Novel mGluR5 Positive Allosteric Modulator Improves Functional Recovery,Attenuates Neurodegeneration,and Alters Microglial Polarization after Experimental Traumatic Brain Injury

Traumatic brain injury (TBI) causes microglial activation and related neurotoxicity that contributes to chronic neurodegeneration and loss of neurological function. Selective activation of metabotropic glutamate receptor 5 (mGluR5) by the orthosteric agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), is neuroprotective in experimental models of TBI, and has potent anti-inflammatory effects in vitro. However, the therapeutic potential of CHPG is limited due to its relatively weak potency and brain permeability. Highly potent, selective and brain penetrant mGluR5 positive allosteric modulators (PAMs) have been developed and show promise as therapeutic agents. We evaluated the therapeutic potential of a novel mGluR5 PAM, VU0360172, after controlled cortical impact (CCI) in mice. Vehicle, VU0360172, or VU0360172 plus mGluR5 antagonist (MTEP), were administered systemically to CCI mice at 3 h post-injury; lesion volume, hippocampal neurodegeneration, microglial activation, and functional recovery were assessed through 28 days post-injury. Anti-inflammatory effects of VU0360172 were also examined in vitro using BV2 and primary microglia. VU0360172 treatment significantly reduced the lesion, attenuated hippocampal neurodegeneration, and improved motor function recovery after CCI. Effects were mediated by mGluR5 as co-administration of MTEP blocked the protective effects of VU0360172. VU0360172 significantly reduced CD68 and NOX2 expression in activated microglia in the cortex at 28 days post-injury, and also suppressed pro-inflammatory signaling pathways in BV2 and primary microglia. In addition, VU0360172 treatment shifted the balance between M1/M2 microglial activation states towards an M2 pro-repair phenotype. This study demonstrates that VU0360172 confers neuroprotection after experimental TBI, and suggests that mGluR5 PAMs may be promising therapeutic agents for head injury.

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The online version of this article (doi:10.1007/s13311-014-0298-6) contains supplementary material, which is available to authorized users.     

Neonatal Fc Receptor Promotes Immune Complex–Mediated Glomerular Disease

The neonatal Fc receptor (FcRn) is a major regulator of IgG and albumin homeostasis systemically and in the kidneys. We investigated the role of FcRn in the development of immune complex–mediated glomerular disease in mice. C57Bl/6 mice immunized with the noncollagenous domain of the α3 chain of type IV collagen (α3NC1) developed albuminuria associated with granular capillary loop deposition of exogenous antigen, mouse IgG, C3 and C5b-9, and podocyte injury. High-resolution imaging showed abundant IgG deposition in the expanded glomerular basement membrane, especially in regions corresponding to subepithelial electron dense deposits. FcRn-null and -humanized mice immunized with α3NC1 developed no albuminuria and had lower levels of serum IgG anti-α3NC1 antibodies and reduced glomerular deposition of IgG, antigen, and complement. Our results show that FcRn promotes the formation of subepithelial immune complexes and subsequent glomerular pathology leading to proteinuria, potentially by maintaining higher serum levels of pathogenic IgG antibodies. Therefore, reducing pathogenic IgG levels by pharmacologic inhibition of FcRn may provide a novel approach for the treatment of immune complex–mediated glomerular diseases. As proof of concept, we showed that a peptide inhibiting the interaction between human FcRn and human IgG accelerated the degradation of human IgG anti-α3NC1 autoantibodies injected into FCRN-humanized mice as effectively as genetic ablation of FcRn, thus preventing the glomerular deposition of immune complexes containing human IgG.The MHC class I–like neonatal Fc receptor (FcRn), a heterodimer comprising a heavy chain and β₂-microglobulin light chain, is the major regulator of IgG and albumin homeostasis.