BC-1215 inhibits ATP-induced IL-1ß secretion via the FBXL2-mediated ubiquitination and degradation of not only NLRP3, but also pro-IL-1ß in LPS-primed THP-1 cells.

BC-1215, bis-pyridinyl benzyl ethanediamine, is an inhibitor of F-box only protein 3 (FBXO3) and exerts anti-inflammatory effects. BC-1215 inhibits interactions between FBXO3-F-box and the leucine rich repeat protein 2 (FBXL2), leading to the upregulation of FBXL2 expression, FBXL2-mediated ubiquitination and the degradation of tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) or NOD-, LRR- and the pyrin domain-containing protein 3 (NLRP3), which subsequently results in the down-regulation of inflammatory cytokine production. In the current study, we investigated the issue of whether or how BC-1215 suppresses the ATP-induced secretion of IL-1ß in LPS-primed human macrophage-like cells, THP-1 cells. Our result show that pre-treatment with BC-1215 attenuated the ATP-induced secretion of IL-1ß in LPS-primed THP-1 cells. Treatment of the LPS-primed THP-1 cells with BC-1215 resulted in a decrease in the level of NLRP3 and pro-IL-1ß at the protein level, but not at the mRNA level. In addition, treatment with MG-132, but not leupeptin, inhibited the BC-1215-induced degradation of NLRP3 and pro-IL-1ß proteins, and restored their levels, suggesting that BC-1215 decreases the stability of NLRP3 and pro-IL-1ß at the protein level via proteasome-dependent degradation. Our results also show that FBXL2, which is increased by BC-1215, bound to and ubiquitinated NLRP3 and pro-IL-1ß, but not pro-caspase-1. These collective results indicate that treatment with BC-1215, an inhibitor of FBXO3, inhibits ATP-induced IL-1ß secretion via the FBXL2-mediated ubiquitination and degradation of pro-IL-1ß as well as NLRP3 in LPS-primed THP-1 cells, suggesting that FBXO3 is a potential therapeutic target for developing agents against inflammatory diseases.

FOSL1 promotes tumorigenesis in colorectal carcinoma by mediating the FBXL2/Wnt/ß-catenin axis via Smurf1.

Colorectal carcinoma (CC), one of the most prevalent digestive cancers with high mortality and morbidity globally, still lacks powerful therapies to improve the prognosis. Here, we established that the expression of fos-like antigen-1 (Fosl1) was elevated in CC tissues versus adjacent tissues. Importantly, high Fosl1 expression was related to dismal prognosis among CC patients. Functional assays displayed that Fosl1 increased the viability, epithelial-to-mesenchymal transition (EMT), migration and invasion of CC cells. Additionally, a xenograft assay showed that silencing of Fosl1 in CC cells retarded lung, liver and kidney metastases in vivo. Further investigation demonstrated that Fosl1 was involved in malignant aggressiveness of CC cells by binding to smad ubiquitination regulatory factor 1 (Smurf1). Mechanistically, Smurf1-induced F-Box and leucine rich repeat protein 2 (FBXL2) ubiquitination resulted in its degradation, while FBXL2 disrupted the activation of the Wnt/ß-catenin signaling. In summary, Fosl1 plays a pro-metastatic and carcinogenetic role in CC, and we provided forceful evidence that Fosl1 inhibition might act as a prognostic and therapeutic option in CC.

O-GlcNAcylation-mediated degradation of FBXL2 stabilizes FOXM1 to induce cancer progression.

