Soluble α-synuclein–antibody complexes activate the NLRP3 inflammasome in hiPSC-derived microglia

Parkinson’s disease is characterized by accumulation of α-synuclein (αSyn). Release of oligomeric/fibrillar αSyn from damaged neurons may potentiate neuronal death in part via microglial activation. Heretofore, it remained unknown if oligomeric/fibrillar αSyn could activate the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome in human microglia and whether anti-αSyn antibodies could prevent this effect. Here, we show that αSyn activates the NLRP3 inflammasome in human induced pluripotent stem cell (hiPSC)-derived microglia (hiMG) via dual stimulation involving Toll-like receptor 2 (TLR2) engagement and mitochondrial damage. In vitro, hiMG can be activated by mutant (A53T) αSyn secreted from hiPSC-derived A9-dopaminergic neurons. Surprisingly, αSyn–antibody complexes enhanced rather than suppressed inflammasome-mediated interleukin-1β (IL-1β) secretion, indicating these complexes are neuroinflammatory in a human context. A further increase in inflammation was observed with addition of oligomerized amyloid-β peptide (Aβ) and its cognate antibody. In vivo, engraftment of hiMG with αSyn in humanized mouse brain resulted in caspase-1 activation and neurotoxicity, which was exacerbated by αSyn antibody. These findings may have important implications for antibody therapies aimed at depleting misfolded/aggregated proteins from the human brain, as they may paradoxically trigger inflammation in human microglia.

Parkinson’s disease (PD) is characterized by accumulation of α-synuclein (αSyn; encoded by the SNCA gene) (1). Release of oligomeric/fibrillar αSyn from damaged neurons may potentiate neuronal cell death in part via microglial activation (2, 3). Moreover, misfolded proteins in general are thought to interact with brain microglia, triggering microglial activation that contributes to neurodegenerative disorders, although microglial phagocytosis may also initially clear aberrant proteins to afford some degree of protection (2, 4). Additionally, in Alzheimer’s disease (AD), amyloid-β peptide (Aβ) is thought to trigger similar processes in microglia (5–7); however, the mechanism for this trigger is still poorly understood.Microglial cells contribute to neuroinflammation, specifically that mediated by the inflammasome. In particular, the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome has been associated with several neurodegenerative disorders, although other types of inflammation may also be important in this regard (8). The NLRP3 inflammasome is a multiprotein complex that responds to cell stress and pathogenic stimuli to promote activation of caspase-1, which in turn mediates maturation and release of proinflammatory cytokines, including interleukin-1β (IL-1β) and IL-18 (9–11). NLRP3 inflammasome activation is a two-step process, involving an initial priming step and a secondary trigger. Priming involves a proinflammatory stimulus, such as endotoxin, a ligand for Toll-like receptor 4 (TLR4), that increases the abundance of NLRP3 and promotes de novo synthesis of pro–IL-1β via nuclear factor κB (11). The secondary trigger promotes inflammasome complex assembly and caspase-1 activation that in turn mediates the cleavage of pro–IL-1β and subsequent release of mature IL-1β. There are various secondary triggers, including adenosine triphosphate (ATP), microparticles, and bacterial toxins, all of which somehow lead to mitochondrial damage and release of oxidized mitochondrial DNA (11). Neuroinflammation has been reported in both human PD and AD brains (12–15), and NLRP3 inflammasome activation in particular has been observed in mouse models of PD and AD (7, 16). Importantly, in these PD models, dopaminergic (DA) neurons in the substantia nigra are resistant to damage in NLRP3-deficient mice compared with wild-type (WT) mice (16). Interestingly, a recent report identified an NLRP3 polymorphism that confers decreased risk in PD (17). Several groups have reported that fibrillar αSyn can activate the NLRP3 inflammasome in mice and in human monocytes (18–22), but it remains unknown if human brain microglia can be activated in this manner. Critically, antibodies targeting misfolded proteins are being tested in human clinical trials for several neurodegenerative diseases, including AD and PD; however, it is still unclear how antibodies to αSyn might affect this inflammatory response. In this study, we characterized the response of human induced pluripotent stem cell (hiPSC)-derived microglia (hiMG) to oligomeric/fibrillar αSyn in vitro and in vivo, using engraftment of hiMG in humanized mice. We used these immunocompromised mice because they prevent human cell rejection and express three human genes that support human cell engraftment (23). We show that αSyn and, even more so, αSyn–antibody complexes activate the NLRP3 inflammasome. Moreover, this process is further sensitized by the presence of Aβ and its cognate antibodies. These observations are of heightened interest because recent studies have shown that both misfolded Aβ and αSyn are present in several neurodegenerative disorders such as AD and Lewy body dementia (LBD), a form of dementia that can occur in the setting of PD (24–26).     

Targeting tumor-derived NLRP3 reduces melanoma progression by limiting MDSCs expansion

Interleukin-1β (IL-1β)–mediated inflammation suppresses antitumor immunity, leading to the generation of a tumor-permissive environment, tumor growth, and progression. Here, we demonstrate that nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing-3 (NLRP3) inflammasome activation in melanoma is linked to IL-1β production, inflammation, and immunosuppression. Analysis of cancer genome datasets (TCGA and GTEx) revealed greater NLRP3 and IL-1β expression in cutaneous melanoma samples (n = 469) compared to normal skin (n = 324), with a highly significant correlation between NLRP3 and IL-1β (P < 0.0001). We show the formation of the NLRP3 inflammasome in biopsies of metastatic melanoma using fluorescent resonance energy transfer analysis for NLRP3 and apoptosis-associated speck-like protein containing a CARD. In vivo, tumor-associated NLRP3/IL-1 signaling induced expansion of myeloid-derived suppressor cells (MDSCs), leading to reduced natural killer and CD8⁺ T cell activity concomitant with an increased presence of regulatory T (Treg) cells in the primary tumors. Either genetic or pharmacological inhibition of tumor-derived NLRP3 by dapansutrile (OLT1177) was sufficient to reduce MDSCs expansion and to enhance antitumor immunity, resulting in reduced tumor growth. Additionally, we observed that the combination of NLRP3 inhibition and anti–PD-1 treatment significantly increased the antitumor efficacy of the monotherapy by limiting MDSC-mediated T cell suppression and tumor progression. These data show that NLRP3 activation in melanoma cells is a protumor mechanism, which induces MDSCs expansion and immune evasion. We conclude that inhibition of NLRP3 can augment the efficacy of anti–PD-1 therapy.

Tumorigenesis is initiated by genomic alterations, leading to cell transformation, proliferation, and resistance to apoptotic signals, which ultimately lead to metastasis and tissue invasion. Tumor progression is also linked to dysregulated inflammation, which is characterized by cytokine signaling between cancer and noncancer cells (1, 2). The proinflammatory cytokine interleukin-1β (IL-1β) mediates several inflammatory diseases and is a pivotal cytokine in initiating inflammatory responses (3). In the context of malignancy, IL-1β is a validated target in mouse models of cancer, including melanoma, where the cytokine contributes to immunosuppression, angiogenesis, metastasis, and regulation of myeloid-derived suppressor cells (MDSCs) (1, 2, 4, 5). In humans, IL-1β is overexpressed in biopsies from metastatic melanoma patients, suggesting a possible role in the melanoma-induced inflammation (6).Processing of IL-1β is largely governed by inflammasomes, cytosolic macromolecular complexes responsible for the conversion of biologically inactive IL-1β and IL-18 precursors into their active forms via caspase-1 cleavage (7). The nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing-3 (NLRP3) is the most studied of the inflammasome sensors driving IL-1β–mediated conditions from sterile inflammation to rare hereditary syndromes (8, 9). NLRP3 is particularly relevant to the processing of IL-1β in melanoma because NLRP3 is constitutively expressed in melanoma cell lines (6) and NLRP3 polymorphisms are linked to increased risk to develop melanoma (10). Whereas these studies indicate a possible role for NLRP3 in melanoma progression, the biological function for NLRP3 in melanoma remains unclear. Furthermore, although it is well known that inflammation participates in the development and progression of melanoma (11–13), the inflammatory pathways that drive this process are still poorly characterized and no therapy developed to date is actually designed to specifically target inflammatory pathways in melanoma. Here, using genetic models of NLRP3 depletion and a specific pharmacological inhibitor of NLRP3 (14), we show that NLRP3 represents a melanoma intrinsic pathway exploited for tumor-mediated immune escape. We demonstrate that tumor-derived NLRP3 activation induces MDSC expansion, which suppresses recruitment and activation of antitumor immunity.From a clinical standpoint, immune checkpoint therapy (ICT) has significantly improved the outcome for melanoma patients, and numerous studies have demonstrated that expression of PD-1/PD-L1 and CTLA4 are often predictors for efficacy of immunotherapy. However, the number of patients that are unresponsive to ICT or relapse continues to rise, and clinical data show that expression of immune checkpoints do not always correlate with responses (15). The limited response to monotherapy in some patients suggests that intrinsic pathways in melanoma cells, such as expression of checkpoint ligands, are not the only mechanisms that drive tumor progression. Identification of other tumor-specific strategies provides an opportunity to interrupt the oncogenic process and improve survival in this population. For example, melanoma-associated inflammation facilitates tumor progression (11, 16) and, specifically, is linked to IL-1β activity (4, 17). An approach for reducing IL-1β activity is via inhibition of NLRP3. Here, we show that disruption of the NLRP3 signaling in combination with ICT increases antitumor activity. The data support the concept that tumor NLRP3 activation represents an intrinsic pathway that favors tumor immune escape. Thus, targeting NLRP3 represents an innovative strategy for treating melanoma, especially in the context of immunotherapy resistance tumors.     

