Significance In the search for effective multiple sclerosis treatment, much effort has been invested in estrogens and estrogen receptor (ER) agonists because of their neuroprotective benefits. However, because estrogens can produce ERα-based feminizing effects and cancer, ERβ agonists represent more desirable therapeutic candidates. The structurally unique ERβ ligand indazole chloride (Ind-Cl), a halogen-substituted phenyl-2H-indazole core, is a preclinical development candidate with a strong dossier. Our results indicate that Ind-Cl is effective in functionally ameliorating disease even when treatment is initiated at peak experimental autoimmune encephalomyelitis clinical disease. Ind-Cl’s immunomodulatory and direct remyelinating effects result in motor dysfunction amelioration. These findings support Ind-Cl's potential to provide unique therapeutic benefits to patients with multiple sclerosis, as well as patients affected by other demyelinating disorders.

Abstract Currently available immunomodulatory therapies do not stop the pathogenesis underlying multiple sclerosis (MS) and are only partially effective in preventing the onset of permanent disability in patients with MS. Identifying a drug that stimulates endogenous remyelination and/or minimizes axonal degeneration would reduce the rate and degree of disease progression. Here, the effects of the highly selective estrogen receptor (ER) β agonist indazole chloride (Ind-Cl) on functional remyelination in chronic experimental autoimmune encephalomyelitis (EAE) mice were investigated by assessing pathologic, functional, and behavioral consequences of both prophylactic and therapeutic (peak EAE) treatment with Ind-Cl. Peripheral cytokines from autoantigen-stimulated splenocytes were measured, and central nervous system infiltration by immune cells, axon health, and myelination were assessed by immunohistochemistry and electron microscopy. Therapeutic Ind-Cl improved clinical disease and rotorod performance and also decreased peripheral Th1 cytokines and reactive astrocytes, activated microglia, and T cells in brains of EAE mice. Increased callosal myelination and mature oligodendrocytes correlated with improved callosal conduction and refractoriness. Therapeutic Ind-Cl-induced remyelination was independent of its effects on the immune system, as Ind-Cl increased remyelination within the cuprizone diet-induced demyelinating model. We conclude that Ind-Cl is a refined pharmacologic agent capable of stimulating functionally relevant endogenous myelination, with important implications for progressive MS treatment.

Multiple sclerosis (MS) is an autoimmune, demyelinating, and neurodegenerative disease of the central nervous system (CNS) that affects 2–2.5 million people worldwide. Currently approved MS drugs reduce relapse rates but fail to reverse or prevent neurodegeneration and disability progression. Disease-modifying drugs capable of restoring neuronal function via axon remyelination (RM) represent a major unmet goal for MS therapeutics.

Oligodendrocyte (OL) progenitor cells (OPCs) are responsible for remyelinating axons, make up at least 3% of all white matter cells, and are present in and around MS lesions; however, they remain largely quiescent in the adult CNS (1). Although endogenous RM can occur in patients with MS, as evidenced by shadow plaques, it is short-lived, incomplete, and relatively ineffective (2). Transition to progressive MS is characterized by increased axon loss, which correlates with RM failure (3). Hence, a treatment that stimulates endogenous OPCs to differentiate and remyelinate axons would reduce axon degeneration and restore neuronal function.

Experimental autoimmune encephalomyelitis (EAE) affords researchers an in-depth, mechanistic understanding of immune-mediated, demyelinating neurodegeneration and anti-inflammatory effects of currently approved MS drugs. Our recent work has demonstrated promising neuroprotective effects of the estrogen receptor (ER) β agonist 2,3-bis(4-hydroxyphenyl)propionitrile (DPN) (4, 5). Although DPN, acting through ERβ, has a desirable palliative effect in EAE, it possesses only 70-fold binding selectivity for ERβ over ERα and lacks anti-inflammatory effects (6, 7). A more selective ERβ agonist capable of immunomodulation would be more efficacious in treating inflammatory demyelinating neurodegeneration.

The structurally unique ERβ ligand indazole chloride (Ind-Cl), based on a halogen-substituted phenyl-2H-indazole core, is a preclinical development candidate with a strong dossier, including in vitro pharmacology using rodent and human cells, selectivity and potency data, promising absorption-distribution-metabolism-excretion findings, and pharmacokinetic profiling that includes confirmation of brain penetrability (mouse brain/plasma: ∼1.0) (7, 8). It is a highly ERβ-selective (>100-fold) small molecule agonist that is administered s.c. and can be developed for oral administration (7).