O-GlcNAcylation is a dynamic and reversible post-translational modification of cytonuclear molecules that regulates cellular signaling. Elevated O-GlcNAcylation is a general property of cancer and plays a critical role in cancer progression. We previously showed that the expression of FOXM1, a critical oncogenic transcription factor widely overexpressed in solid tumors, was elevated in MKN45 cells, a human gastric cancer cell line, by the O-GlcNAcase inhibitor Thiamet G (TMG), which induces augmented O-GlcNAcylation. Here, we identified FBXL2 E3 ubiquitin ligase as a new target of O-GlcNAcylation. Consistent with the results in MKN45 cells, FOXM1 expression was increased, accompanied by its decreased ubiquitination and degradation by TMG in the other gastric cancer cell lines, including NUGC-3 cells. We found that FBXL2 ubiquitinated FOXM1, and the interaction with FBXL2 and ubiquitination of FOXM1 were reduced by TMG in NUGC-3 cells. Interestingly, FBXL2 was also ubiquitinated, which was promoted by TMG in the cells. Moreover, FOXM1 expression and cell proliferation were reduced in FBXL2-induced NUGC-3 cells, and the reductions were attenuated by TMG, indicating that FOXM1 was stabilized by O-GlcNAcylation-mediated degradation of FBXL2 to induce cancer progression. These data suggest that elevated O-GlcNAcylation contributes to cancer progression by suppressing FBXL2-mediated degradation of FOXM1.

Spinal Fbxo3-Dependent Fbxl2 Ubiquitination of Active Zone Protein RIM1α Mediates Neuropathic Allodynia through CaV2.2 Activation.

UNLABELLED: Spinal plasticity, a key process mediating neuropathic pain development, requires ubiquitination-dependent protein turnover. Presynaptic active zone proteins have a crucial role in regulating vesicle exocytosis, which is essential for synaptic plasticity. Nevertheless, the mechanism for ubiquitination-regulated turnover of presynaptic active zone proteins in the progression of spinal plasticity-associated neuropathic pain remains unclear. Here, after research involving Sprague Dawley rats, we reported that spinal nerve ligation (SNL), in addition to causing allodynia, enhances the Rab3-interactive molecule-1α (RIM1α), a major active zone protein presumed to regulate neural plasticity, specifically in the synaptic plasma membranes (SPMs) of the ipsilateral dorsal horn. Spinal RIM1α-associated allodynia was mediated by Fbxo3, which abates Fbxl2-dependent RIM1α ubiquitination. Subsequently, following deubiquitination, enhanced RIM1α directly binds to CaV2.2, resulting in increased CaV2.2 expression in the SPMs of the dorsal horn. While exhibiting no effect on Fbxo3/Fbxl2 signaling, the focal knockdown of spinal RIM1α expression reversed the SNL-induced allodynia and increased spontaneous EPSC (sEPSC) frequency by suppressing RIM1α-facilitated CaV2.2 expression in the dorsal horn. Intrathecal applications of BC-1215 (a Fbxo3 activity inhibitor), Fbxl2 mRNA-targeting small-interfering RNA, and ω-conotoxin GVIA (a CaV2.2 blocker) attenuated RIM1α upregulation, enhanced RIM1α expression, and exhibited no effect on RIM1α expression, respectively. These results confirm the prediction that spinal presynaptic Fbxo3-dependent Fbxl2 ubiquitination promotes the subsequent RIM1α/CaV2.2 cascade in SNL-induced neuropathic pain. Our findings identify a role of the presynaptic active zone protein in pain-associated plasticity. That is, RIM1α-facilitated CaV2.2 expression plays a role in the downstream signaling of Fbxo3-dependent Fbxl2 ubiquitination/degradation to promote spinal plasticity underlying the progression of nociceptive hypersensitivity following neuropathic injury. SIGNIFICANCE STATEMENT: Ubiquitination is a well known process required for protein degradation. Studies investigating pain pathology have demonstrated that ubiquitination contributes to chronic pain by regulating the turnover of synaptic proteins. Here, we found that the spinal presynaptic active zone protein Rab3-interactive molecule-1α (RIM1α) participates in neuropathic pain development by binding to and upregulating the expression of CaV2.2. In addition, Fbxo3 modifies this pathway by inhibiting Fbxl2-mediated RIM1α ubiquitination, suggesting that presynaptic protein ubiquitination makes a crucial contribution to the development of neuropathic pain. Research in this area, now in its infancy, could potentially provide a novel therapeutic strategy for pain relief.