Structural insights into α-synuclein monomer–fibril interactions

Protein aggregation into amyloid fibrils is associated with multiple neurodegenerative diseases, including Parkinson’s disease. Kinetic data and biophysical characterization have shown that the secondary nucleation pathway highly accelerates aggregation via the absorption of monomeric protein on the surface of amyloid fibrils. Here, we used NMR and electron paramagnetic resonance spectroscopy to investigate the interaction of monomeric α-synuclein (α-Syn) with its fibrillar form. We demonstrate that α-Syn monomers interact transiently via their positively charged N terminus with the negatively charged flexible C-terminal ends of the fibrils. These intermolecular interactions reduce intramolecular contacts in monomeric α-Syn, yielding further unfolding of the partially collapsed intrinsically disordered states of α-Syn along with a possible increase in the local concentration of soluble α-Syn and alignment of individual monomers on the fibril surface. Our data indicate that intramolecular unfolding critically contributes to the aggregation kinetics of α-Syn during secondary nucleation.

Synucleinopathies, including Parkinson’s disease (PD), are associated with the accumulation of intracellular neuronal aggregates termed as Lewy bodies and Lewy neuritis, which contain high concentration of the protein α-synuclein (α-Syn) in an aggregated state (1, 2). The disease-relevant role of α-Syn is further highlighted by mutations in the α-Syn gene (SNCA) causing familial PD [i.e., A30P (3), E46K (4), H50Q (5), G51D (6), A53E (7), and A53T (8)] and the duplication or triplication of the SNCA leading to early-onset PD in affected families (9, 10). α-Syn is a 140-residue intrinsically disordered protein (IDP) in solution (11) but adopts a helical structure in the presence of acidic lipid surfaces (12, 13). The positively charged N terminus (residues 1 to 60) is rich in lysine residues and contains KTKEGV binding repeats associated with vesicle binding (14). Moreover, the N-terminal domain includes all known SNCA familial PD mutations. The central region (residues 61 to 95) defines the non-amyloid-β component (NAC) (15), which is essential for α-Syn aggregation (16), while the C terminus (residues 96 to 140) is highly negatively charged.In vitro, α-Syn forms polymorphic amyloid fibrils (17–19) with unique arrangements of cross-β-sheet motifs (20–22). When injected into model animals, these fibrils induce a PD-like pathology (23) where the aggregation pathway of α-Syn plays a key role in the development of the disease (24). A detailed analysis of the aggregation kinetics of α-Syn into amyloids is therefore important toward understanding the toxic mechanisms relevant for synucleinopathies.Amyloid formation of α-Syn is very sensitive to solution conditions, including pH (25), temperature (26), and salt concentration (27). It further requires the presence of an air–water interface (28) or negatively charged lipid membranes (29) for which α-Syn has a high affinity. Previous studies suggest that amyloid fibril growth of α-Syn occurs via a nucleation-dependent polymerization reaction (30). Following a fairly slow primary nucleus formation, α-Syn fibrils are elongated by addition of single monomers. In a next step, the amyloid fibrils multiply by fragmentation or can catalyze the formation of new amyloids from monomers on their surface—a process known as secondary nucleation that was first described for sickle cell anemia 40 y ago (31). Fragmentation and secondary nucleation critically depend on the fibril mass and accelerate the aggregation kinetics (30). In the case of α-Syn aggregation under quiescent condition fragmentation does not exist and only the described secondary nucleation process occurs. While detailed kinetic experiments showed no significant secondary nucleation at pH 7, it strongly contributes at pH values lower than 6 (25, 30). However, mechanistic or structural information of the secondary nucleation process in α-Syn aggregation has been lacking so far.In this study we investigated the structural properties of α-Syn monomer–fibril interactions by NMR and electron paramagnetic resonance (EPR) spectroscopy. Our results provide insights into how monomeric α-Syn transiently interacts in vitro via its positively charged N terminus with the negatively charged C-terminal residues of the α-Syn fibrils, giving detailed insights into the mechanism of the secondary nucleation process.     

The NLRP3 inflammasome inhibitor OLT1177 rescues cognitive impairment in a mouse model of Alzheimer’s disease

Numerous studies demonstrate that neuroinflammation is a key player in the progression of Alzheimer’s disease (AD). Interleukin (IL)-1β is a main inducer of inflammation and therefore a prime target for therapeutic options. The inactive IL-1β precursor requires processing by the the nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome into a mature and active form. Studies have shown that IL-1β is up-regulated in brains of patients with AD, and that genetic inactivation of the NLRP3 inflammasome improves behavioral tests and synaptic plasticity phenotypes in a murine model of the disease. In the present study, we analyzed the effect of pharmacological inhibition of the NLRP3 inflammasome using dapansutrile (OLT1177), an oral NLRP3-specific inhibitor that is safe in humans. Six-month-old WT and APP/PS1 mice were fed with standard mouse chow or OLT1177-enriched chow for 3 mo. The Morris water maze test revealed an impaired learning and memory ability of 9-mo-old APP/PS1 mice (P = 0.001), which was completely rescued by OLT1177 fed to mice (P = 0.008 to untreated APP/PS1). Furthermore, our findings revealed that 3 mo of OLT1177 diet can rescue synaptic plasticity in this mouse model of AD (P = 0.007 to untreated APP/PS1). In addition, microglia were less activated (P = 0.07) and the number of plaques was reduced in the cortex (P = 0.03) following NLRP3 inhibition with OLT1177 administration. We also observed an OLT1177 dose-dependent normalization of plasma metabolic markers of AD to those of WT mice. This study suggests the therapeutic potential of treating neuroinflammation with an oral inhibitor of the NLRP3 inflammasome.

Alzheimer’s disease (AD) and other related neurodegenerative diseases leading to dementia represent an enormous burden for the society and health economies. AD patients suffer progressive cognitive and functional deficits often for many years, which result in a heavy burden to patients, families, and the public health system. In fact, in 2015 an estimated 46.8 million people worldwide were living with dementia, which could extend to 131.5 million by 2050 (1). Rising prevalence and mortality rates in combination with a lack of effective treatments lead to enormous costs to society. Research on AD in the last decades has focused on the pathological hallmarks and cellular deposits of amyloid-β (Aβ) peptides and neurofibrils (2). Recently, there has been increased evidence supporting a central role of the immune system in the progression or even the origin of the disease (3–5). In this respect, it is noteworthy that it has been known since 1989 that levels of interleukin (IL)-1β, one of the main mediators of innate immune response, are elevated in brains of patients with AD and can be associated with the progression and onset of AD (6–11). Additionally, it was shown that the nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome (12, 13), a multisubunit complex important for the maturation of IL-1β, is activated by Aβ peptides, leading to an overproduction of IL-1β, neuroinflammation, and cognitive impairment (14, 15). Inhibition of the NLRP3 inflammasome and the subsequent reduced IL-1β production can be linked to a change in the phenotype of microglia, the innate immune cells in the brain. Heneka et al. (16) pointed out the important role of the NLRP3 inflammasome/caspase-1 axis in AD pathogenesis by demonstrating significant improvements (e.g., in cognition) in APP/PS1 mice (a mouse model for AD) when crossed with NLRP3^−/− animals. The APP/PS1 mice express a human amyloid precursor protein (APP) and human presenilin-1 (PS1), leading to the accumulation of Aβ peptides, neuroinflammation, and cognitive impairment (17).OLT1177 (rINN: dapansutrile) is a new chemical entity small molecule that specifically targets the NLRP3 inflammasome and prevents the activation of caspase-1 and the maturation and release of IL-1β (18). OLT1177 has been shown to be well tolerated in animals and humans (18) and is currently in phase 2 clinical studies for the treatment of inflammatory conditions, such as osteoarthritis (topical gel dosage form) and inflammatory diseases, such as acute gout flare (oral capsule dosage form), among other diseases (19).In this study, we used the APP/PS1 mouse model of AD to investigate the effects of OLT1177 as an acute, oral pharmacological intervention (17). Six-month-old WT and APP/PS1ΔE9 mice consumed ad libitum OLT1177 in feed pellets (∼0, 500, or 1,000 mg/kg/d based on feed concentrations of 0, 3.75 or 7.5 g of OLT1177 per kilogram of feed; hereafter referred to as 3.75 or 7.5 g/kg OLT1177) for the treatment duration of 3 mo. APP/PS1 mice treated with OLT1177 showed rescue effects in various assessments, ranging from improved cognitive function to overall reduction in proinflammatory cytokines in the brain, suggesting the potential benefits of pharmaceutically blocking NLRP3 signaling in AD.     

