GraB induced apoptotic pathway in certain cells. The first Specific aim is to continue characterization of the Ich-3 gene products and analyze the interaction of two products of Ich-3 locus. The second specific aim is to determine the mechanism of pro-ICH-3 activation. The members of the ICE family are synthesized as precursors and proteolytic activation is a critical regulatory step. Activation of ICH-3 in apoptosis will be determined by western blot analysis. The third specific Aim is to determine the mechanism of Ich-3 induction. Expression of Ich-3 is very low in normal condition and is highly induced upon stimulation by cellular stresses such lipoplysacharide (LPS) treatment and heat shock. The hypothesis that induction of Ich-3 is mediated by stress-activated MAP kinases JNK and p38 will be directly examined using dominant negative and constitutive active mutants of the JNK and p38 pathway. The fourth specific aim is to determine the substrates of ICH-3. Since expression of Ich-3 potentiates the ability of ICE in processing pro-IL-1b, the hypothesis is that one of the substrates of ICH-3 is an inhibitor of ICE. The fifth specific aim is to determine the role of ICH-3 in inflammatory responses. Ich-3-/- mice are resistant to lethality induced by LPS. The hypothesis is that the resistance of Ich-3 mutant mice to LIP lethality is caused at least in part by the resistance of the Ich-3 mutant cells to apoptosis. Vital organs of Ich-3-/- and wild type mice injected with LPS will be examined for apoptosis. These experiments will elucidate the ICH-3 pathway from signal tranduction, regulation of expression, mechanism of activation to the substrates of ICH-3. These works will have direct implication in control of apoptosis in normal and inflammatory conditions.

The long-term objective of this proposal is to illustrate the mechanisms and functions of tumor suppressor gene p33ING1 in regulation of cellular senescence and organismal aging. This work will be a collaboration with the laboratories of Ruvkun, Avruch and Alexander-Bridges. The work from Ruvkun's lab showed that the longevity of C. elegans is at least partly regulated by a pathway involving daf-2, an insulin receptor-like gene, and age-1, a homologue of the mammalian phosphatidylinositol-3-OH kinase (PI3 kinase) catalytic subunit. We hypothesize that the aging of mammalian animals is regulated by a similar pathway involving homologues the genes in the C. elegans age-1 pathway. In a screen designed to identify phosphatidylinositol (3,4,5)- triphosphate (PtdIns(3,4,5)P3 binding proteins, we found that PtdIns(3,4,5)P3 can bind specifically to p33ING1, a mammalian tumor suppressor gene product. p33ING1 has been shown to interact physically with p53 in mediating cellular growth control and senescence. We propose that regulation of p33ING1 by PI3 kinase plays an important role in regulating cell proliferation and senescence. p33ING1 may be a critical missing link between the growth factor signal transduction pathways mediated through PI3 kinase and cell proliferation and senescence regulated by tumor suppressor genes. Specific Aim 1 is to determine the functional interaction between PI3 kinase and p33ING1 in mediating cellular growth control and senescence. Our hypothesis is that the activation of Pi3 kinase suppresses p33ING1 activity and its ability to induce cellular senescence and apoptosis. Specific Aim 2 is to characterize the p53/p33ING1 complex and the role of PtdIns(3,4,5)P3 binding in complex formation. We would like to determine the proteins associated with p53/p33ING1 complex and the role of PI3 kinase in the interaction. Specific Aim 3 is to determine the downstream events regulated by p33ING1. We would like to determine whether p33ING1 acts upstream or in a parallel pathway of the mammalian homologues of DAF-16. Specific Aims 4 is to determine the in vivo functions of p33ING1 by generation and characterization p33ING1 mutant mice generated by gene-targeting. We would like to generate mutant mice expressing a null allele or a constitutively active mutant allele of p33ING1. These works will illustrate the role of p33ING1 in mediating cellular senescence in culture and organismal aging in vivo.