¹ Perinatally, FcRn mediates the transfer of IgG from mother to offspring, across the placenta in primates and trans-intestinally in suckling rodents. Throughout life, FcRn protects IgG and albumin from catabolism, explaining the unusually long t_1/2 and high serum levels of these proteins. IgG and albumin taken up by cells by pinocytosis bind strongly to FcRn at pH 6.0–6.5 in endosomes. FcRn-bound ligands are then recycled to the plasma membrane, where they dissociate at pH 7.4, whereas IgG and albumin not bound to FcRn are targeted to lysosomes for degradation. FcRn is thought to promote some autoimmune diseases because it protects pathogenic IgG from degradation. For instance, Fcrn^−/− mice are resistant to passive transfer of arthritis by K/BxN sera and autoimmune skin pathology induced by antibodies targeting autoantigens at the dermal–epidermal junction, although this protection can be overcome by excess autoantibodies.^2–4In kidneys, FcRn is expressed in podocytes and proximal tubular epithelial cells.⁵ Overall, renal FcRn reclaims albumin but facilitates elimination of IgG.⁶ Tubular FcRn mediates IgG transcytosis.⁷ Podocytes use FcRn to clear IgG from the glomerular basement membrane (GBM).⁸ IgG accumulates in the glomeruli of aged Fcrn^−/− mice due to impaired clearance of IgG from the GBM, and saturating this clearance mechanism by excess ligand potentiates the pathogenicity of nephrotoxic sera in wild-type mice. Podocyte FcRn has been postulated to be involved in the clearance of immune complexes (ICs) present in pathologic conditions such as membranous nephropathy.⁵ Expression of FcRn in human podocytes is increased in various immune-mediated glomerular diseases.⁹ Given its role in IgG and albumin handling in the kidneys and systemically, FcRn can be expected to influence the development of immune-mediated kidney diseases at multiple levels. This conjecture awaits experimental verification.To determine the role of FcRn in IgG-mediated glomerular disease, we asked how FcRn deficiency alters the course of disease in mice immunized with the NC1 domain of α3 type IV collagen (α3NC1). We chose this antigen because of its reported ability to induce disease in C57Bl/6 (B6) mice,¹⁰ corroborated in pilot studies (Supplemental Figure 1). Fcrn^−/− mice are hypoalbuminemic due to impaired albumin recycling,¹¹ and also exhibit reduced urinary albumin excretion.¹² As a control for this potential confounder, we used FCRN-humanized mice, which have normal serum albumin because human FcRn recycles mouse albumin but not mouse IgG.¹³All mice immunized with α3NC1 developed circulating mouse IgG anti-α3NC1 antibodies, which reached the maximum titer about 6 weeks later and gradually declined thereafter. At all times, the levels of mouse IgG anti-α3NC1 antibodies in sera from Fcrn^−/− mice and FCRN-humanized mice were approximately 50%–70% lower than those in wild-type mouse sera (Figure 1A). The results were similar for mouse IgG1, IgG2b, and IgG2c anti-α3NC1 antibodies (Supplemental Figure 2). Wild-type B6 mice immunized with α3NC1 started developing progressive albuminuria 8–10 weeks later (Figure 1B). By week 14, the urinary albumin creatinine ratio increased approximately 100-fold, and hypoalbuminemia developed (Figure 1C). Urinary albumin excretion in Fcrn^−/− mice and FCRN-humanized mice immunized with α3NC1 was not significantly higher than in adjuvant-immunized control mice. No mice developed renal failure (Supplemental Figure 3).Open in a separate windowFigure 1.FcRn ablation reduces serum levels of mouse IgG anti-α3NC1 antibodies and prevents the development of albuminuria in α3NC1-immunized mice. (A) The left panel shows circulating mIgG anti-α3NC1 antibodies from C57Bl6 wild-type mice (○), Fcrn^−/− mice (□), FCRN-humanized (hFCRN) mice (◇), and the control CFA group (△), which are assayed by indirect ELISA in plates coated with α3NC1 (100 ng/well). Mouse sera are diluted 1:5000. The right panel shows the significance of circulating mIgG anti-α3NC1 antibody differences among groups at week 12, as assessed by one-way ANOVA followed by Bonferroni post tests for pairwise comparisons. (B) The left panel shows that the urinary albumin creatinine ratio (mean±SEM) time course is monitored in C57Bl6 wild-type mice (○), Fcrn^−/− mice (□), and hFCRN mice (◇) immunized with α3NC1 (n=5–8 mice in each group, from two separate experiments). Mice in the control group (△) are immunized with adjuvant alone (n=9). The right panel shows the urinary albumin creatinine ratio (mean±SEM) at 14 weeks, when mice are euthanized. The significance of differences among groups is assessed by one-way ANOVA followed by Bonferroni post tests for pairwise comparisons. (C) The left panel shows SDS-PAGE analysis of serum (0.5 µl/lane) and urine samples (2 µl/lane) from CFA-immunized control mice (a) and α3NC1-immunized wild-type mice (b), Fcrn^−/− mice (c), and hFCRN mice (d) collected at week 14. The right panel presents a densitometric analysis of the relative levels of albumin in mouse serum samples showing that α3NC1-immunized wild-type mice developed hypoalbuminemia. *P<0.05 by two-tailed t test versus CFA-immunized wild-type mice; **P<0.01; ***P<0.001. ns, not significant; WT, wild type.At 14 weeks after α3NC1 immunization, kidneys examined by light microscopy showed mild glomerular pathology, with few crescents and relatively little inflammation (Figure 2A), similar to α3NC1-immunized DBA/1 mice with comparable albuminuria.