Fbxo3-Dependent Fbxl2 Ubiquitination Mediates Neuropathic Allodynia through the TRAF2/TNIK/GluR1 Cascade.

Emerging evidence has indicated that the pathogenesis of neuropathic pain is mediated by spinal neural plasticity in the dorsal horn, which provides insight for analgesic therapy. Here, we report that the abundance of tumor necrosis factor receptor-associated factor 2 and NcK-interacting kinase (TNIK), a kinase that is presumed to regulate neural plasticity, was specifically enhanced in ipsilateral dorsal horn neurons after spinal nerve ligation (SNL; left L5 and L6). Spinal TNIK-associated allodynia is mediated by downstream TNIK-GluR1 coupling and the subsequent phosphorylation-dependent trafficking of GluR1 toward the plasma membrane in dorsal horn neurons. Tumor necrosis factor receptor-associated factor 2 (TRAF2), which is regulated by spinal F-box protein 3 (Fbxo3)-dependent F-box and leucine-rich repeat protein 2 (Fbxl2) ubiquitination, contributes to SNL-induced allodynia by modifying TNIK/GluR1 phosphorylation-associated GluR1 trafficking. Although exhibiting no effect on Fbxo3/Fbxl2/TRAF2 signaling, focal knockdown of spinal TNIK expression prevented SNL-induced allodynia by attenuating TNIK/GluR1 phosphorylation-dependent subcellular GluR1 redistribution. In contrast, intrathecal administration of BC-1215 (N1,N2-Bis[[4-(2-pyridinyl)phenyl]methyl]-1,2-ethanediamine) (a novel Fbxo3 inhibitor) prevented SNL-induced Fbxl2 ubiquitination and subsequent TFAF2 de-ubiquitination to ameliorate behavioral allodynia via antagonizing TRAF2/TNIK/GluR1 signaling. By targeting spinal Fbxo3-dependent Fbxl2 ubiquitination and the subsequent TRAF2/TNIK/GluR1 cascade, spinal application of a TNF-α-neutralizing antibody ameliorated SNL-induced allodynia, and, conversely, intrathecal TNF-α injection into naive rats induced allodynia via a spinal Fbxo3/Fbxl2-dependent modification of the TRAF2/TNIK/GluR1 cascade. Together, our results suggest that spinal TNF-α contributes to the development of neuropathic pain by upregulating TRAF2/TNIK/GluR1 signaling via Fbxo3-dependent Fbxl2 ubiquitination and degradation. Thus, we propose a potential medical treatment strategy for neuropathic pain by targeting the F-box protein or TNIK. SIGNIFICANCE STATEMENT: TNF-α participates in neuropathic pain development by facilitating the spinal TRAF2-dependent TNIK-GluR1 association, which drives GluR1-containing AMPA receptor trafficking toward the plasma membrane. In addition, F-box protein 3 modifies this pathway by inhibiting F-box and leucine-rich repeat protein 2-mediated TRAF2 ubiquitination, suggesting that protein ubiquitination contributes crucially to the development of neuropathic pain. These results provide a novel therapeutic strategy for pain relief.

Methylation regulation for FUNDC1 stability in childhood leukemia was up-regulated and facilitates metastasis and reduces ferroptosis of leukemia through mitochondrial damage by FBXL2.