DESCRIPTION (appended verbatim from investigator's abstract): The objective of this proposal is to determine the mechanism of LKB1/STK11, a serine/threonine kinase, in mediating apoptosis. The mutations in LKB1 are responsible for majority of Peutz-Jegher syndrome cases (PJS; MIM 175200), an autosomal dominant disease characterized by melanocytic macules of the lips, multiple benign gastrointestinal hamartomatous polyps in early life and a dramatically increased risk for various cancers later in life. The discovery that the PJ wild-type allele was lost in the hamartomas has led to the suggestion that the target of the deletion was a tumor suppressor gene. LKB1 is the first and so far the only kinase whose loss-of-function mutations predispose to tumorigenesis, which makes it very interesting to explore its mechanism of action. Through our preliminary studies, we found that LKB1 may play an important role in regulating apoptosis. Overexpression of wild type LKB1 but not a kinase dead mutant induces apoptosis. Endogenous LKB1 is present both in cytoplasm and nucleus; upon induction of apoptosis by paclitaxel or Fas, endogenous LKB1 is translocated to mitochondria. The LKB1 kinase domain alone induces apoptosis and translocates to the mitochondria more efficiently than that of wild type, whereas the kinase dead LKB1 mutant can neither induce apoptosis nor translocate to mitochondria, suggesting that both apoptosis and mitochondrial translocation require its kinase activity and the regulatory domain of LKB1 plays a negative role in regulating its apoptotic activity. Furthermore, a dominant negative mutant of LKB1 inhibits apoptosis induced by paclitaxel and to a weaker extent by etoposide but not that by Fas. We propose that LKB1 is an important apoptotic signal transducer and is required for inducing apoptosis through mitochondrial damage through a p53-dependent mechanism. This proposal is to determine the functional domains of LKB1 required for induction of apoptosis in HT1O8O cells and in the small intestine epithelium cells. We propose that autophosphorylation plays an important role in negative regulation of LKB1 activity. We will determine the specificity of LKB1 mediated apoptotic pathway using the dominant negative LKB1 mutant. To illustrate the apoptotic pathway mediated by LKB1, we will identify the downstream targets of LKB1 in mitochondrial translocation. To establish the in vivo function of LKB1, we will examine chimeric transgenic and transgenic mice expressing the dominant negative LKB1 mutant using an intestinal epithelium-specific promoter. We will also search for evidence of apoptosis inhibition in the polyps regions of the intestinal samples from Peutz-Jegher patients.

The long term objective of this proposal is to illustrate the molecular mechanism of aponecrosis. Aponecrosis is defined as caspase-independent cell death induced by the death receptors such as Fas and TNFalpha receptors. Aponecrotic cell death, which occurs in certain cell types when induced with Fas or TNFalpha in the presence of pan caspase inhibitors, lacks the typical apoptotic feature such as cytoplasm and nuclear condensation and DNA cleavage, and is not associated with caspase activation. Instead, aponecrotic cell death exhibit nuclear and cytoplasm swelling which is typically associated with necrosis. To illustrate this receptor-mediated caspase-independent necrosis pathway, we developed a high throughput screen for small molecule inhibitors of caspase-independent cell death. Such screens allowed us to identify three small molecules, MTHtrp, U2 and U3, which effectively block Fas-mediated caspase-independent cell death. Specifically, MTHtrp blocks TNF/zVAD and Fas/zVAD induced cell death in all cell types tested; whereas U2 blocks TNF/zVAD and Fas/zVAD induced cell death only in certain cell types. Thus, MTHtrp may define a common regulator of necrosis whereas U2 defines a cell -type/pathway-specific control point. This proposal is to use cellular, molecular and chemical genetic approaches to characterize the molecular pathway of aponecrosis. This work is a close collaboration between John Porco, an organic chemist at Boston University, and Junying Yuan, a cell biologist at Harvard Medical School. The Specific Aim 1 is to evaluation of the roles of Bcl-xL, caspases and stress kinases in necrotic cell death. Antiapoptotic members of the Bcl-2 family inhibit apoptosis by preventing mitochondrial damage; but their roles in aponecrosis are not well characterized. Our preliminary data indicate that caspases may normally act to inhibit the caspase- independent necrosis pathway. We would determine the identity of the caspases involved by expressing cellular caspase inhibitors, dominant negative mutants and caspase knockout EF cells. We showed in our preliminary results that p38 may be a cell type/signal-specific necrosis mediator. We would like to further characterize the roles of p38 and other stress kinases in regulating aponecrosis. The Specific Aim 2 is to use chemical genetic approach to identify the key molecules involved in regulating aponecrosis. We would synthesize affinity reagent of MTHtrp to identify its cellular target involved in regulating aponecrosis. We would use parallel synthesis to generate derivatives of MTHtrp to identify more effective inhibitors of aponecrosis. As an alternative approach, we would generate derivatives of U2 using combinatorial method to develop better cell type/pathway-specific inhibitors of necrosis and synthesize U3 to analyze its activity. The Specific Aim 3 is to identify critical mediators of caspase-independent cell death. We will characterize the cellular MTHtrp target. We will test the hypothesis that caspases may cleave certain critical regulators to promote apoptosis and inhibit necrosis. We will analyze the difference in gene expression pattern of normal BalbC3T3 cells and the necrosis-resistant/apoptosis-sensitive subline. Our work may lead to the identification of critical regulators of aponecrosis which plays a similar role as that of caspases in apoptosis.