Wild-type α-synuclein inherits the structure and exacerbated neuropathology of E46K mutant fibril strain by cross-seeding

Heterozygous point mutations of α-synuclein (α-syn) have been linked to the early onset and rapid progression of familial Parkinson’s diseases (fPD). However, the interplay between hereditary mutant and wild-type (WT) α-syn and its role in the exacerbated pathology of α-syn in fPD progression are poorly understood. Here, we find that WT mice inoculated with the human E46K mutant α-syn fibril (hE46K) strain develop early-onset motor deficit and morphologically different α-syn aggregation compared with those inoculated with the human WT fibril (hWT) strain. By using cryo-electron microscopy, we reveal at the near-atomic level that the hE46K strain induces both human and mouse WT α-syn monomers to form the fibril structure of the hE46K strain. Moreover, the induced hWT strain inherits most of the pathological traits of the hE46K strain as well. Our work suggests that the structural and pathological features of mutant strains could be propagated by the WT α-syn in such a way that the mutant pathology would be amplified in fPD.

α-Synuclein (α-Syn) is the main component of Lewy bodies, which serve as the common histological hallmark of Parkinson’s disease (PD) and other synucleinopathies (1, 2). α-Syn fibrillation and cell-to-cell transmission in the brain play essential roles in disease progression (3–5). Interestingly, WT α-syn could form fibrils with distinct polymorphs, which exhibit disparate seeding capability in vitro and induce distinct neuropathologies in mouse models (6–10). Therefore, it is proposed that α-syn fibril polymorphism may underlie clinicopathological variability of synucleinopathies (6, 9). In fPD, several single-point mutations of SNCA have been identified, which are linked to early-onset, severe, and highly heterogeneous clinical symptoms (11–13). These mutations have been reported to influence either the physiological or pathological function of α-syn (14). For instance, A30P weakens while E46K strengthens α-syn membrane binding affinity that may affect its function in synaptic vesicle trafficking (14, 15). E46K, A53T, G51D, and H50Q have been found to alter the aggregation kinetics of α-syn in different manners (15–17). Recently, several cryogenic electron microscopy (cryo-EM) studies revealed that α-syn with these mutations forms diverse fibril structures that are distinct from the WT α-syn fibrils (18–26). Whether and how hereditary mutations induced fibril polymorphism contributes to the early-onset and exacerbated pathology in fPD remains to be elucidated. More importantly, most fPD patients are heterozygous for SNCA mutations (12, 13, 27, 28), which leads to another critical question: could mutant fibrils cross-seed WT α-syn to orchestrate neuropathology in fPD patients?E46K mutation is one of the eight disease-causing mutations on SNCA originally identified from a Spanish family with autosomal-dominant PD (11). E46K-associated fPD features early-onset motor symptoms and rapid progression of dementia with Lewy bodies (11). Studies have shown that E46K mutant has higher neurotoxicity than WT α-syn in neurons and mouse models overexpressing α-syn (29–32). The underlying mechanism is debatable. Some reported that E46K promotes the formation of soluble species of α-syn without affecting the insoluble fraction (29, 30), while others suggested that E46K mutation may destabilize α-syn tetramer and induce aggregation (31, 32). Our previous study showed that E46K mutation disrupts the salt bridge between E46 and K80 in the WT fibril strain and rearranges α-syn into a different polymorph (33). Compared with the WT strain, the E46K fibril strain is prone to be fragmented due to its smaller and less stable fibril core (33). Intriguingly, the E46K strain exhibits higher seeding ability in vitro, suggesting that it might induce neuropathology different from the WT strain in vivo (33).In this study, we found that human E46K and WT fibril strains (referred to as hE46K and hWT strains) induced α-syn aggregates with distinct morphologies in mice. Mice injected with the hE46K strain developed more α-syn aggregation and early-onset motor deficits compared with the mice injected with the hWT strain. Notably, the hE46K strain was capable of cross-seeding both human and mouse WT (mWT) α-syn to form fibrils (named as hWT_cs and mWT_cs). The cross-seeded fibrils replicated the structure and seeding capability of the hE46K template both in vitro and in vivo. Our results suggest that the hE46K strain could propagate its structure as well as the seeding properties to the WT monomer so as to amplify the α-syn pathology in fPD.     

Homeostatic regulation of T follicular helper and antibody response to particle antigens by IL-1Ra of medullary sinus macrophage origin

Hepatitis B virus (HBV) vaccines are composed of surface antigen HBsAg that spontaneously assembles into subviral particles. Factors that impede its humoral immunity in 5% to 10% of vaccinees remain elusive. Here, we showed that the low-level interleukin-1 receptor antagonist (IL-1Ra) can predict antibody protection both in mice and humans. Mechanistically, murine IL-1Ra–inhibited T follicular helper (Tfh) cell expansion and subsequent germinal center (GC)-dependent humoral immunity, resulting in significantly weakened protection against the HBV challenge. Compared to soluble antigens, HBsAg particle antigen displayed a unique capture/uptake and innate immune activation, including IL-1Ra expression, preferably of medullary sinus macrophages. In humans, a unique polymorphism in the RelA/p65 binding site of IL-1Ra enhancer associated IL-1Ra levels with ethnicity-dependent vaccination outcome. Therefore, the differential IL-1Ra response to particle antigens probably creates a suppressive milieu for Tfh/GC development, and neutralization of IL-1Ra would resurrect antibody response in HBV vaccine nonresponders.