Leukemia, the most common malignant tumor in childhood, can be categorized into acute leukemia and chronic leukemia. However, the role of FUNDC1 in childhood leukemia (CL) remains unknown. This study aims to investigate the effects of FUNDC1 on patients with CL and its underlying mechanism both in vivo and in vitro. The mRNA expression levels of FUNDC1 were found to be up-regulated in serum samples from CL patients as well as in leukemia cell lines. Furthermore, it was observed that the mRNA expression of FUNDC1 was lower in stage I-II CL patients compared to stage III-IV patients. The up-regulation of FUNDC1 was found to promote leukemia metastasis. Additionally, it was discovered that FUNDC1 up-regulation reduces ferroptosis by inhibiting mitochondrial damage. In a leukemia model, FUNDC1 up-regulation induces the expression of FBXL2. Moreover, FUNDC1 up-regulation reduces FBXL2 ubiquitination, thus maintaining FBXL2 protein expression in leukemia. By inducing FBXL2, FUNDC1 reduces ferroptosis in leukemia through the inhibition of mitochondrial damage. The stability of FUNDC1 is controlled by METTL3 methylation. Overall, this study sheds light on the role of FUNDC1 in CL and provides insights into its underlying mechanisms.

FUNDC1 mediated mitochondria-dependent ferroptosis of epithelial cells in model of asthma by FBXL2/ar/GPX4 signaling pathway of SUMO1 at K136.

This study aimed to explore the critical role of FUNDC1 on epithelial cells in model of asthma. Patients with asthma and normal healthy volunteers were obtained from our hospital. The serum of FUNDC1 mRNA expression was down-regulated in patients with asthma. Meanwhile, the serum of FUNDC1 mRNA expression was positive correlation with IgE and anti-HDM IgE protein. FUNDC1 expression in lung tissue of mice model was decreased in mice model of asthma. Sh-FUNDC1 enhanced asthma in mice model of asthma. FUNDC1 up-regulation reduced IL-4, IL-5, IL-10 and IL-13 activity levels in vitro model of asthma.FUNDC1 down-regulation promoted IL-4, IL-5, IL-10 and IL-13 activity levels in vitro model of asthma. FUNDC1 reduced ferroptosis of epithelial cells in model of asthma through the inhibition of mitochondrial damage. FUNDC1 induced FBXL2 and AR protein expression in model of asthma. FUNDC1 interlinked with FBXL2 is modified by SUMO1 at K136. FBXL2, ASN-205, GLN-204, ARG-235, and GLN-237 form hydrogen bonds with FUNDC1's ASP-15, ASP-16, GLU-25, and ARG-29, with lengths of 2.3, 3.1, 2.9, 2.3, and 2.9 Å, respectively. The induction of FBXL2 reduced the effects of Sh-FUNDC1 on asthma in mice model of asthma. The inhibition of AR reduced the effects of Sh-FUNDC1 on asthma in mice model of asthma Overall, FUNDC1 prevents ferroptosis of airway epithelial cells of asthma through FBXL2/AR/GPX4 signaling pathway of SUMO1 at K136. FUNDC1 might benefit the treatment of asthma or other pulmonary disease.

FUNDC1 prevents ferroptosis of airway epithelial cells of asthma through FBXL2/AR/GPX4 signaling pathway of SUMO1 at K136. FUNDC1 might benefit the treatment of asthma or other pulmonary disease.

MiR-346-5p promotes colorectal cancer cell proliferation in vitro and in vivo by targeting FBXL2 and activating the ß-catenin signaling pathway.