[unreadable] DESCRIPTION (provided by applicant): The long-term objective of this application is to understand the roles and mechanisms of caspase-11 in regulating patho-physiological processes. Caspase-11, originally named ICH-3, is a member of the caspase-1 subfamily of cysteine proteases. Caspase-11 has been shown to play critical roles in innate immune responses by regulating cytokine maturation and in apoptosis. Recently it was found that caspase-11 also plays an important role in regulating cell migration during acquired immune responses. Thus, caspase-11 may serve as a novel link between innate and acquired immune responses. This proposal is to explore the mechanism by which caspase-11 regulates cell migration and the mechanism that specifies the activation of different downstream pathways of caspase-11 in patho-physiological responses. Specific Aim I is to elucidate the molecular mechanism by which caspase-11 regulates activated lymphocyte and macrophage migration in immune responses. This is to determine if caspase-11 regulates cell migration through a cell autonomous mechanism by regulating levels of key signal transduction molecules or cell non-autonomous mechanism by regulating key cytokine secretion. Specific Aim II is to determine the molecular mechanism of caspase-11 activation and specification in regulating cytokine release, activated immune cell migration and apoptosis. The hypothesis is that caspase-11 exists in different protein complexes in a concentration, time or cell type-dependent manner during immune and inflammatory responses depending on the levels of caspase-11 and the interacting proteins that dictate whether to activate apoptosis, cytokine release or limit lymphocyte migration. Specific Aim Ill is to determine the molecular mechanism of caspase-11 induction. The expression of caspase-1 I may be regulated by NF-kB and STAT1 pathways. This Specific Aim will test the hypothesis that STATI regulates the basal expression of caspase-11, which is required for the induction of caspase-11, while the latter is regulated mainly through the NF-kB pathway. These studies will provide new molecular insights into the mechanism by which innate immune response participates in acquired immune responses and create new therapeutic options for infectious and autoimmune diseases. This work will also provide novel insights into the non-apoptotic function of caspases, which are directly relevant for development of caspase inhibitors as drugs for treatment of human diseases.

Abstract Not Provided

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] The objective of this proposal is to develop necrostatins that inhibit necroptosis, a type of programmed necrosis, as a novel therapy for acute brain injury. Apoptosis plays a critical role in physiological neuronal cell death and also contributes to pathological neuronal cell death. However, evidence is accumulating that cells possess an alternative mechanism of cell death which when activated induces cell death with features of necrosis which we termed "necroptosis". Nine structurally distinct classes of small molecule inhibitors of necroptosis, termed necrostatins, have been identified from screening approximately 100,000 compounds. Preliminary "proof-of-concept" work has demonstrated in vivo efficacy of a necrostatin in reducing ischemic brain injury with a prolonged time window. The plan is to expand this effort by screening an additional 100,000 compounds (Specific Aim 1). The pool of necrostatins will be analyzed in primary neurons for protection against oxygen and glucose deprivation and in mouse model of middle cerebral artery occlusion in vivo by icv delivery to select at least two lead compounds for further optimization (Specific Aim 2). Medicinal chemistry will be carried out to improve the efficacy and bio-availability of the lead compounds and to reduce toxicity (Specific Aim 3). The efficacy of lead compounds in inhibiting acute brain injury will be analyzed systematically using rodent models of ischemic brain injury and in a large animal model of ischemic brain injury (Specific Aim 4). The lead compound series will be analyzed for their pharmacokinetic, ADME and toxicology profiles. Finally, a pre-clinical candidate with efficacy in cerebral ischemia in a large animal and clean safety pharmacology will be selected for clinical development. Drug product for a Phase 1 study will be manufactured under GMP conditions with GLP analytics, and further toxicology (GLP) and safety pharmacology studies will be conducted (Specific Aim 5). This project will culminate with the filing of an IND application with the FDA in order to enter a Phase I human clinical trial. [unreadable] [unreadable] [unreadable]