Follicular helper T (Tfh) cells are antigen-experienced CD4⁺ T cells within B cell follicles of secondary lymphoid organs, such as lymph nodes (LN), spleens, and Peyer’s patches, that constitutively express the B cell follicle homing receptor CXCR5 (1). Upon cellular interaction and cross-signaling with their cognate follicular B (FoB) cells in the presence of follicular dendritic cells (FDCs), Tfh cells trigger the formation and maintenance of germinal centers (GCs) through the expression of CD40 ligand and the secretion of IL-21 and IL-4 (2–4). Tfh-dependent paracrine activation of CD40 results in B cell survival and differentiation in the GC (5), whereas isotype class switching is believed to occur predominantly outside GCs. Therefore, Tfh cells play a critical role in mediating the selection of high-affinity B cells that differentiate either into plasma cells or memory B cells (6–11).Besides the inducible T cell costimulator (ICOS) that activates Tfh cells to secrete IL-21, other cytokines [e.g., IL-2 (12), IL-6 (13), and IL-7 (14)] also signal for Tfh cell differentiation. The role of IL-1 signaling remained puzzling until recently: Tfh cells can be primed by IL-1β, whose production is licensed by IFN-β in response to infectious agents (15). Such featured innate response of IFN-β and IL-1β relies on the activation of TLR and inflammasomes by live, but not dead, bacteria or recombinant vaccines (16, 17). Therefore, OVA antigen augments Tfh cell response in mice only when IL-1β is exogenously applied at a nonphysiological high concentration (18), whereas endogenous IL-1β/IL-1R1 signaling may not be required for antibody responses to T-dependent or -independent antigens (19–21). We reasoned that IL-1Ra (encoded by IL-1rn), which can compete with IL-1 for binding to IL-1R1 in the homeostatic inflammatory response (22–24), would intrinsically antagonize IL-1β/IL-1R1 signaling for Tfh/GC development. For example, IL-1rn^−/− mice exhibit an excessive antibody response to sheep red blood cells immunization (25, 26). A thorough investigation is required to dissect how IL-1 and IL-1Ra mutually regulate a homeostatic Tfh/GC response.LN macrophages are conventionally divided into two subtypes. Subcapsular sinus (SCS, CD169⁺F4/80⁻) macrophages are specialized antigen presenting cells that capture certain particle or opsonized antigens and display them intact for cognate recognition by FoB cells (27–30). SCS macrophages also relay immune complex to noncognate B cells for antibody affinity maturation (30). Macrophages in medullary sinus (MSM, CD169⁺F4/80⁺), in contrast, are potent in phagocytosis (31) for clearance of pathogens and particulate antigens from the lymph. It has been postulated 10 y ago that SCS may capture particle antigens, such as hepatitis B virus (HBV) vaccine, and migrate to follicles to facilitate more effective activation of B cells and FDCs (32). In this work, we found that murine antibody response inversely correlated to IL-1Ra level and clearly distinguished responders from nonresponders in volunteers receiving HBV vaccination. Further studies showed that LN macrophages subsets exhibited different capture and activation kinetics for particle and soluble antigens, and IL-1Ra expression by MSM could critically modulate IL-1R1 potentiation of Tfh cells and, hence, the specific antibody response to particle antigens. Therefore, mice lacking IL-1Ra or with IL-1Ra being neutralized yielded more robust antibody response to HBV vaccine and enables protection against chronic HBV infection.     

Pharmacological inhibition of MDA-9/Syntenin blocks breast cancer metastasis through suppression of IL-1β

Melanoma differentiation associated gene-9 (MDA-9), Syntenin-1, or syndecan binding protein is a differentially regulated prometastatic gene with elevated expression in advanced stages of melanoma. MDA-9/Syntenin expression positively associates with advanced disease stage in multiple histologically distinct cancers and negatively correlates with patient survival and response to chemotherapy. MDA-9/Syntenin is a highly conserved PDZ-domain scaffold protein, robustly expressed in a spectrum of diverse cancer cell lines and clinical samples. PDZ domains interact with a number of proteins, many of which are critical regulators of signaling cascades in cancer. Knockdown of MDA-9/Syntenin decreases cancer cell metastasis, sensitizing these cells to radiation. Genetic silencing of MDA-9/Syntenin or treatment with a pharmacological inhibitor of the PDZ1 domain, PDZ1i, also activates the immune system to kill cancer cells. Additionally, suppression of MDA-9/Syntenin deregulates myeloid-derived suppressor cell differentiation via the STAT3/interleukin (IL)-1β pathway, which concomitantly promotes activation of cytotoxic T lymphocytes. Biologically, PDZ1i treatment decreases metastatic nodule formation in the lungs, resulting in significantly fewer invasive cancer cells. In summary, our observations indicate that MDA-9/Syntenin provides a direct therapeutic target for mitigating aggressive breast cancer and a small-molecule inhibitor, PDZ1i, provides a promising reagent for inhibiting advanced breast cancer pathogenesis.

Breast cancer remains the second leading cause of death among women in the United States (1). Prognosis for early-stage disease is favorable, whereas late-stage disease with tumor cell spread beyond the primary site (i.e., metastasis) frequently heralds poorer outcomes (1). Therapy of metastatic disease usually involves systemic chemotherapy combined with radiation, providing mostly palliative options to reduce metastatic outgrowth (2). Multiple unique and distinct biological steps and an interplay between transformed and nontransformed cells highlight complexities of the metastatic process, which habitually thwarts clinical intervention. In principle, targeting these processes independently or collectively could culminate in effective antimetastatic therapies.Melanoma differentiation-associated gene-9 (mda-9), also known as Syntenin-1 or syndecan binding protein (SDCBP), was cloned in our laboratory using subtraction hybridization from terminal differentiating metastasis-derived human melanoma cells treated with interferon (IFN)-β and the protein kinase C activator, mezerein (3, 4) (designated as mda-9/Syntenin). Preferential elevated expression of mda-9/Syntenin is evident in histologically distinct tumors and contributes to several steps in the metastatic process (5). These include tumor cell invasion and migration (6, 7), induction of angiogenesis through secretion of proangiogenic factors (8–10), enhancement of epithelial–mesenchymal transition (EMT) (11, 12), regulation of the expression of integrins affecting cell-adhesion processes (13), exosome biogenesis and exosome-mediated signaling in cell–cell communication (14), and recently immune-modulation suppressing host-immune surveillance (15). Cancer cell-independent functions of MDA-9/Syntenin also contribute to metastatic progression by regulating immunosuppressive cell infiltration in the metastatic niche (16). Based on its relevance to the invasive and metastatic phenotype of cancers, MDA-9/Syntenin represents a prospective target for rational design of antimetastatic drugs.Differential expression of MDA-9/Syntenin in cancer versus adjacent normal tissue is often a predictor of poor clinical outcomes (8). A relationship exists between MDA-9/Syntenin (SDCBP) and breast cancer in rat mammary tumors (genomic localization) (17) and in metastasis and clinical situations in human triple negative and other human breast cancers (11, 15, 18). MDA-9/Syntenin plays a pivotal role in EMT induction that includes initiation of Smad-dependent EMT through interaction with TGF-βR1, disrupting receptor internalization (11). Physical interaction between MDA-9/Syntenin and TGF-β activates small GTPases, Rho A, and CDC 42 (12). In addition, MDA-9/Syntenin enhances primary tumor growth and lung metastasis through immune evasion by up-regulating PD-L1 (program death ligand 1) through STAT3 activation, causing T cell apoptosis (15). In breast cancer, MDA-9/Syntenin affects tumor cell proliferation in estrogen receptor-negative breast cancer, causing cells to bypass the G1/S checkpoint promoting S-phase entry (19). MDA-9/Syntenin is also considered a potential antigen in breast cancer (20). These observations endorse MDA-9/Syntenin as a prospective target for the therapy of breast cancer metastasis.Disturbing MDA-9/Syntenin protein:protein interactions is viewed as a viable strategy to disrupt key downstream signaling pathways regulating cancer cell invasion and metastasis (reviewed in ref. 5). Fragment-based drug discovery guided by NMR identified a first in-class interaction inhibitor of the PDZ1 domain of MDA-9/Syntenin, PDZ1i (21), displaying efficacy against glioblastoma multiforme, neuroblastoma, and prostate cancer (5, 13, 22, 23). PDZ1i suppresses cancer cell-autonomous and nonautonomous functions of MDA-9/Syntenin, culminating in strong antiinvasive and antimetastatic properties in vitro and in vivo, without inducing overt cytostatic or toxic effects in normal or most cancer cells. Informed by the crystal structure of MDA-9/Syntenin, peptide-based inhibitory molecules have been developed and validated in cell-based assays (24). Additionally, a genetic approach using adenovirus-mediated delivery of shmda-9 has shown efficacy in xenografted human melanoma (8) and prostate cancer (25) in nude mice. These investigations confirm MDA-9/Syntenin as a viable target for suppressing both primary and metastatic tumor growth and support further evaluation of PDZ1i on breast cancer metastasis.Evidence from both experimental models and clinical studies show a relationship between abundance of tumor-infiltrating immune cells and metastasis (26). To create a permissive environment in a secondary site, disseminated tumor cells employ multiple strategies, including reducing host immune surveillance (27). In several mouse tumor models, myeloid-derived suppressor cells (MDSCs), a heterogeneous population of myeloid cells with immunosuppressive properties, are expanded in the blood, lymph nodes, and spleen (28). They help shape the microenvironment and metastatic niches by regulating both innate and adaptive immunity (29). In breast cancer mouse models, MDSCs accumulate in the lungs prior to metastatic spread (30) and promote immune suppression by producing reactive oxygen species and arginase (Arg-1) (31). Not surprisingly, various chemotherapeutic agents, such as gemcitabine (32), 5-flurouracil (33), and docetaxel (34) decrease MDSC accumulation in the tumors’ stroma, thereby enhancing antitumor immune responses (29). Similar soluble factors are operational in primary tumors and metastases, including granulocyte-macrophage colony-stimulating factor, interleukins (e.g., IL-6, IL-1β), and vascular endothelial growth factor (VEGF), causing MDSC infiltration. Tumor cells in the metastatic niche that produce various cytokines or growth factors also regulate this process.We have now explored a potential role of MDA-9/Syntenin in breast cancer progression with specific emphasis on a relevant interleukin, IL-1β, representing an important inflammatory cytokine mediating cancer pathogenesis and tumor progression (35). Inflammation regulates fundamental pathways that are causative of the cancer phenotype, including proliferation, survival, and migration (36). IL-1β regulates tumor initiation/progression, angiogenesis, Th17 cell differentiation, and expansion of MDSCs (35). Additionally, IL-1β controls macrophage recruitment and invasion, and metastasis of cancer cells (35). Based on these seminal roles in orchestrating the neoplastic process, IL-1β represents a potential therapeutic target and its regulation deserves further analysis. We now confirm that MDA-9/Syntenin, which can be obstructed by the small-molecule inhibitor PDZ1i, regulates IL-1β, thereby directly controlling breast cancer pathogenesis.     