^14,15 Electron microscopy showed extensive subepithelial IC deposits surrounded by an expanded GBM and effacement of podocyte foot processes in α3NC1-immunized B6 mice, whereas Fcrn^−/− mice had fewer subepithelial deposits (Figure 2B, Supplemental Figure 4). Immunofluorescence staining showed granular capillary loop deposition of mouse IgG, exogenous antigen, C3, and C5b-9, more intense in wild-type mice than in Fcrn^−/− mice and FCRN-humanized mice (Figure 2, Ca–Cp, Supplemental Figure 5). A loss of nephrin staining, indicative of podocyte injury, occurred in α3NC1-immunized B6 mice but not in Fcrn^−/− mice or FCRN-humanized mice (Figure 2, Cq–Ct).Open in a separate windowFigure 2.FcRn deficiency reduces formation of pathogenic subepithelial ICs. (A) Light microscopic evaluation of kidneys from adjuvant-immunized control mice (a) and α3NC1-immunized wild-type mice (b) and Fcrn^−/− mice (c) revealed few pathogenic changes and the absence of glomerular inflammation (periodic acid–Schiff staining). (B) Transmission electron microscopy shows normal GBM (arrow) and podocyte foot processes in control mice (a), extensive subepithelial electron dense deposits (arrowhead), thickened GBM, and podocyte foot process effacement in α3NC1-immunized wild-type mice (b), and fewer IC deposits in the Fcrn^−/− mice (c). (C) Immunofluorescence analysis of kidneys from adjuvant-immunized control mice (a, e, i, m, and q) and α3NC1-immunized wild-type mice (b, f, j, n, and r), FcRn^−/− mice (c, g, k, o, and s), and hFCRN mice (d, h, l, p, and t) evaluate the deposition of mouse IgG (a–d), exogenous α3NC1 antigen stained by mAb RH34 (e–h), mouse C3c (i–l), C5b-9 (m–p), and nephrin staining (q–t) at 14 weeks. Wild-type mice exhibit linear-granular GBM deposition of mouse IgG and granular GBM deposition of exogenous antigen, C3, and C5b-9, which are attenuated in Fcrn^−/− mice and hFCRN mice and essentially absent in control mice. Compared with control mice, α3NC1-immunized wild-type mice but not Fcrn^−/− or hFCRN mice exhibit a loss of nephrin staining, indicative of podocyte injury. WT, wild type; EM, electron microscopy, PAS, periodic acid–Schiff. Original magnification, ×400 in A; ×2850 in B; ×200 in C.Because B6 mice immunized with bovine GBM NC1 hexamers have normal kidney function and histology despite linear GBM deposition of IgG autoantibodies binding to mouse α345(IV) collagen (Supplemental Figure 1), the question arises as to what causes proteinuria in α3NC1-immunized mice. Because the clinical presentation, morphology, and effector mechanisms depend on where ICs are localized in the capillary wall, we compared IgG distribution in α3NC1-immunized mice and mice injected with anti-α3NC1 antibodies modeling anti-GBM autoantibodies. The distribution and relative abundance of mouse IgG, as imaged by immunoperoxidase immunoelectron microscopy and stochastic optical reconstruction microscopy (STORM), a method for super-resolution fluorescence microscopy, were concordant. In α3NC1-immunized mice, IgG deposition was abundant in the areas of expanded GBM and especially in regions corresponding to the subepithelial dense deposits seen by routine electron microscopy. By contrast, in mice injected with α3NC1-specific anti-GBM mAb, the IgG was confined to an ultrastructurally normal GBM that lacked subepithelial deposits (Figure 3).Open in a separate windowFigure 3.Localization of IgG by high-resolution imaging. The localization of mouse IgG in glomerular capillary walls of wild-type mice immunized with α3NC1 (A, C–E), or intravenously injected with anti-mouse α3NC1 IgG mAb 8D1 (B, F–H) is determined by immunoperoxidase electron microscopy (A and B) and STORM imaging (C–H). In A, the GBM is irregularly thickened, and abundant electron dense peroxidase reaction product is present in discontinuous, subepithelial patterns beneath broadly effaced podocyte foot processes (arrows). In