The goal of this proposal is to test the hypothesis that the reduction of key autophagy gene expression and[unreadable] autophagy function as a result of DNA damage during the aging process plays a key role in mediating the[unreadable] onset of Huntington's disease (HD). Mouse, Drosophila and C. elegans models of HD suggest that the[unreadable] cytotoxicity of expanded polyglutamine is highly dependent upon protein context and protein expression[unreadable] levels of mutant Htt. Autophagy plays an important role in regulating the intracellular accumulation of mutant[unreadable] Htt with expanded polyQ. The expression of beclin 1, a key gene involved in autophagy, decreased in an[unreadable] age-dependent fashion in human brains. Since beclin 1 gene is haploid insufficient in regulating[unreadable] autophagosome function, age-dependent decrease of beclin 1 expression may lead to a reduction of[unreadable] autophagic activity during aging. The hypothesis is that reduction of autophagy function in aging results in[unreadable] both increased oxidative stress induced DNA damage and reduced long-lived protein turnover which[unreadable] promotes the accumulation of mutant Htt. Increased accumulation of mutant Htt and oxidative stress may[unreadable] play an important role in promoting the onset of HD. This hypothesis will be tested in the following specific[unreadable] aims. Specific Aim 1 is to test,the hypothesis that the reduction of beclin 1 expression in aging human brains[unreadable] contributes to the onset of HD by generating beclin 1+/-; HdhQ111 mice and examining if 50% reduction of[unreadable] beclin 1 expression led to an increased accumulation of mutant Htt as well as to determine the[unreadable] consequence of autophagy deficiency on neuronal survival and functions. Specific Aim 2 is to examine the[unreadable] mechanism which led to the age-dependent reduction of beclin 1 expression by testing if the promoter of[unreadable] beclin 1 is preferentially damaged in aging human brains and particularly susceptible to oxidative damage in[unreadable] cellular models. The contribution of reduced expression of transcriptional factors regulating beclin 1[unreadable] expression will also be considered. Specific Aim 3 is to test the hypothesis that the reduction of autophagy[unreadable] function exacerbates the DNA damage during aging by increasing the accumulation of damaged[unreadable] mitochondria which further promotes the levels of intracellular ROS by examining aging beclin 1+/- mice and[unreadable] autophagy deficient cells for evidence of increased damaged mitochondria. Specific Aim 4 is to investigate[unreadable] the functional role of autophagy to oxidative DNA damage using CK-p25 mice as a model and to examine[unreadable] the roles of of DNA damage and autophagy deficiency to the accumulation of mutant Htt in HdhQ111; CKp25[unreadable] mice. The ability of SIRT1 activating molecules (STACs) to restore the autophagy function in CK-p25[unreadable] mice and to delay the onset of motor dysfunction in HD models will be determined. Understanding the[unreadable] mechanism by which DNA damage negatively regulates autophagy during aging would allow us to develop[unreadable] strategies to maintain normal autophagy function during aging process which may delay or prevent the onset[unreadable] of HD and other aging related neurodegenerative diseases.