Polymerase δ promotes chromosomal rearrangements and imprecise double-strand break repair

Recent studies have implicated DNA polymerases θ (Pol θ) and β (Pol β) as mediators of alternative nonhomologous end-joining (Alt-NHEJ) events, including chromosomal translocations. Here we identify subunits of the replicative DNA polymerase δ (Pol δ) as promoters of Alt-NHEJ that results in more extensive intrachromosomal mutations at a single double-strand break (DSB) and more frequent translocations between two DSBs. Depletion of the Pol δ accessory subunit POLD2 destabilizes the complex, resulting in degradation of both POLD1 and POLD3 in human cells. POLD2 depletion markedly reduces the frequency of translocations with sequence modifications but does not affect the frequency of translocations with exact joins. Using separation-of-function mutants, we show that both the DNA synthesis and exonuclease activities of the POLD1 subunit contribute to translocations. As described in yeast and unlike Pol θ, Pol δ also promotes homology-directed repair. Codepletion of POLD2 with 53BP1 nearly eliminates translocations. POLD1 and POLD2 each colocalize with phosphorylated H2AX at ionizing radiation-induced DSBs but not with 53BP1. Codepletion of POLD2 with either ligase 3 (LIG3) or ligase 4 (LIG4) does not further reduce translocation frequency compared to POLD2 depletion alone. Together, these data support a model in which Pol δ promotes Alt-NHEJ in human cells at DSBs, including translocations.

Translocations are genetic rearrangements involving the fusion of heterologous chromosomes (1) and can be initiated by two or more DNA double-strand breaks (DSBs) (2, 3). DSBs in human cells are repaired by multiple pathways with distinct genetic requirements. The first pathway, homology-directed repair (HDR), is active during the S/G2 phase of the cell cycle, when sister chromatids are present to template DNA synthesis. In the first step of HDR, the DSB ends undergo 5′-to-3′ single-strand resection that involves the Mre11/Rad50/Nbs1 (MRN) complex and the endonuclease CtIP (4). The RPA complex binds the exposed single-stranded DNA and is then exchanged for RAD51 by BRCA2 (5). The RAD51 nucleoprotein filament then facilitates strand invasion into a homologous duplex that serves as a repair template. Multiple DNA polymerases, including the replicative polymerases (Pol δ and Pol ε) and translesion DNA polymerases can participate in DNA synthesis during HDR in mammalian cells (6, 7), which is followed by ligation of the ends.The second pathway, classic nonhomologous end-joining (C-NHEJ), is active throughout the cell cycle and therefore responsible for the repair of most DSBs in somatic cells (5). In C-NHEJ, the DSB is bound by the Ku70/Ku80 (Ku) heterodimer, resulting in recruitment of DNA-dependent protein kinase and the end-bridging factors XLF and PAXX (8). End-processing factors, including Artemis and DNA polymerases μ and λ, can also be recruited for end-resection and gap-filling (9, 10). The XRCC4/LIGIV complex is recruited and ligates both strands (11).The third type of repair, alternative NHEJ (Alt-NHEJ), is often described as a back-up end-joining process, as it resolves a greater fraction of DSBs when C-NHEJ is compromised (12). Alt-NHEJ is also more error-prone than C-NHEJ and appears to contribute to mutagenesis in many types of cancer cells (13, 14). A large number of potentially redundant factors may participate in Alt-NHEJ, including CtIP, MRN, Pol θ (encoded by POLQ), poly(ADP-ribose) polymerase 1 (PARP1), and Ligases 1 and 3 (Lig1/3) (4, 12, 15–21). The large number of factors suggests that the “pathway” is actually a combination of potential mediators that compete at the break. As a result, context-specific, lineage-driven, and stochastic effects may each influence which factors ultimately mediate Alt-NHEJ at a given DSB.Similar to HDR, the first step of Alt-NHEJ can involve resection of the DNA ends to single-strands by MRN and CtIP. During HDR, single-strand resection typically extends for kilobases but the extent of resection is limited in G0/G1 phases of the cell cycle by 53BP1. Thus, Alt-NHEJ occurring during G0/G1 involves short 3′ overhangs that may anneal at sites of microhomology (4). Previous studies with mammalian cells have described varying lengths of microhomology characteristic of Alt-NHEJ junctions [e.g., ≥2 bp (22), ≥3 bp (23), ≥5 bp (24), and 2 to 6 bp (25)], suggesting that there is no absolute requirement for a given length across contexts.After annealing, DNA polymerases and ligases are needed to fill the gaps before end ligation. The role of polymerases in Alt-NHEJ (and translocation formation) remains poorly understood. Pol θ is thought to play a role in Alt-NHEJ by facilitating DNA synthesis from annealed microhomologies, and loss of Pol θ led to reduced frequency of Cas9-induced translocations (17) and increased frequency of spontaneous IgH/Myc translocations in mouse cells (26). A recent study showed that loss of Pol β can also reduce translocation frequency between endonuclease-induced breaks in human cells (27).Translocation junctions in mammalian cells tend to have longer deletions and increased use of microhomology compared to repair at single DSBs, suggesting that Alt-NHEJ is involved. Initial studies on the mechanisms of translocation formation were performed in mouse embryonic stem cells (mESCs) containing a translocation reporter that reconstitutes a neomycin resistance gene after cleavage of chromosomes 14 and 17 by the I-SceI meganuclease (22, 28, 29). mESCs lacking either Ku or Xrcc4/Lig4 had increased translocation frequencies, suggesting that these factors suppress translocations rather than promoting them (28). Furthermore, in the absence of C-NHEJ factors, translocation junctions contained longer deletions and an increased usage of microhomology in the final repair products, consistent with Alt-NHEJ mediating translocations in these cells. Additional studies in mESCs demonstrated that loss of CtIP, Parp1, Lig3, and Lig1 can each result in reduced translocation frequency, further implicating these factors in the Alt-NHEJ that mediates translocation formation (22, 29).A subsequent study in human cells painted a more complex picture. Human cell lines depleted of LIG4 had reduced translocation frequency and depletion of LIG3 only decreased translocations in LIG4-depleted cells (23). The mechanisms and cell type-specificity behind these differential end-joining requirements for translocation formation in mouse and human cells remain poorly defined (23).We previously reported a screen of short hairpin RNA (shRNA) against 169 DNA repair-related genes in human cells to identify factors that modulate translocation frequency (30). We used stringent criteria to define “hits” and validated each in multiple human cell lines. Knockdown of the SUMO E2 enzyme UBC9 or RAD50 increased translocation frequency, so we categorized these factors as translocation suppressors. Conversely, knockdown of 53BP1, DNA damage-binding protein 1 (DDB1), or PARP3 decreased translocation frequency, so we categorized these as translocation promoters (30).We noted that POLD2, an accessory subunit of the replicative polymerase Pol δ, nearly scored in the screen as a promoter of chromosomal translocations. In budding yeast, Pol δ promotes both Alt-NHEJ and microhomology-mediated chromosomal translocations (31) but this has not been assessed in mammalian cells. Here, we demonstrate that Pol δ plays a role during Alt-NHEJ in human cells and that Pol δ subunits promote translocations. Based on these findings, we propose a model in which Pol δ exonuclease and polymerase activity promote Alt-NHEJ after annealing of sequences with microhomology.     

