Analogues of ERβ ligand chloroindazole exert immunomodulatory and remyelinating effects in a mouse model of multiple sclerosis

Pharmaceutical agents currently approved for the treatment of multiple sclerosis reduce relapse rates, but do not reverse or prevent neurodegeneration nor initiate myelin repair. The highly selective estrogen receptor (ER) β ligand chloroindazole (IndCl) shows particular promise promoting both remyelination while reducing inflammatory cytokines in the central nervous system of mice with experimental autoimmune encephalomyelitis. To optimize these benefits, we developed and screened seven novel IndCl analogues for their efficacy in promoting primary oligodendrocyte (OL) progenitor cell survival, proliferation, and differentiation in vitro by immunohistochemistry. Two analogues, IndCl-o-chloro and IndCl-o-methyl, induced proliferation and differentiation equivalent to IndCl and were selected for subsequent in vivo evaluation for their impact on clinical disease course, white matter pathology, and inflammation. Both compounds ameliorated disease severity, increased mature OLs, and improved overall myelination in the corpus callosum and white matter tracts of the spinal cord. These effects were accompanied by reduced production of the OL toxic molecules interferon-γ and chemokine (C-X-C motif) ligand, CXCL10 by splenocytes with no discernable effect on central nervous system-infiltrating leukocyte numbers, while IndCl-o-methyl also reduced peripheral interleukin (IL)−17. In addition, expression of the chemokine CXCL1, which is associated with developmental oligodendrogenesis, was upregulated by IndCl and both analogues. Furthermore, callosal compound action potential recordings from analogue-treated mice demonstrated a larger N1 component amplitude compared to vehicle, suggesting more functionally myelinated fibers. Thus, the o-Methyl and o-Chloro IndCl analogues represent a class of ERβ ligands that offer significant remyelination and neuroprotection as well as modulation of the immune system; hence, they appear appropriate to consider further for therapeutic development in multiple sclerosis and other demyelinating diseases.


2-(2-chloro-4-methoxyphenyl)-2H-indazol-5-ol
To the suspension of 2-chloro-4-methoxyaniline (628 mg, 4.00 mmol) in cold DI water (10 mL) was added c-HCl (1.2 mL) and subsequently sodium nitrite (284 mg, 4.11 mmol) added portionwise a in ice bath by maintaining the temperature below 5 o C. After 1.5 hr stirring of the reaction mixture at below 5 o C, 3-hydroxymethylphenol (496 mg, 4.00 mmol) in a water-acetone (1:1, v/v, 5 ml) mixture was added into the reaction mixture in ice bath, followed by adjusting the pH up to 7.5 with 1N NaOH to precipitate a yellowish solid. During the addition of the NaOH aq.
solution, a massive yellowish solid precipitated from solution as the pH reached ~7.5.
To form the Indazole ring: Method 1) the solid was collected by filtration, washed with DI water, and dried in an oven at 60 ~80 o C (1.1 g yellowish solid). (It is not necessary to adjust pH acidic in this case to collect a solid, but in some case the pH of the solution needs to be acidifies to collect more solid.). The collected solid used without further purification. Hz, 1H), 7.77 (d, J = 8.5 Hz, 1H). 13

Method 2)
The diazo compound in aqueous solution was extracted with ethyl acetate (50 mL x 3).
To the extract was added 1 mL of c-HCl, and the resulting solution was refluxed until the yellowish diazo compound disappeared on silica gel TLC (20 % ethyl acetate in n-hexane, v/v, Rf ~0.8) and new blue fluorescent spot (Rf ~0.65) appeared. The reaction mixture was washed with sat. aq.
sodium bicarbonate solution, brine, water, dried over sodium sulfate, followed by filtration and evaporation to afford an enough pure title compound (680 mg) as a pale brownish solid.

Primary OPC Cultures
Primary OPCs, isolated from postnatal day P1 C57BL6 male and female mouse cortices as described earlier 3 . Primary OPC were cultured into 8-well chamber slides (three wells per condition, 2.5x10 5 cells/well) for three days to attach and five days in differentiating media Software (La Jolla, CA). One-way ANOVA with Tukey's posthoc test for multiple comparisons was used to generate p-values, and data are presented as mean ± SEM (with α ≤0.05).