No Abstract provided

The goal of this proposal is to test the hypothesis that the reduction of key autophagy gene expression and[unreadable] autophagy function as a result of DNA damage during the aging process plays a key role in mediating the[unreadable] onset of Huntington's disease (HD). Mouse, Drosophila and C. elegans models of HD suggest that the[unreadable] cytotoxicity of expanded polyglutamine is highly dependent upon protein context and protein expression[unreadable] levels of mutant Htt. Autophagy plays an important role in regulating the intracellular accumulation of mutant[unreadable] Htt with expanded polyQ. The expression of beclin 1, a key gene involved in autophagy, decreased in an[unreadable] age-dependent fashion in human brains. Since beclin 1 gene is haploid insufficient in regulating[unreadable] autophagosome function, age-dependent decrease of beclin 1 expression may lead to a reduction of[unreadable] autophagic activity during aging. The hypothesis is that reduction of autophagy function in aging results in[unreadable] both increased oxidative stress induced DNA damage and reduced long-lived protein turnover which[unreadable] promotes the accumulation of mutant Htt. Increased accumulation of mutant Htt and oxidative stress may[unreadable] play an important role in promoting the onset of HD. This hypothesis will be tested in the following specific[unreadable] aims. Specific Aim 1 is to test,the hypothesis that the reduction of beclin 1 expression in aging human brains[unreadable] contributes to the onset of HD by generating beclin 1+/-; HdhQ111 mice and examining if 50% reduction of[unreadable] beclin 1 expression led to an increased accumulation of mutant Htt as well as to determine the[unreadable] consequence of autophagy deficiency on neuronal survival and functions. Specific Aim 2 is to examine the[unreadable] mechanism which led to the age-dependent reduction of beclin 1 expression by testing if the promoter of[unreadable] beclin 1 is preferentially damaged in aging human brains and particularly susceptible to oxidative damage in[unreadable] cellular models. The contribution of reduced expression of transcriptional factors regulating beclin 1[unreadable] expression will also be considered. Specific Aim 3 is to test the hypothesis that the reduction of autophagy[unreadable] function exacerbates the DNA damage during aging by increasing the accumulation of damaged[unreadable] mitochondria which further promotes the levels of intracellular ROS by examining aging beclin 1+/- mice and[unreadable] autophagy deficient cells for evidence of increased damaged mitochondria. Specific Aim 4 is to investigate[unreadable] the functional role of autophagy to oxidative DNA damage using CK-p25 mice as a model and to examine[unreadable] the roles of of DNA damage and autophagy deficiency to the accumulation of mutant Htt in HdhQ111; CKp25[unreadable] mice. The ability of SIRT1 activating molecules (STACs) to restore the autophagy function in CK-p25[unreadable] mice and to delay the onset of motor dysfunction in HD models will be determined. Understanding the[unreadable] mechanism by which DNA damage negatively regulates autophagy during aging would allow us to develop[unreadable] strategies to maintain normal autophagy function during aging process which may delay or prevent the onset[unreadable] of HD and other aging related neurodegenerative diseases.

[unreadable] DESCRIPTION (provided by applicant): The objective of this proposal is to isolate additional necrostatins for understanding the molecular mechanism of necroptosis. Apoptotic pathways have been studied extensively during the past decade. However, it has become increasingly clear that apoptosis is not the only cellular suicide mechanism. For example, in a subset of cell types, inhibition of caspases when cells are stimulated by FasL or TNFa lead to inhibition of apoptosis but cells die with necrotic morphology through a cellular process termed necroptosis. Necroptosis has been shown to be a promising target for the treatment of stroke with an extended time window. Necrostatins are small molecules that specifically inhibits necroptosis but not apoptosis. This application is to carry out a mechanistic study of necroptosis by utilizing unique chemical resources at NIH to generate small molecule affinity reagents for target identification, and to identify new necrostatins in order to meet the highly challenging goal of developing an anti-stroke drug. This project is to identify small molecule inhibitors of a novel cell death pathway, termed necroptosis. Necroptosis has been shown to be a promising target for the treatment of stroke as it represents a type of delayed cell death in stroke and offers an extended time window for therapy. [unreadable] [unreadable] [unreadable]