Autoregulation of insulin receptor signaling through MFGE8 and the αvβ5 integrin

The role of integrins, in particular αv integrins, in regulating insulin resistance is incompletely understood. We have previously shown that the αvβ5 integrin ligand milk fat globule epidermal growth factor like 8 (MFGE8) regulates cellular uptake of fatty acids. In this work, we evaluated the impact of MFGE8 on glucose homeostasis. We show that acute blockade of the MFGE8/β5 pathway enhances while acute augmentation dampens insulin-stimulated glucose uptake. Moreover, we find that insulin itself induces cell-surface enrichment of MFGE8 in skeletal muscle, which then promotes interaction between the αvβ5 integrin and the insulin receptor leading to dampening of skeletal-muscle insulin receptor signaling. Blockade of the MFGE8/β5 pathway also enhances hepatic insulin sensitivity. Our work identifies an autoregulatory mechanism by which insulin-stimulated signaling through its cognate receptor is terminated through up-regulation of MFGE8 and its consequent interaction with the αvβ5 integrin, thereby establishing a pathway that can potentially be targeted to improve insulin sensitivity.

Acute insulin resistance can be viewed as a protective response under specific physiological conditions that necessitate increased insulin secretion. Nevertheless, the increasing prevalence of chronic insulin resistance (1) in the current obesity epidemic hastens the development of type 2 diabetes (T2D) and induces compensatory hyperinsulinemia. Hyperinsulinemia can produce potentially maladaptive consequences at least in part, due to the mitogenic roles of insulin (2–4). As such, there remains a critical need for new therapies to improve insulin sensitivity in order to prevent T2D, avoid the need for insulin treatment in patients with T2D, or reduce the insulin dose required to normalize blood glucose in such individuals.Insulin binding to the alpha subunit of the insulin receptor induces a conformational change that triggers activation of insulin receptor beta subunit (IRβ) tyrosine kinase activity (5–7). The activated insulin receptor phosphorylates target molecules that mediate downstream signaling leading to glucose uptake and other metabolic effects (8, 9). Dephosphorylation of IRβ and insulin receptor substrate-1 (IRS-1) aids in termination of insulin signaling pathways (10, 11) and is the basis of clinical trials targeting putative phosphatases to treat diabetes (12). Despite their potential therapeutic relevance, there is a relative paucity of knowledge regarding molecular mechanisms that lead to termination of insulin receptor signaling.The integrin families of cell surface receptors mediate bidirectional signaling between the cell and its external environment. Previous work has identified interactions between integrin receptors and other growth factor receptor tyrosine kinases (13–16) that lead to modulation of downstream signaling (17–19). For example, the αvβ3 and α6β4 integrins function as coreceptors for insulin-like growth factor-1 and 2 (IGF1 and 2) and potentiate IGF1 receptor (IGF1R)-mediated signaling (19–23). Immunoprecipitation studies have demonstrated a physical association between the αv integrins and IRβ (24, 25). The impact of these associations on glucose homeostasis has not been evaluated. A role for β1 integrins in the regulation of glucose homeostasis is well established. This class of integrins appears to be particularly important in regulating insulin-mediated glucose homeostasis in the obese state. The effect of β1 integrins on glucose homeostasis appears to be primarily due to obesity-associated matrix remodeling (26–30) rather than a direct effect secondary to a physical association between β1 integrins and the insulin receptor.Milk fat globule epidermal growth factor like 8 (MFGE8) is a secreted integrin ligand which binds the αvβ3, αvβ5, and α8β1 integrins (31, 32). Several recent observations suggest a role for MFGE8 in modulating insulin resistance. In humans, serum MFGE8 levels are increased in the context of diabetes and correlate positively with the extent of hemoglobin glycosylation (33, 34). Indeed, serum MFGE8 levels correlate with indices of insulin resistance in two independent cohorts of patients with T2D or gestational diabetes from China (35, 36). A missense variation in the gene encoding MFGE8, present in South Asian Punjabi Sikhs, is associated with increased circulating MFGE8 levels and increased risk of developing T2D (37). Increased circulating levels of MFGE8 in diabetic patients may impact T2D through effects on inflammation and cardiovascular disease. Humans with increased MFGE8 expression have a greater risk of developing coronary artery disease (38). In contrast, in murine models, MFGE8 deficiency exacerbates cardiac hypertrophy and atherosclerosis (39, 40). MFGE8 also improves wound healing responses in diabetic foot ulcers (41, 42) by triggering apoptotic cell clearance and promoting resolution of inflammation (43–45).Despite the notable links between MFGE8, insulin resistance, and T2D pathology, the biology underlying these associations has not been investigated. We therefore evaluated the effect of acute antibody-mediated disruption of the MFGE8/β5 pathway on glucose homeostasis in wild-type (WT) mice. We report here that MFGE8 markedly attenuates the effect of insulin on skeletal muscle glucose uptake. Antibody-mediated blockade of MFGE8 or αvβ5 enhances while recombinant MFGE8 (rMFGE8) reduces insulin-stimulated glucose uptake in vitro and in vivo. Mechanistically, insulin acts to promotes cell-surface enrichment of skeletal muscle MFGE8, which then binds to cell surface αvβ5 and increases the interaction between the integrin and the insulin receptor. This interaction subsequently aids in terminating insulin receptor signaling.     

The high-affinity immunoglobulin receptor FcγRI potentiates HIV-1 neutralization via antibodies against the gp41 N-heptad repeat

The HIV-1 gp41 N-heptad repeat (NHR) region of the prehairpin intermediate, which is transiently exposed during HIV-1 viral membrane fusion, is a validated clinical target in humans and is inhibited by the Food and Drug Administration (FDA)-approved drug enfuvirtide. However, vaccine candidates targeting the NHR have yielded only modest neutralization activities in animals; this inhibition has been largely restricted to tier-1 viruses, which are most sensitive to neutralization by sera from HIV-1–infected individuals. Here, we show that the neutralization activity of the well-characterized NHR-targeting antibody D5 is potentiated >5,000-fold in TZM-bl cells expressing FcγRI compared with those without, resulting in neutralization of many tier-2 viruses (which are less susceptible to neutralization by sera from HIV-1–infected individuals and are the target of current antibody-based vaccine efforts). Further, antisera from guinea pigs immunized with the NHR-based vaccine candidate (ccIZN36)₃ neutralized tier-2 viruses from multiple clades in an FcγRI-dependent manner. As FcγRI is expressed on macrophages and dendritic cells, which are present at mucosal surfaces and are implicated in the early establishment of HIV-1 infection following sexual transmission, these results may be important in the development of a prophylactic HIV-1 vaccine.