MiR-346-5p is overexpressed in several cancers, including colorectal cancer (CRC). However, the effects of miR-346-5p on CRC progression have not yet been clarified. In our study, miR-346-5p levels in four CRC cell lines and normal human colon epithelial cells were determined by real-time PCR. SW620 and HCT116 cells were selected and then transfected with miR-346-5p mimic, miR-346-5p inhibitor, or specific siRNAs targeting F-box/LRR-repeat protein 2 (FBXL2). Cell proliferation, cell cycle distribution and cell cycle regulators were examined by CCK-8 assay, flow cytometry, and western blot. The binding of miR-346-5p on 3' untranslated region (UTR) of FBXL2 were verified by dual-luciferase reporter assay. CRC cells were co-transfected with miR-346-5p inhibitor and siFBXL2 to investigate the involvement of FBXL2. Interaction of FBXL2 with forkhead box M1 (FoxM1) was examined by co-immunoprecipitation (Co-IP) assay. The effect of miR-346-5p knockdown on CRC tumorigenesis in vivo was investigated. Here, we found that miR-346-5p overexpression promoted, while miR-346-5p knockdown inhibited cell proliferation and G1-S transition. Inhibition of FBXL2 showed similar effects as miR-346-5p overexpression. Moreover, we verified that FBXL2 was a direct target of miR-346-5p. FBXL2 interacted with FoxM1, and then negatively regulated both FoxM1 and nuclear ß-catenin levels. Additionally, FBXL2 knockdown reversed the effects of miR-346-5p inhibitor. In xenograft models, miR-346-5p knockdown significantly inhibited tumor growth, increased FBXL2 expression, and downregulated the levels of FoxM1 and nuclear ß-catenin. In conclusion, miR-346-5p may promote CRC growth by targeting FBXL2 and activating the ß-catenin signaling pathway. MiR-346-5p may be a novel target in cancer therapy.

Tumor Necrosis Factor Alpha Regulates Skeletal Myogenesis by Inhibiting SP1 Interaction with cis-Acting Regulatory Elements within the Fbxl2 Gene Promoter.

FBXL2 is an important ubiquitin E3 ligase component that modulates inflammatory signaling and cell cycle progression, but its molecular regulation is largely unknown. Here, we show that tumor necrosis factor alpha (TNF-α), a critical cytokine linked to the inflammatory response during skeletal muscle regeneration, suppressed Fbxl2 mRNA expression in C2C12 myoblasts and triggered significant alterations in cell cycle, metabolic, and protein translation processes. Gene silencing of Fbxl2 in skeletal myoblasts resulted in increased proliferative responses characterized by activation of mitogen-activated protein (MAP) kinases and nuclear factor kappa B and decreased myogenic differentiation, as reflected by reduced expression of myogenin and impaired myotube formation. TNF-α did not destabilize the Fbxl2 transcript (half-life [t1/2], ∼10 h) but inhibited SP1 transactivation of its core promoter, localized to bp -160 to +42 within the proximal 5' flanking region of the Fbxl2 gene. Chromatin immunoprecipitation and gel shift studies indicated that SP1 interacted with the Fbxl2 promoter during cellular differentiation, an effect that was less pronounced during proliferation or after TNF-α exposure. TNF-α, via activation of JNK, mediated phosphorylation of SP1 that impaired its binding to the Fbxl2 promoter, resulting in reduced transcriptional activity. The results suggest that SP1 transcriptional activation of Fbxl2 is required for skeletal muscle differentiation, a process that is interrupted by a key proinflammatory myopathic cytokine.IMPORTANCE Skeletal muscle regeneration and repair involve the recruitment and proliferation of resident satellite cells that exit the cell cycle during the process of myogenic differentiation to form myofibers. We demonstrate that the ubiquitin E3 ligase subunit FBXL2 is essential for skeletal myogenesis through its important effects on cell cycle progression and cell proliferative signaling. Further, we characterize a new mechanism whereby sustained stimulation by a major proinflammatory cytokine, TNF-α, regulates skeletal myogenesis by inhibiting the interaction of SP1 with the Fbxl2 core promoter in proliferating myoblasts. Our findings contribute to the understanding of skeletal muscle regeneration through the identification of Fbxl2 as both a critical regulator of myogenic proliferative processes and a susceptible gene target during inflammatory stimulation by TNF-α in skeletal muscle. Modulation of Fbxl2 activity may have relevance to disorders of muscle wasting associated with sustained proinflammatory signaling.

Targeting F-Box Protein Fbxo3 Attenuates Lung Injury Induced by Ischemia-Reperfusion in Rats.