The objective of this proposal is to study the molecular mechanism of necroptosis, a cellular necrotic cell death pathway. Apoptosis has been established as a cellular mechanism that regulates programmed cell death. However, it has become increasingly clear that apoptosis is not the only cellular mechanism that regulates cell death. Apoptosis triggered by the activation of death receptors represents a prototypic apoptosis pathway. Interestingly, stimulation of certain apoptotic deficient cells with death receptor ligands such as FasL or TNF[unreadable] has been shown to lead to necrotic cell death termed necroptosis. Cell death with necrotic features (necrosis) is prevalent in cancers, viral infections and other acute pathologies. Furthermore, excessive necrosis in cancers has long been noted as a indicator for poor prognosis. However, very little attention has been directed towards studying necrosis because necrosis is believed to be an unregulated process caused by overwhelming external stress. The discovery of necroptosis offers the possibility that a subset of pathologic necrotic cell death is regulated by a distinct cellular mechanism, and therefore is amenable to therapeutic intervention. A genome-wide siRNA screen of 16,873 genes was carried out to identify genes involved in regulating necroptosis. A set of genes was identified as common mediators of necroptosis that are downstream of RIP1, an essential kinase for the activation of necroptosis. This screen identified a tumor suppressor network previously known to regulate DNA damage response and a BH3-only member of Bcl-2 family in the signaling of necroptosis. This proposal is to characterize the role of necroptosis and RIPI kinase in DFJA damage induced cell death and to determine the mechanism by which RIPI mediates the activation of BH3-only Bcl-2 family member in the execution of necroptosis. This study will provide important molecular insights into a cellular necrotic cell death pathway. Understanding the mechanism by which cells regulate necroptotic cell death may provide unique opportunities for developing novel therapies of major human diseases with little or no known treatment.

DESCRIPTION (provided by applicant): The goal of this proposal is to explore the molecular mechanism as to why oligodendrocytes (OGs) prefer necroptosis, a regulated necrotic cell death pathway, as the primary cell death mechanism and the contribution of this pathway to progressive demyelination, inflammation and neurodegeneration in animal models of multiple sclerosis (MS). MS, an inflammatory demyelinating disease of the central nervous system (CNS), is the most common chronic neurodegenerative disease for young adults during their most productive ages. While the immunological basis of MS has been studied extensively, we still know very little about the mechanism that leads to the degeneration of OGs, the myelin producing cells that play a critical role in the maintenance of activity and integrity of neuronal axons. Preventing the death of OGs might be able to inhibit demyelination and axonal degeneration, the major cause of irreversible neurological disability in patients with progressive MS. Activation of TNFR1 by TNFalpha has recently been shown to mediate two alternative cell death pathways: caspase-dependent apoptosis and caspase-independent RIP1 kinase-dependent necroptosis (programmed necrosis). However, for most cell types analyzed so far, necroptosis is only activated when the activation of caspases is inhibited by chemical inhibitors or by genetic mutation. Interestingly, we found that OGs undergo necroptosis upon stimulation by TNFalpha alone which can be effectively blocked by Nec-1 or by RIP3 deficiency. We have shown that 7-Cl-O-necrostatin-1 (7-Cl-O-Nec-1), a highly specific inhibitor of RIP1 kinase, protects against TNFalpha-induced oligodendrocyte death in vitro and two mouse models of MS in vivo [cuprizone model and experimental autoimmune encephalomyelitis model (EAE)]. In addition, RIP3-/- mice are also resistant to cuprizone model and RIP3-/- OGs are protected against TNFalpha. We propose to investigate as to why OGs prefer to use necroptosis as the primary cell death pathway and the role and mechanism of RIP1 kinase in mediating the death of OGs. Specific Aim 1 is to investigate the role and mechanism by which cellular metabolism and redox state control the sensitivity of OGs to TNFalpha. This is to test the hypothesis that the cell-cell interaction regulated high metabolic activity in OGs provides a critical mechanism that controls redox state and the sensitivity of OGs to TNFalpha mediated necroptosis. Specific Aim 2 is to investigate the role of S-nitrosylation in regulating the sensitivity of OGs to TNFalpha induced cell death. This is to test the hypothesis that elevated nitrosylation stress in TNFalpha stimulated OGs leads to the inhibition of caspases and sensitization of OGs to necroptosis. Specific Aim 3 is to examine the involvement of RIP1 kinase in mediating necroptosis of OGs in vivo and in vitro using RIP1 kinase dead knockin mutant mice. Our study may provide a strong rationale for the development of RIP1 kinase inhibitors as an OG protective strategy for the treatment of MS, and an orally available, highly specific and nontoxic RIP1 kinase inhibitor, 7-Cl-O-Nec-1, as a lead compound.