Membrane fusion between HIV-1 and host cells is mediated by the viral envelope glycoprotein (Env), a trimer consisting of the gp120 and gp41 subunits. Upon interaction with cellular receptors, Env undergoes a dramatic conformational change and forms the prehairpin intermediate (PHI) (1–3), in which the fusion peptide region at the amino terminus of gp41 inserts into the cell membrane. In the PHI, the N-heptad repeat (NHR) region of gp41 is exposed and forms a stable, three-stranded α-helical coiled coil. Subsequently, the PHI resolves when the NHR and the C-heptad repeat (CHR) regions of gp41 associate to form a trimer-of-hairpins structure that brings the viral and cell membranes into proximity, facilitating membrane fusion (Fig. 1).Open in a separate windowFig. 1.HIV-1 membrane fusion. The surface protein of the HIV-1 envelope is composed of the gp120 and gp41 subunits. After Env binds to cell-surface receptors, gp41 inserts into the host cell membrane and undergoes a conformational change to form the prehairpin intermediate. The N-heptad repeat (orange) region of gp41 is exposed in the PHI and forms a three-stranded coiled coil. To complete viral fusion, the PHI resolves to a trimer-of-hairpins structure in which the C-heptad repeat (blue) adopts a helical conformation and binds the NHR region. Fusion inhibitors such as enfuvirtide bind the NHR, preventing viral fusion by inhibiting formation of the trimer of hairpins (1–3). The membrane-proximal external region (red) is located adjacent to the transmembrane (TM) region of gp41.The NHR region of the PHI is a validated therapeutic target in humans: the Food and Drug Administration (FDA)-approved drug enfuvirtide binds the NHR and inhibits viral entry into cells (4, 5). Various versions of the three-stranded coiled coil formed by the NHR have been created and used as vaccine candidates in animals (6–10). The neutralization potencies of these antisera, as well as those of anti-NHR monoclonal antibodies (mAbs) (11–15), are modest and mostly limited to HIV-1 isolates that are highly sensitive to antibody-mediated neutralization [commonly referred to as tier-1 viruses (16)]. These results have led to skepticism about the PHI as a vaccine target.Earlier studies showed that the neutralization activities of mAbs that bound another region of gp41, the membrane-proximal external region (MPER) (Fig. 1), were enhanced as much as 5,000-fold in cells expressing FcγRI (CD64) (17, 18), an integral membrane protein that binds the Fc portion of immunoglobulin G (IgG) molecules with high (nanomolar) affinity (19, 20). This effect was not attributed to phagocytosis and occurred when the cells were preincubated with antibody and washed before adding virus (17, 18). Since the MPER is a partially cryptic epitope that is not fully exposed until after Env engages with cellular receptors (21, 22), these results suggest that by binding the Fc region, FcγRI provides a local concentration advantage for MPER mAbs at the cell surface that enhances viral neutralization (17, 18). While not expressed on T cells, FcγRI is expressed on macrophages and dendritic cells (23), which are present at mucosal surfaces and are implicated in sexual HIV-1 transmission and the early establishment of HIV-1 infection (22–34).Here we investigated whether FcγRI expression also potentiates the neutralizing activity of antibodies targeting the NHR, since that region, like the MPER, is preferentially exposed during viral fusion. We found that D5, a well-characterized anti-NHR mAb (11, 12), inhibits HIV-1 infection ∼5,000-fold more potently in TZM-bl cells expressing FcγRI (TZM-bl/FcγRI cells) than in TZM-bl cells that do not. Further, while antisera from guinea pigs immunized with (ccIZN36)₃, an NHR-based vaccine candidate (7), displayed weak neutralizing activity in TZM-bl cells, they exhibited enhanced neutralization in TZM-bl/FcγRI cells, including against some tier-2 HIV-1 isolates that are more resistant to antibody-mediated neutralization (16) and that serve as benchmarks for antibody-based vaccine efforts. These results indicate that FcγRI can play an important role in neutralization by antibodies that target the PHI. Since these receptors are expressed on cells prevalent at mucosal surfaces thought to be important for sexual HIV-1 transmission, our results motivate vaccine strategies that harness this potentiating effect.     

The Parkinson’s disease-associated gene ITPKB protects against α-synuclein aggregation by regulating ER-to-mitochondria calcium release

Inositol-1,4,5-triphosphate (IP₃) kinase B (ITPKB) is a ubiquitously expressed lipid kinase that inactivates IP₃, a secondary messenger that stimulates calcium release from the endoplasmic reticulum (ER). Genome-wide association studies have identified common variants in the ITPKB gene locus associated with reduced risk of sporadic Parkinson’s disease (PD). Here, we investigate whether ITPKB activity or expression level impacts PD phenotypes in cellular and animal models. In primary neurons, knockdown or pharmacological inhibition of ITPKB increased levels of phosphorylated, insoluble α-synuclein pathology following treatment with α-synuclein preformed fibrils (PFFs). Conversely, ITPKB overexpression reduced PFF-induced α-synuclein aggregation. We also demonstrate that ITPKB inhibition or knockdown increases intracellular calcium levels in neurons, leading to an accumulation of calcium in mitochondria that increases respiration and inhibits the initiation of autophagy, suggesting that ITPKB regulates α-synuclein pathology by inhibiting ER-to-mitochondria calcium transport. Furthermore, the effects of ITPKB on mitochondrial calcium and respiration were prevented by pretreatment with pharmacological inhibitors of the mitochondrial calcium uniporter complex, which was also sufficient to reduce α-synuclein pathology in PFF-treated neurons. Taken together, these results identify ITPKB as a negative regulator of α-synuclein aggregation and highlight modulation of ER-to-mitochondria calcium flux as a therapeutic strategy for the treatment of sporadic PD.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by a variety of motor symptoms (including unbalanced gait, resting tremor, and bradykinesia) that are accompanied by psychosis and dementia at later stages of disease. The onset of motor symptoms is largely caused by the selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the corresponding depletion of dopamine innervation in the striatum. Although the cause of neuron loss is unknown, the hallmark pathological feature of PD is the presence of intraneuronal inclusions composed of misfolded and fibrillar α-synuclein (α-syn) in the neurites and soma, termed Lewy neurites and Lewy bodies, respectively (1). Protein-coding single-nucleotide polymorphisms (SNPs), duplications, and triplications in the gene encoding α-syn (SNCA) all cause early-onset, familial forms of PD and result in accelerated aggregation of α-syn protein into insoluble, fibrillar aggregates (1, 2). Recent evidence suggests that these aggregates can spread from cell to cell, leading to the propagation of pathology to neuroanatomically connected brain regions (3, 4). Therefore, therapeutic approaches that reduce the aggregation or spreading of pathological α-syn species represent potential disease-modifying therapies for PD.In addition to SNCA, mutations in several other genes have been identified that cause rare, familial forms of PD. These genes are primarily involved in the autophagic clearance of intracellular aggregates or damaged organelles, especially mitochondria (5–8). Genome-wide association studies (GWAS) have also identified common SNPs in several genes related to endolysosomal function, such as GBA and LRRK2, that are associated with increased risk of PD (8–11). GBA and LRRK2 mutations have been shown to affect lysosomal and mitochondrial phenotypes (12–14), which contribute to the accumulation of PD-like neuropathology in mouse models, primary neurons, and human iPSC-derived cells (15–18). These findings highlight the role of the lysosomal and mitochondria quality control pathways in PD and demonstrate that perturbations in these pathways are sufficient to increase α-syn aggregation. Despite this, the etiology of sporadic PD is not fully understood, and the specific genes and pathways that are tractable for therapeutic modulation remain elusive. Therefore, the discovery of new genes associated with sporadic PD may be critical for both understanding disease pathogenesis and identifying novel therapeutic approaches.Recently, Chang et al. conducted a GWAS analysis that identified 17 novel gene loci significantly associated with sporadic PD in a European population including more than 26,000 patients across three independent cohorts (19). The lead GWAS SNP in the novel 1q42 locus was rs4653767. This is an intronic SNP in the gene encoding inositol-1,4,5-triphosphate kinase B (ITPKB) and produces a thymine-to-cytosine nucleotide substitution that is protective against developing PD (odds ratio [OR] = 0.92, P = 2.4 × 10⁻¹⁰). A follow up meta-analysis study of 37,688 PD patients, which included the discovery cohort, and 18,618 proxy cases strengthened the GWAS finding at this locus (OR = 0.92, P = 1.4 × 10⁻¹⁵). Furthermore, this locus was still significant when the analysis was performed on only the new independent cases and proxy cases (OR = 0.92, P = 2.8 × 10⁻⁵) (20). The rs4653767-C allele is present in similar frequencies across populations (27% in non-Finnish European and 29% in East Asian populations) and was found to have the same direction of effect (OR = 0.87, P = 0.016) in a targeted replication study of the European PD loci in an East Asian cohort (21). ITPKB is also highly expressed in several brain regions related to PD, including the SNpc, striatum, and cerebral cortex (22).ITPKB is one of three ubiquitously expressed kinases known to phosphorylate inositol-1,4,5-triphosphate (IP₃), an intracellular messenger produced from phosphatidylinositol-4,5-bisphosphate by phospholipase C (23, 24). IP₃ binds to IP₃ receptors (IP₃Rs) in the endoplasmic reticulum (ER) to stimulate the release of calcium ions from the ER into the cytosol to mediate various downstream signaling pathways. IP₃ kinases (ITPKA, ITPKB, and ITPKC) add a fourth phosphate group to IP₃ producing inositol-1,3,4,5-tetrakisphosphate (IP₄), which has no activity on IP₃Rs. Thus, IP₃ kinases negatively regulate IP₃-mediated calcium release from the ER. While the role of this pathway in peripheral cell types under normal physiological conditions is well understood (25, 26), whether ITPKB is involved in the pathogenesis of PD is unknown. Here, we investigate whether the modulation of ITPKB expression or kinase activity impacts the accumulation of α-syn pathology in cellular models of PD.     