B, the peroxidase reaction product is diffusely present throughout the GBM (arrowhead), but less abundant compared with A. Electron dense deposits are absent, and podocyte foot process architecture appears normal. (C–E) By STORM imaging, anti-agrin (blue) identifies both normal and thickened areas of the GBM, both of which contain dense accumulations of mouse IgG throughout (red). The electron microscopy correlation in E shows GBM staining with respect to the podocytes and endothelial cells. (F–H) IgG mAb 8D1 (red) is present in the GBM, which shows no evidence of thickening. CL, capillary lumen; EM, electron microscopy En, endothelium;Po, podocyte.Subepithelial ICs, a hallmark of human membranous nephropathy (MN), form when IgG antibodies bind to podocyte antigens, such as phospholipase A2 receptor (PLA2R) and neutral endopeptidase (NEP), or to planted antigens, such as cationic BSA.^16–18 Subsequent expansion of the GBM, complement activation, and podocyte injury by C5b-9 cause proteinuria. Although it is unexpected, formation of subepithelial ICs in α3NC1-immunized mice may be explained by exogenous α3NC1 deposited in glomeruli acting as a planted antigen.¹⁹ Alternatively, anti-α3NC1 antibodies in complex with α3NC1 antigen may act as surrogate antipodocyte antibodies, because α3NC1-containing ICs bind to podocytes.²⁰ After four immunizations with α3NC1 monomers, B6 mice and DBA/1 mice eventually develop crescentic GN by 26 and 10 weeks, respectively.^10,14 The combination of subepithelial ICs and crescentic anti-GBM antibody GN was most recently described in a series of eight patients with circulating anti-α3NC1 autoantibodies but undetectable anti-PLA2R autoantibodies.²¹In contrast to wild-type B6 mice, congenic Fcrn^−/− mice and FCRN-humanized mice did not develop albuminuria after α3NC1 immunization. Their resistance to proteinuria was associated with lower serum titers of anti-α3NC1 IgG antibodies and reduced glomerular deposition of IgG, antigen, C3, and C5b-9. Because C5b-9 is an essential mediator of podocyte damage and proteinuria by subepithelial ICs,^22,23 reduced complement activation potentially explains the attenuated glomerular pathology in FcRn-deficient mice. The resistance of FCRN-humanized mice indicates that FcRn promotes IC-mediated glomerular disease due to its interaction with IgG rather than albumin. We propose that FcRn promotes the development of subepithelial ICs and subsequent glomerular injury primarily by maintaining higher serum levels of pathogenic IgG (Supplemental Figure 6). However, we cannot formally exclude a possible pathogenic role of podocyte FcRn, whose stimulation by ICs may induce maladaptive signaling.⁹ Future studies in mice with podocyte-specific ablation of FcRn would address this possibility.Our findings identify FcRn as a potential target for therapeutic intervention in IC-mediated glomerular diseases, typically treated with nonspecific immunosuppressants that are toxic and sometimes ineffective. More specific therapies include ablation of B cells by rituximab. In patients with idiopathic MN who respond to rituximab therapy, serum levels of anti-PLA2R IgG autoantibodies decline over a period of many months, and their disappearance is followed by resolution of proteinuria.²⁴ The slow decline in proteinuria is problematic for patients already suffering from complications of nephrotic syndrome, who would benefit from ancillary therapies that reduce pathogenic IgG antibodies more rapidly. This may be achieved by inhibiting FcRn.One implementation of this concept is therapy with high-dose intravenous Ig (HD-IVIG). HD-IVIG accelerates the degradation of IgG by saturating FcRn,²⁵ one of the mechanisms that explain the beneficial effects of HD-IVIG therapy in some autoimmune diseases.³ In pregnant women with circulating anti-NEP alloantibodies mediating antenatal MN, treatment with HD-IVIG reduces the titers of IgG alloantibodies by approximately 30% within 2–3 weeks.²⁶ However, HD-IVIG is inefficient, because large amounts of IgG (1–2 g/kg) cause relatively modest reductions in pathogenic IgG titers. Specific FcRn inhibitors recapitulate this activity of HD-IVIG more effectively at lower doses. By reducing pathogenic IgG levels, function-blocking anti-FcRn mAbs ameliorate experimental myasthenia gravis in rats,²⁷ and engineered IgG “Abdegs” that bind with high affinity to FcRn ameliorate arthritis transferred by K/BxN serum.