DESCRIPTION (provided by applicant): The goal of this proposal is to validate the role and explore the molecular mechanism of RIP1 kinase as a mediator of inflammatory response in Alzheimer's disease (AD), a devastating neurodegenerative disorder and the leading cause of dementia of the elderly. Chronic brain inflammation, characterized by the presence of an increased number of microglia and elevated levels of proinflammatory cytokines, is a hallmark of AD. Increased levels of cerebral spinal TNF? were found in patients with mild cognitive impairment (MCI) at risk to develop AD, suggesting that CNS inflammation is an early event during the pathogenesis of AD. The role of inflammation in the pathogenesis of AD was further highlighted by a recent network-based integrative analysis of a large collection of gene expression profiles from patients of late-onset Alzheimer's disease (LOAD) which discovered the immune/microglia system, including multiple TLR receptors and TNF?, as the molecular system most strongly associated with the pathophysiology of the LOAD. When activated, microglia may release proinflammatory cytokines to drive the chronic progression of AD by exacerbating A? deposition and neuronal death. Identification of the molecular targets in microglia that can be safely modulated to inhibit their inflammatory response may provide new options for the treatment of AD. However, there is a lack of knowledge about the neuroinflammatory mechanism that can be specifically and effectively modulated. We have developed a highly specific and potent inhibitor of RIP1 kinase, 7-Cl-O-Nec-1, a small molecule with excellent oral availability and safety profile, and highly CNS permeable. RIP1 kinase, a death-domain containing Ser/Thr kinase, has an established role in mediating multiple downstream signaling pathways downstream of TNFR1. We found that RIP1 kinase also plays an important role in mediating the production of TNF? by microglia induced by A? in vitro and in PSAPP transgenic mice in vivo which can be effectively inhibited by 7-Cl-O-Nec-1. Furthermore, oral administration of 7-Cl-O-Nec-1 led to the reduction of amyloid plaques and improved behavior and memory of B6.Cg-Tg(APPswe, PSEN1dE9) 85Dbo/J mice (PSAPP) mice, a model for AD. Our study suggests that RIP1 kinase is an important target for inhibiting neuroinflammatory response in AD. This proposal is to test this hypothesis and investigate the mechanism by which RIP1 kinase mediates neuroinflammatory responses in microglia. Specific Aim 1: Investigating the role and mechanism by which RIP1 kinase mediates inflammatory response in microglia activated by oligomeric A? by testing the possible involvement of MKK7 and TLR signaling as downstream mediators of RIP1 signaling. Specific Aim 2: Investigating the role and mechanism of p62 in A? mediated RIP1 kinase activation in microglia by testing the hypothesis that oligomerized p62 provides a platform for mediating RIP1 activation. Specific Aim 3: Genetic confirmation of the role of RIP1 kinase in mediating inflammatory response in AD transgenic mice using a RIP1 kinase dead knockin mouse line.