Primary Astrocyte Cultures and OPC/OL Astrocyte Conditioned Media Treatment
Astrocyte cultures were prepared by chemical dissociation of the cerebral cortex from p0-p4

Experimental Autoimmune Encephalomyelitis (EAE)
Active EAE was induced in eight-week-old female C57BL/6 and Thy1-YFP mice as previously

Spleen Isolation and Cytokine Analysis
On day 20-21 after induction of EAE, spleens were harvested prior to transcardial perfusion.

Rotarod behavioral assay
Motor behavior was tested up to two times per week for each mouse using a rotarod apparatus (Med Associates, Inc., St. Albans, VT). Briefly, animals were placed on a rotating horizontal cylinder for a maximum of 200 seconds. The amount of time the mouse remained walking on the cylinder without falling was recorded. Each mouse was tested on a speed of 3-30 rpm and given three trials for any given day. The three trials were averaged to report a single value for an individual mouse, and averages were then calculated for all animals within a given treatment group

Histological Preparation of Tissues
Mice were deeply anesthetized by isoflurane (Piramal Healthcare) inhalation and perfused transcardially with 1x PBS followed by 10% formalin (Thermo Fisher Scientific) to fix tissues.
Brains and spinal cords were dissected and post-fixed in 10% formalin (Thermo Fisher Scientific) for 24 hours, then cryoprotected in 30% sucrose (EMD Millipore, Darmstadt, Germany) for 48 hours and embedded in gelatin for sectioning. Embedded brains and spinal cords were then cut into 40-m coronal sections using an HM525 NX cryostat (Thermo Fisher Scientific). Sections were collected serially and stored in PBS with 1% sodium azide at 4°C until staining by immunohistochemistry, following a previously described protocol 9,10 .

Immunohistochemistry
Before histological staining, 40-m free-floating sections were thoroughly washed with PBS to remove residual sodium azide 10

Quantification and Microscopy
Thoracic spinal cord dorsal and ventral column sections, as well as CC were imaged using an Olympus BX61 confocal microscope (Olympus America Inc., Center Valley, PA) using a 10x and 40x objective. Z-stack projections were compiled using SlideBook 6 software (Intelligent Imaging Innovations, Inc., Denver, CO). Immunostaining was quantified using unbiased stereology 10 . All images (RGB) were converted to grayscale, split, and separated by color channel using imageJ version 2.2.0-rc-46/1.50g (NIH). To avoid experimenter bias, auto-adjustment of brightness and contrast, as well as threshold of staining signal, was carried out by ImageJ. MBP + , GFAP + , CD45 + , and CXCL1 + , staining intensity was measured as percent area of positive immunoreactivity within the region of interest and intensity of signal determined by ImageJ. CC1 + cell numbers were automatically counted within a region of interest using ImageJ.

Electrophysiology
To assess functional conductivity across the corpus callosum (CC), electrophysiological recordings of compound action potentials (CAPs) were measured as previously described 10,11 . placed approximately 1 mm away across from a recoding electrode (glass micropipette filled with ACSF) with a resistance of 1-3 MΩ. To elicit CAPs, an episodic stimulation protocol was created consisting of 8 consecutive sweeps, each 12 ms long, with a 5-sec delay between sweeps and an immediate stimulus (0.01 ms duration) after the start of each sweep 14 . Stimulus intensity was adjusted manually using an ISO-Flex stimulator (A.M.P.I). Standardized input-output plots were generated in current clamp mode for each slice by averaging at least 4 consecutive sweeps together to reduce the signal-to-noise ratio. Brain slices that exhibited near zero voltage even when stimulated with the maximal current were not included in the analysis.

Statistical Analysis
All statistics were performed using Prism 6 software (GraphPad Software, La Jolla, CA).
Differences in EAE clinical scores were determined by two-way unbalanced ANOVA with Dunnett's multiple comparisons test 6 . Luminex data and immunohistochemistry data were GraphPad Prism 6 (GraphPad Software). The averaged mean amplitude was compared using oneway ANOVA with post hoc tests using Tukey's multiple comparison test. All data are presented as mean ± SEM for two independent experiments. Differences were considered significant at * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001, **** p ≤ 0.0001.