²⁸To assess the translational potential of our findings, we asked whether pharmacologic blockade of human FcRn can reproduce the effects of genetic FcRn deficiency. To this end, FCRN-humanized and Fcrn^−/− mice were passively immunized with human IgG containing anti-α3NC1 (Goodpasture) autoantibodies. To inhibit human FcRn, we used a lysine analog of SYN1436 (Figure 4A),²⁹ a peptide that binds with subnanomolar affinity to human FcRn, thus preventing IgG binding.³⁰ In vivo, SYN1436 reduces IgG levels in cynomolgus monkeys by 80%.³⁰ Serum anti-α3NC1 autoantibodies in FCRN-humanized mice treated with anti-FcRn peptide, but not with control peptide, sharply decreased to the same levels as in Fcrn^−/− mice (Figure 4B), and were no longer detected after 4 days. In mice, human IgG elicits murine anti-human IgG antibodies, forming ICs that can deposit in glomeruli, as shown in active serum sickness models. Glomerular deposition of ICs containing human IgG was abolished in mice treated with anti-FcRn peptide, but not with control peptide (Figure 4C). Linear GBM deposition of human anti-GBM IgG was not observed, because the epitopes recognized by Goodpasture autoantibodies are completely inaccessible in the mouse GBM.³¹ These results provide proof of concept that therapies targeting human FcRn effectively lower serum levels of pathogenic human IgG autoantibodies, which could be beneficial in patients with IgG-mediated kidney diseases. Because FcRn also mediates the trans-placental transfer of IgG from mother to the fetus, FcRn inhibition may be particularly attractive for preventing antenatal MN caused by maternal anti-NEP alloantibodies.Open in a separate windowFigure 4.Pharmacologic blockade of human FcRn accelerates the catabolism of human IgG autoantibodies in FCRN-humanized mice. (A) Structure of a peptide that binds with high affinity to human FcRn, competitively inhibiting its interaction with human IgG (top). The control peptide (bottom) containing D-amino acids does not bind to human FcRn. Pen, Sar, and NMeLeu denote penicillamine, sarcosine, and N-methyl-leucine, respectively. (B) Serum level of human IgG anti-α3NC1 antibodies in FCRN-humanized mice treated with anti-FcRn peptide (▪) or control peptide (●) and in Fcrn^−/− (▲) mice sera (n=3 in each group) is analyzed by indirect ELISA in plates coated with α3NC1 (100 ng/well). Mouse sera are diluted 1:500. (C) Kidney deposition of human IgG (a and b) and mouse IgG (c and d) in FCRN-humanized mice treated with control peptide (a and c) or anti-FcRn peptide (b and d) is evaluated by direct immunofluorescence staining. Treatment with anti-FcRn peptide prevents the glomerular deposition of ICs containing human IgG.     

Tamoxifen stimulates calcitonin-producing thyroid C-cells and prevents trabecular bone loss in a rat model of androgen deficiency

Thyroid C-cells produce calcitonin (CT), a hypocalcemic hormone, that acts as an inhibitor of bone resorption. In this study, we investigated the effects of tamoxifen (TAM) as a selective estrogen receptor modulator on thyroid C-cells, trabecular bone and biochemical markers of bone metabolism in an animal model of androgen deficiency, represented by middle-aged orchidectomized (Orx) rats. Fifteen-month-old male Wistar rats were divided into: Orx and sham-operated (SO) groups. Rats from one Orx group were injected subcutaneously with TAM citrate (Orx + TAM; 0.3 mg kg⁻¹ b.w.), while the rats from SO and a second Orx group received vehicle alone, once a day for 3 weeks. The peroxidase–antiperoxidase method was applied for localization of CT in C-cells. Thyroid C-cells were morphometrically and ultrastructurally analyzed. An ImageJ image-processing program was used to measure bone histomorphometric parameters. Blood serum samples were analyzed for CT, osteocalcin (OC), calcium (Ca²⁺) and phosphorus (P). Urinary Ca²⁺ concentrations were measured. TAM treatment significantly increased thyroid C-cell volume (V_c) and serum CT when compared with vehicle-treated Orx rats. Analysis of trabecular microarchitecture of the tibia showed that administration of TAM significantly increased cancellous bone area, trabecular thickness and trabecular number, whereas trabecular separation was significantly decreased compared with vehicle-treated Orx rats. Serum OC and urinary Ca²⁺ concentrations were significantly lower in comparison with the control Orx group. These results indicate that in our rat model of androgen deficiency, TAM stimulated calcitonin-producing thyroid C-cells and increased trabecular bone mass.