The goal of this study is to use quantitative single-cell RNA-seq to define the molecular signature of RIPK1- mediated inflammatory signaling in glial cells, with a focus on microglia and oligodendrocytes, during aging and investigate the contribution of this pathway to the aging-dependent risk to Alzheimer's disease (AD). AD is the most common age-related neurodegenerative disease that currently affects more than five million Americans ? a number that is expected to nearly triple by 2050. Low-grade, unresolved molecular inflammation may provide an underlying mechanism of aging and age-related diseases, bridging normal aging with age-related pathological processes. Circulating levels of TNF?, a strong proinflammatory cytokine, are elevated in elderly and implicated in the development of AD. Thus, a key question is how chronic neuroinflammation, predominantly mediated by cells of glial lineages, during aging might eventually lead to neuronal cell death and onset of AD. However, the mechanism by which various cell types of the CNS interact to promote the onset of AD is not well understood. The use of single cell RNA-seq may provide an exciting opportunity to explore the mechanism at transcriptomic levels to understand how various glial cells of the CNS, including microglia, astrocytes and oligodendrocytes, interact during aging to mediate inflammation and eventually lead to the development of AD. RIPK1, a key mediator of the innate immune response that regulates both inflammation and cell death, represents an ideal target for reducing cell death and inflammation in the CNS. A RIPK1 inhibitor developed by us has been advanced into a human Phase I clinical trial for the treatment of ALS and AD. Our hypothesis is that the RIPK1-mediated inflammatory process plays an important role in mediating ?microglial priming? for inflammation and in promoting degeneration of oligodendrocytes during aging which set the stage for axonal loss, neural dysfunction and the eventual onset of AD. Specific Aim 1: Investigating the molecular signatures of RIPK1 for age-related changes in the CNS glial lineages at single cell levels using quantitative RNA-seq to explore the mechanisms as to why aging constitutes the biggest risk factor for AD. Specific Aim 2: Identifying aging-related molecular signatures of RIPK1-mediated inflammatory process in microglia of WT and AD mouse models at single cell levels and comparison with human AD. The goal here is to identify key biomarkers for RIPK1-dependent inflammatory processes to be used in the ongoing clinical trial on RIPK1 inhibitor as a new drug for AD. Specific Aim 3: Identifying the molecular signature of RIPK1-mediated age-related changes in astrocytes and oligodendrocytes of WT and AD animal models. Understanding the molecular signature of RIPK1-mediated inflammatory response and cell death and the role of RIPK1 on glial subtypes will provide important biomarkers for guiding the ongoing clinical trial on RIPK1 inhibitor as a treatment for AD.

The goal of this study is to investigate the mechanism by which RIPK1 mediates the pathology of endothelial cells and its contribution to the pathogenesis of Alzheimer's disease (AD). AD is a multifactorial and multifaceted disease with majority of demented patients display mixed AD and vascular pathology. While amyloid pathology has been recognized as a hallmark of AD, there is an increasing appreciation of the contribution from cerebrovascular pathology to AD. However, the mechanism that mediates the cerebrovascular dysfunction in AD is unclear. Most importantly, we have scarce knowledge regarding druggable targets that can be safely targeted to remedy the cerebrovascular defects for the treatment of AD. Recent studies have demonstrated that endothelial cells are able to undergo necroptosis, a form of programmed necrotic cell death that requires RIP1 Kinase (RIPK1) activity as well as RIP Kinase 3 (RIPK3) and MLKL. Furthermore, necroptosis has been implicated in mediating atherosclerosis and plaque rupture and can be targeted for both therapeutic and diagnostic interventions. RIPK1 is a key mediator of the innate immune response that regulates both inflammation and necroptosis. This study will provide important new insights into the mechanisms by which RIPK1 mediated inflammation and necroptosis contribute to the cerebrovascular pathology in AD as well as important biomarkers for the human clinical trials on RIPK1 inhibitor targeting AD. The specific aims are: Specific Aim 1: To investigate if pharmacological inhibition of RIPK1 activity can rescue cerebral microvascular pathology and functional deficits in APP/PS1 mice as that of genetic inactivation of RIPK1 that we have shown. Specific Aim 2: To investigate the mechanism of RIPK1 in mediating cerebrovascular pathology in AD. This is to investigate the cell-non-autonomous role of RIPK1 mediated inflammation and cytokine release in microglia, and the cell-autonomous role of RIPK1 in regulating the gene transcription and the release of cyclophilin A (CypA) from endothelial cells in mediating cerebrovascular pathology of APP/PS1 mice. Specific Aim 3. To investigate if blocking necroptosis in brain endothelial cells can ameliorate cerebral vascular pathology in APP/PS1 mice. This is to knockdown the expression of RIPK3 and MLKL in cerebral vascular endothelial cells of APP/PS1 mice by shRNA using an endothelial specific AAV delivery system and examine their impact on the release of CypA and vascular pathology mediated by amyloid ?.