Background: Increasing evidence suggests that Fbxo3 signaling has an important impact on the pathophysiology of the inflammatory process. Fbxo3 protein inhibition has reduced cytokine-driven inflammation and improved disease severity in animal model of Pseudomonas-induced lung injury. However, it remains unclear whether inhibition of Fbxo3 protein provides protection in acute lung injury induced by ischemia-reperfusion (I/R). In this study, we investigated the protective effects of BC-1215 administration, a Fbxo3 inhibitor, on acute lung injury induced by I/R in rats. Methods: Lung I/R injury was induced by ischemia (40 min) followed by reperfusion (60 min). The rats were randomly assigned into one of six experimental groups (n = 6 rats/group): the control group, control + BC-1215 (Fbxo3 inhibitor, 0.5 mg/kg) group, I/R group, or I/R + BC-1215 (0.1, 0.25, 0.5 mg/kg) groups. The effects of BC-1215 on human alveolar epithelial cells subjected to hypoxia-reoxygenation (H/R) were also examined. Results: BC-1215 significantly attenuated I/R-induced lung edema, indicated by a reduced vascular filtration coefficient, wet/dry weight ratio, lung injury scores, and protein levels in bronchoalveolar lavage fluid (BALF). Oxidative stress and the level of inflammatory cytokines in BALF were also significantly reduced following administration of BC-1215. Additionally, BC-1215 mitigated I/R-stimulated apoptosis, NF-κB, and mitogen-activated protein kinase activation in the injured lung tissue. BC-1215 increased Fbxl2 protein expression and suppressed Fbxo3 and TNFR associated factor (TRAF)1-6 protein expression. BC-1215 also inhibited IL-8 production and NF-κB activation in vitro in experiments with alveolar epithelial cells exposed to H/R. Conclusions: Our findings demonstrated that Fbxo3 inhibition may represent a novel therapeutic approach for I/R-induced lung injury, with beneficial effects due to destabilizing TRAF proteins.

NS5A Promotes Constitutive Degradation of IP3R3 to Counteract Apoptosis Induced by Hepatitis C Virus.

FBXL2 targets IP3R3 for ubiquitin-mediated degradation to limit Ca2+ flux to mitochondria and, consequently, apoptosis. Efficient replication of hepatitis C virus (HCV) requires geranylgeranylation of FBXL2. Here, we show that the viral protein NS5A forms a trimeric complex with IP3R3 and FBXL2, unmasking IP3R3's degron in the absence of inositol 1,4,5-trisphosphate (IP3) stimulation. FBXL2 knockdown or expression of a stable IP3R3 mutant causes persistent Ca2+ flux and sensitizes cells to apoptosis, resulting in the inhibition of viral replication. Importantly, the effect of FBXL2 silencing is rescued by depleting IP3R3, but not p85ß, another established FBXL2 substrate, indicating that the anti-HCV effect of FBXL2 knockdown is largely due to IP3R3 stabilization. Finally, disruption of the FBXL2-NS5A-IP3R3 complex using somatic cell genetics or pharmacologic inhibition results in IP3R3 stabilization and suppression of HCV replication. This study reveals an IP3-independent molecular mechanism through which HCV promotes IP3R3 degradation, thereby inhibiting virus-induced apoptosis and establishing chronic infection.

Crystal structure and interaction studies of the human FBxo3 ApaG domain.