Here, we explored pathologic, functional, and behavioral consequences of prophylactic and therapeutic (after onset of peak EAE) Ind-Cl in chronic EAE mice. Importantly, our recent finding of Ind-Cl-induced RM was confirmed, using the chronic cuprizone (CPZ)-induced demyelinating model (9), supporting Ind-Cl’s remyelinating capabilities independent of its effects on primary inflammation. Our results demonstrate that prophylactic and therapeutic Ind-Cl have significant beneficial effects in a murine model of progressive MS. Specifically, Ind-Cl attenuates clinical disease, and its functional immunomodulatory, remyelinating, and neuroprotective effects manifest in axon conduction and myelination improvements. Importantly, these effects correlate with improved motor function. Thus, Ind-Cl could impart much-needed, unique therapeutic benefits in progressive MS and other demyelinating disorders.

Discussion In MS, demyelinated areas containing damaged axons are associated with inflammatory reactions orchestrated by activated T cells, macrophages, and endogenous glia, which produce proinflammatory and neurotoxic factors and attenuate repair/RM of damaged/demyelinated axons (22). This manifests as clinical deficits (23, 24). Thus, these cell types are immunomodulation targets in MS. Currently approved immunomodulators are only modestly effective in reducing relapses by slowing disability accumulation; these treatments fail to stop axon loss and/or stimulate RM. OL and myelin rescue and sustenance, with immunomodulation, are of high priority for effective MS therapy development. In the search for effective MS treatments, much has been invested in estrogens and ER agonists because of their neuroprotective benefits (5, 10, 25, 26). Different ERβ ligand analogs have distinct effects on gene transcription in signaling pathways for chromosome replication, cell death, and OPC differentiation (27). The therapeutic potential of ERβ-selective compounds is particularly favorable because beneficial effects of ERβ activation are independent of undesired proliferative effects on breast and uterine tissue, which are principally ERα-mediated (28). Certain haloindazoles, synthetic ERβ-specific ligands based on a halogen-substituted phenyl-2H-indazole core (8), potently inhibit transcriptional activation of inflammatory response genes in microglia and astrocytes (7). Our study demonstrates that the haloindazole Ind-Cl ameliorates chronic EAE even when treatment is initiated at peak clinical disease. We analyzed callosal white matter integrity in addition to spinal cord, as MS CC reflects demyelinating lesions, diffuse tissue damage, and neural connectivity abnormalities (29, 30). Specifically, Ind-Cl inhibits ongoing demyelination and axon damage in EAE, leading to substantial recovery of axon conduction, a functional indicator of axon myelination and neuroprotection. Ind-Cl increased BDNF, decreased cell death markers, and activated the PI3K/Akt/mTOR signaling pathway required for OPC proliferation and OL differentiation. Furthermore, therapeutic Ind-Cl reversed ongoing motor deficit. In contrast to DPN’s effects, the present study confirms a reduction of reactive astrocytes by Ind-Cl. Reactive astrocytes respond to and magnify ongoing inflammatory response. We report that both EAE-induced peripheral immune response and CNS immune cell increases are reduced with prophylactic Ind-Cl, which is further evidence of this drug’s promising immunomodulatory properties. ER β is present in various cell types within the peripheral immune system and CNS, including neurons, astrocytes, microglia, OLs, and immune cells (31). Using conditional gene knockout mice, we have shown that the functional beneficial effects of the less-selective ERβ agonist DPN in EAE mice are largely attributable to its action on ERβ in OL lineage cells (6). Further, increased BDNF expression in DPN-administered mice lacking ERβ in OLs is not sufficient to reduce clinical disease or demyelination or to increase the PI3K/AKT/mTOR pathway activation, although it may explain partial improvement of axonal loss and conduction (5, 6). It would follow that the functional benefits of therapeutic Ind-Cl, a more selective ERβ agonist, are at least partly attributable to the drug’s actions on ERβ in OL lineage cells. However, unlike DPN, Ind-Cl exhibits immunomodulatory capabilities in both the peripheral immune system and CNS (7). Ind-Cl may concurrently act on ERβ in multiple cell types. An effect of Ind-Cl on peripheral cells does not exclude a direct effect on the CNS. Time of treatment initiation may contribute to predominant Ind-Cl mechanism of action in EAE. For example, immunomodulatory capabilities may yield indirect neuroprotection (i.e., prevention of neurodegeneration caused by immunomodulatory response) if treatment is initiated early in disease, whereas treatment initiation late in disease may rely on the direct neuroprotective (i.e., restoration of neuronal components, including myelinating OLs, and function) capabilities of Ind-Cl, which we have demonstrated here in the CPZ diet-induced demyelination, a model with an intact blood–brain barrier and no primary inflammatory response. The difference in immunomodulatory plus remyelinating properties of Ind-Cl and the solely remyelinating properties of DPN raises questions about ERβ binding affinity, selectivity, gene regulation, and mechanisms of action of various ERβ ligands. Thus, it is not surprising that structurally related ERβ ligands, even ones having similar ERβ versus ERα binding affinity, selectivity, and efficacy in standard reporter gene assays, have distinct patterns of endogenous gene regulation. In light of such findings, one may understand why the actions of Ind-Cl through ERβ are different (i.e., more favorable) in terms of amelioration and reversal of MS-like symptoms compared with DPN and other ERβ ligands (5, 7, 32). Furthermore, although estradiol is a more potent ligand for ERβ than Ind-Cl, it reverses some of the anti-inflammatory effects of Ind-Cl (7), so its therapeutic efficacy in MS (irrespective of increased risk of breast and uterine cancer) is questionable. Ind-Cl’s distinct immunomodulating and regenerative/remyelinating effects support its potential to provide unique therapeutic benefits to patients with secondary and progressive MS, as well as patients with other demyelinating disorders. Because MS is a multifocal disorder, systemic delivery of Ind-Cl, a brain-penetrable small-molecule compound, is expected to provide greater therapeutic benefit compared with other ER ligands and cell-based therapies.