Molecular basis of the dual role of the Mlh1-Mlh3 endonuclease in MMR and in meiotic crossover formation

In budding yeast, the MutL homolog heterodimer Mlh1-Mlh3 (MutLγ) plays a central role in the formation of meiotic crossovers. It is also involved in the repair of a subset of mismatches besides the main mismatch repair (MMR) endonuclease Mlh1-Pms1 (MutLα). The heterodimer interface and endonuclease sites of MutLγ and MutLα are located in their C-terminal domain (CTD). The molecular basis of MutLγ’s dual roles in MMR and meiosis is not known. To better understand the specificity of MutLγ, we characterized the crystal structure of Saccharomyces cerevisiae MutLγ(CTD). Although MutLγ(CTD) presents overall similarities with MutLα(CTD), it harbors some rearrangement of the surface surrounding the active site, which indicates altered substrate preference. The last amino acids of Mlh1 participate in the Mlh3 endonuclease site as previously reported for Pms1. We characterized mlh1 alleles and showed a critical role of this Mlh1 extreme C terminus both in MMR and in meiotic recombination. We showed that the MutLγ(CTD) preferentially binds Holliday junctions, contrary to MutLα(CTD). We characterized Mlh3 positions on the N-terminal domain (NTD) and CTD that could contribute to the positioning of the NTD close to the CTD in the context of the full-length MutLγ. Finally, crystal packing revealed an assembly of MutLγ(CTD) molecules in filament structures. Mutation at the corresponding interfaces reduced crossover formation, suggesting that these superstructures may contribute to the oligomer formation proposed for MutLγ. This study defines clear divergent features between the MutL homologs and identifies, at the molecular level, their specialization toward MMR or meiotic recombination functions.

During the first meiotic division, in most organisms, each pair of homologous chromosomes (homologs) needs to experience at least one crossover to ensure their accurate segregation and increase genetic diversity of the progeny (1, 2). Crossovers are generated after programmed DNA double-strand break (DSB) formation, and their subsequent repair by homologous recombination (3). Failure to achieve at least one crossover per homolog pair results in aneuploid gametes. These dysfunctions are frequent causes of spontaneous miscarriages and birth defects in humans (1). DSBs are generated by the meiosis-specific Spo11 protein and resected to form 3′ single-stranded tails that are directed to invade and pair with an unbroken homologous template, preferentially on the homolog (3). Invasion intermediates are a substrate for DNA synthesis. After the capture of the second DSB end, a subset of the intermediates is converted into double Holliday junctions (dHJs), which in meiosis are primarily resolved into crossovers (4, 5). The remaining recombination intermediates are repaired as noncrossovers.MutLγ (Mlh1-Mlh3 in yeast and MLH1-MLH3 in human) is essential for the formation of meiotic crossovers in many organisms. The MutLγ heterodimer possesses, similarly to MutLα (Mlh1-Pms1 in yeast, MLH1-PMS2 in human), a latent endonuclease activity (6–10). It has been proposed that the MutLγ endonuclease activity catalyzes the resolution of the dHJ intermediates and promotes the formation of crossovers (8, 11). In agreement with this, Mlh1 and Mlh3 form foci on pachytene chromosomes in different organisms at future crossover sites (12–14). In yeast mlh3 mutants, the crossover rates are reduced to 50 to 70% of the wild-type level (8, 15–17). These mutants exhibit failure in chromosome disjunction and consequently a decrease of spore viability. In Mlh3^-/- mice, males and females present a crossover defect that leads to aneuploidy (18). In agreement with the proposed resolvase role of MutLγ, a mutant of the active site within the conserved DQHAX₂EX₄E motif of Saccharomyces cerevisiae Mlh3, D523N, results in a loss of activity and confers a phenotype similar to the mlh3∆ mutant with decreased crossover frequencies (8). A mutation in the endonuclease domain of mouse MLH3 leads to infertile males and strongly reduced crossover numbers, despite a correct loading of factors essential for crossover resolution (19). In budding yeast and mammals, MLH1 deletion is also associated with severe dysfunctions in meiosis due to crossover defects, combined with high genetic instability due to its additional central role with Pms1 (as part of the MutLα complex) in mutation avoidance (20).In budding yeast, the majority of meiotic crossovers are formed by MutLγ, requiring in addition other proteins including the ZMM proteins (Zip1-4, Spo16, Mer3, and Msh4-Msh5), Exo1, and the Sgs1-Top3-Rmi1 complex (11, 21). The ZMM factors are proposed to stabilize the recombination intermediates and protect them from the action of helicases (reviewed in ref. 22). The 5′-3′ exonuclease, Exo1, is important for crossover formation independently of its nuclease activity and likely acts as scaffold factor in particular through its interaction motif with Mlh1 (17). We recently reported that MutLγ-Exo1 associates with recombination intermediates, followed by direct Cdc5 recruitment that triggers MutLγ crossover activity (23). Despite its central role in the formation of crossovers, to date, the MutLγ endonuclease does not show any specific activity on either a single HJ or dHJ DNA substrate in vitro, in contrast to other resolvases or structure-specific endonucleases (24). Recently, it was shown that human MutLγ is part of an ensemble with MutSγ, EXO1, RFC, and PCNA that preferentially cleaves plasmid DNA containing a HJ, although it does not cleave symmetrically across the junction, as would be expected for a canonical resolvase (25, 26).MutLγ is also an accessory factor of the postreplicative mismatch repair (MMR). In S. cerevisiae, MutLα is the major MutL homolog involved in MMR. It is targeted to DNA mismatches by the MutS homologs and introduces DNA nicks to initiate the excision of the strand containing the mismatch. MutLα contains an endonuclease site formed by three conserved motifs in Pms1 and the last conserved amino acids of Mlh1 (6, 7, 27). Mutation of this active site (e.g., pms1E707K in yeast) is associated with high mutations rates. A minor role of MutLγ has been reported in the repair of a subset of DNA mismatches recognized by Msh2-Msh3 (28, 29). The third MutL heterodimer, Mlh1-Mlh2 (MLH1-PMS1 in mammals) or MutLβ, has no endonuclease motif or activity. In yeast, mlh2Δ cells present only a slight defect in the repair of a subset of frameshift mutations (30–32). Apart from this function in MMR, we recently identified an interaction of MutLβ with Mer3 helicase that limits gene conversion tract lengths (31). A major challenge is to further characterize the molecular basis of the specific interactions and regulations of the three MutL homologs with their DNA substrates and protein partners in MMR and in meiotic recombination.MutLγ and MutLα present an overall common organization with a N-terminal domain (NTD) bearing an ATPase function, connected through a long linker to a C-terminal domain (CTD). The CTD mediates the dimerization of the complexes (33, 34) and possesses the endonuclease site (6–8). Upon ADP and ATP binding, the NTD undergoes large asymmetric conformational changes that can position it into a close proximity to the CTD (35, 36). The heterodimers MutLα and MutLγ can slide on double-stranded DNA (dsDNA) with linear diffusion modes (37, 38) and control strand excision during MMR (39). However, both heterodimers differ in their DNA-binding properties. MutLα has very low affinity for short dsDNA in physiological salt concentrations and can bind cooperatively to long dsDNA (>200 bp), forming long continuous protein tracts (40). In contrast, MutLγ binds short-branched DNA substrates with a higher affinity (in the nanomolar range) and with a marked preference for HJs (9). MutLγ also binds, albeit more weakly, short and long dsDNA and is proposed to form oligomers on long dsDNA (9, 41). The Pms1 and Mlh3 subunits present a moderate sequence identity precluding correct modeling of MutLγ from the MutLα crystal structures and thus limiting our understanding of the molecular basis of the specificity of MutLα and MutLγ heterodimers in MMR and meiosis, respectively.Here, we present the crystal structure of the S. cerevisiae MutLγ(CTD), and we compare it with the three-dimensional structure of the S. cerevisiae MutLα(CTD) that we previously reported (27). We reveal differences between the two complexes with regard to the size of the heterodimerization interface, the regulatory domain position, and the shape of the cavity surrounding the endonuclease site. We characterize the role of the last amino acids of Mlh1 using mutant alleles and identify a central role of the last three conserved residues of Mlh1 in vivo for both MMR and meiotic recombination. We analyze the DNA-binding specificities of the MutLα(CTD) and Mut-Lγ(CTD) and compare to the properties of full-length proteins. We then identify positions in Mlh3(NTD) and Mlh3(CTD) that can participate to the positioning of the NTD close to the CTD and characterize the corresponding mutants in meiosis. Finally, we report a filament arrangement of the MutLγ(CTD) in the crystal in agreement with oligomers proposed in previous studies (25, 41), and we characterize an allele mutated in this oligomerization region.