UNLABELLED: Transcriptional activation of proinflammatory cytokines, mediated by tumor necrosis factor receptor-associated factors (TRAFs), is in part triggered by the degradation of the F-box protein, FBxl2, via an E3 ligase that contains another F-box protein, FBxo3. The ApaG domain of FBxo3 is required for the interaction with and degradation of FBxl2 [Mallampalli RK et al., (2013) J Immunol 191, 5247-5255]. Here, we report the X-ray structure of the human FBxo3 ApaG domain, residues 278-407, at 2.0 Å resolution. Like bacterial ApaG proteins, this domain is characterized by a classic Immunoglobin/Fibronectin III-type fold, comprising a seven-stranded ß-sheet core, surrounded by four extended loops. Although cation binding had been proposed for bacterial ApaG proteins, no interactions with Mg(2+) or Co(2+) were detected for the human ApaG domain. In addition, dinucleotide polyphosphates, which have been reported to be second messengers in the inflammation response and targets of the bacterial apaG-containing operon, are not bound by the human ApaG domain. In the context of the full-length protein, loop 1, comprising residues 294-303, is critical for the interaction with FBxl2. However, titration of the individual ApaG domain with a 15-mer FBxl2 peptide that was phosphorylated on the crucial T404, as well as the inability of the ApaG domain to interact with full-length FBxl2, assessed by coimmunoprecipitation, indicate that the ApaG domain alone is necessary, but not sufficient for binding and degradation of FBxl2. DATABASE: PDB ID (5HDW).

Calmodulin protects Aurora B on the midbody to regulate the fidelity of cytokinesis.

Aurora B kinase is an integral regulator of cytokinesis as it stabilizes the intercellular canal within the midbody to ensure proper chromosomal segregation during cell division. Here we identified an E3 ligase subunit, F box protein FBXL2, that by recognizing a calmodulin binding signature within Aurora B, ubiquitinates and removes the kinase from the midbody. Calmodulin, by competing with the F box protein for access to the calmodulin binding signature, protected Aurora B from FBXL2. Calmodulin co-localized with Aurora B on the midbody, preserved Aurora B levels in cells, and stabilized intercellular canals during delayed abscission. Genetic or pharmaceutical depletion of endogenous calmodulin significantly reduced Aurora B protein levels at the midbody resulting in tetraploidy and multi-spindle formation. The calmodulin inhibitor, calmidazolium, reduced Aurora B protein levels resulting in tetraploidy, mitotic arrest, and apoptosis of tumorigenic cells and profoundly inhibiting tumor formation in athymic nude mice. These observations indicate molecular interplay between Aurora B and calmodulin in telophase and suggest that calmodulin acts as a checkpoint sensor for chromosomal segregation errors during mitosis.

Beta-amyloid toxicity modifier genes and the risk of Alzheimer's disease.

Late-onset Alzheimer's disease (LOAD) is a complex and multifactorial disease. So far ten loci have been identified for LOAD, including APOE, PICALM, CLU, BIN1, CD2AP, CR1, CD33, EPHA1, ABCA7, and MS4A4A/MS4A6E, but they explain about 50% of the genetic risk and thus additional risk genes need to be identified. Amyloid beta (Aß) plaques develop in the brains of LOAD patients and are considered to be a pathological hallmark of this disease. Recently 12 new Aß toxicity modifier genes (ADSSL1, PICALM, SH3KBP1, XRN1, SNX8, PPP2R5C, FBXL2, MAP2K4, SYNJ1, RABGEF1, POMT2, and XPO1) have been identified that potentially play a role in LOAD risk. In this study, we have examined the association of 222 SNPs in these 12 candidate genes with LOAD risk in 1291 LOAD cases and 958 cognitively normal controls. Single site and haplotype analyses were performed using PLINK. Following adjustment for APOE genotype, age, sex, and principal components, we found single nucleotide polymorphisms (SNPs) in PPP2R5C, PICALM, SH3KBP1, XRN1, and SNX8 that showed significant association with risk of LOAD. The top SNP was located in intron 3 of PPP2R5C (P=0.009017), followed by an intron 19 SNP in PICALM (P=0.0102). Haplotype analysis revealed significant associations in ADSSL1, PICALM, PPP2R5C, SNX8, and SH3KBP1 genes. Our data indicate that genetic variation in these new candidate genes affects the risk of LOAD. Further investigation of these genes, including additional replication in other case-control samples and functional studies to elucidate the pathways by which they affect Aß, are necessary to determine the degree of involvement these genes have for LOAD risk.