Materials and Methods Treatment. Ind-Cl [synthesized by J.A.K.’s laboratories (8)] was dissolved in 10% ethanol + 90% (vol/vol) Miglyol 812N (vehicle; Sasol) and administered s.c. daily at 5 mg/kg body weight. Control groups received (s.c.) either 0.04 mg/kg/d 17β-estradiol (E2) or 8 mg/kg/48 h DPN (4, 10). Treatment was initiated at EAE postinduction day 0 (preEAE) or day 21 (post/peakEAE) and continued until day 40. For CPZ experiments, animals received Ind-Cl or vehicle during RM only (n = 10–15 per group; two to three experiments). EAE. Active EAE was induced in 8-wk-old male and female PLP_EGFP C57BL/6 mice (4, 10, 13). All procedures were conducted in accordance with the NIH and approved by the Animal Care and Use Committee at the University of California, Los Angeles,. CPZ. Male PLP_EGFP mice were randomly assigned to one of two groups. The normal myelination group received normal chow. The demyelination group (n = 32) received 0.2% CPZ-milled chow for 9 wk (33). DM animals (n = 10) were killed, and the remaining 24 animals were returned to normal chow (RM), during which they received daily Ind-Cl or vehicle (n = 12 per group). Rotorod. Each mouse was tested for motor function twice a week, using a rotorod (6). Peripheral cytokines. Cytokine levels were assessed from MOG 35–55 -stimulated splenocyte supernatant by Searchlight (Aushon Biosciences) (34). Histopathology and immunohistochemistry. Formalin-fixed CNS sections were examined by immunohistochemistry (10). In parallel, CC was examined using EM (4). Microscopy and quantification. Immunostaining was quantified using unbiased stereology (34). For EM, serial ultrathin sections of Epon-embedded CC stained with uranyl acetate-lead citrate were analyzed (4). Electrophysiology. Coronal brain slices corresponding to plates 40–48 (35) were used for recordings (4, 9). Western blot. Dissected CC (some cortex, no hippocampus) was rapidly frozen and stored at −80 °C. Polyacrylamide gel electrophoresis and immunoblotting were performed (5). Lanes represent individual animals (graphs, n = 4–8 animals per group over the course of two to three experiments). Statistical analysis. Statistical analysis of mean values was carried out using one-way ANOVA or Friedman Test (for clinical scores), where *P < 0.05 was considered significant. Western blot data are presented as mean ± SEM and analyzed by t test for independent samples or two-way ANOVA. MicroCal Origin or Prism 4 (GraphPad Prism Software Inc.) were used.

Acknowledgments We thank Mr. Jonathan Hasselmann for taking care of EAE mice and performing Ind-Cl injections in a blind manner. This work was generously supported by the National Multiple Sclerosis Society Grants NMSS-RG-4853A3/2 and R01-NS081141-0​1A1, and NIH Grants NIH-R21NS075198 (all to S.K.T.-W.) and NIH-R01DK015556 (to J.A.K.).

Footnotes Author contributions: S.K.T.-W. designed research; S.M.M., A.J.K., S.K., Z.W., J.Y., T.Y., L.M.-T., N.Y., J.A.K., and S.K.T.-W. performed research; N.Y. and J.A.K. contributed new reagents/analytic tools; S.M.M., A.J.K., S.K., Z.W., and S.K.T.-W. analyzed data; and S.M.M., A.J.K., and S.K.T.-W. wrote the paper.

The authors declare no conflict of interest.

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