Extra-skeletal manifestations in mice affected by Clcn7-dependent autosomal dominant osteopetrosis type 2 clinical and therapeutic implications

Autosomal dominant osteopetrosis type 2 (ADO2) is a high-density brittle bone disease characterized by bone pain, multiple fractures and skeletal-related events, including nerve compression syndrome and hematological failure. We demonstrated that in mice carrying the heterozygous Clcn7G213R mutation, whose human mutant homolog CLCN7G215R affects patients, the clinical impacts of ADO2 extend beyond the skeleton, affecting several other organs. The hallmark of the extra-skeletal alterations is a consistent perivascular fibrosis, associated with high numbers of macrophages and lymphoid infiltrates. Fragmented clinical information in a small cohort of patients confirms extra-skeletal alterations consistent with a systemic disease, in line with the observation that the CLCN7 gene is expressed in many organs. ADO2 mice also show anxiety and depression and their brains exhibit not only perivascular fibrosis but also β-amyloid accumulation and astrogliosis, suggesting the involvement of the nervous system in the pathogenesis of the ADO2 extra-skeletal alterations. Extra-skeletal organs share a similar cellular pathology, confirmed also in vitro in bone marrow mononuclear cells and osteoclasts, characterized by an impairment of the exit pathway of the Clcn7 protein product, ClC7, through the Golgi, with consequent reduced ClC7 expression in late endosomes and lysosomes, associated with high vesicular pH and accumulation of autophagosome markers. Finally, an experimental siRNA therapy, previously proven to counteract the bone phenotype, also improves the extra-skeletal alterations. These results could have important clinical implications, supporting the notion that a systematic evaluation of ADO2 patients for extra-skeletal symptoms could help improve their diagnosis, clinical management, and therapeutic options.

RevertAid H Minus First Strand cDNA Synthesis Kit. Real time PCR reaction was performed loading 0.1μg of cDNA using the SensiMix TM SYBR ® kit. Gene expression data were represented as fold change over the control and normalized by gapdh, unless otherwise stated. Primers sequences are listed in Supplemental information (Supplemental Table 6).

Histology and histomorphometry
Soft tissues were fixed in 4% paraformaldehyde for at least 48 hours and processed for paraffin or Optimal Cutting Temperature (OCT) embedding. Microtome or cryotome sectioning were used to obtain tissue slices of 5 and 10 µm thickness, respectively. Hematoxylin and Eosin and Masson's trichrome staining were used to evaluate tissue morphology and fibrosis. Histomorphometry was performed using Fiji® software to evaluate tissue fibrosis and cellular infiltrates.

Immunostaining in mouse tissues
For immunohistochemistry, sections were deparaffinized, endogenous peroxidases were quenched with 3% H2O2 and the non-specific binding sites blocked with 5% BSA. Sections were incubated for 1 hour at room temperature and then overnight at 4°C with anti-ClC7 goat or rabbit polyclonal antibody (Santa Cruz Biotechnology). Vectastain Elite ABC HRP Kit was used to develop the immunostaining following the manufacture's instruction and signal was detected by DAB. Section were counterstained with hematoxylin, then dehydrated and mounted.
For immunofluorescence on paraffin-embedded samples, sections were deparaffinized, permeabilized with PBS with Tween 20 (PBS-T) 0.1%, blocked with 5% Bovine Serum Albumin (BSA) and incubated with a single or multiple primary antibodies mixture 1 hour at room temperature and then overnight at 4°C. Secondary incubations were for 1 hour at room temperature with corresponding secondary antibody at dilution 1:1,000.
For immunofluorescence on OCT-embedded samples, cryosections were rehydrated in PBS for 30 minutes and incubated for 2 hours with blocking/permeabilization solution (5% BSA in 0.1% PBS-Triton X-100) at room temperature. Sections were then incubated with a single or multiple primary antibodies mixture for 1 hour at room temperature and then overnight at 4°C. Secondary incubations were for 1 hour at room temperature with corresponding secondary antibody at dilution 1:1,000. Sections were mounted with DAPI antifade mounting medium.
The list of the antibodies and the dilutions used are reported in the Supplemental materials (Supplemental Table 3).

Serological tests
Biomarkers of kidney disease were measured in the sera of 12-month-old CD1, using specific colorimetric assays for calcium and phosphate, and diagnostic Reflotron® strips for urea and uric acid, the following the manufacturer's instructions 2 .

Primary bone marrow mononuclear cell (BMMCs) and osteoclast cultures
For BMMCs isolation, bone marrow was flushed out from the bone cavity of the long bones of 10-day-old mice (C57BL6/J background) and diluted 1:1 in HBBS, layered over Histopaque 1077 solution and centrifuged at 400g for 30 minutes. Cells were washed twice with HBBS, re-suspended in DMEM + 10% FBS and plated in culture dishes. After 12 minutes, the non-adherent cells were harvested and counted. BMMCs were plated in 6 or 24 well-plate dishes, with or without round glass coverslips, to perform protein or RNA isolation and immunofluorescence staining, respectively. Cells were maintained for 7 days in DMEM + 10% FBS, supplemented with 50 ng/ml rhM-CSF.
For osteoclast generation, BMMCs obtained as described in the previous paragraph were plated in 10 cm or 24 well-plate dishes, with or without round glass coverslips, to perform RNA isolation and immunofluorescence staining, respectively. The cells were maintained for 7 days in DMEM + 10% FBS supplemented with 50 ng/ml human recombinant (rh)M-CSF and 120 ng/ml rhRANKL.

Neutral red uptake assay
Lysosomes were isolated from bone marrow flushed out from long bones of 2-month-old WT and ADO2 mice using the lysosome isolation kit (LYSISO1) provided by Sigma Aldrich, according to the manufacturer's instructions. After isolation, lysosomes were exposed to Neutral Red dye (Catalog Number N2537) and the uptake was followed by a spectrophotometer in real time for 10 minutes. The maximum absorbance of the Neutral Red dye shifts from 460 nm to 510 nm in acidic pH conditions. The Neutral Red uptake was calculated using the following formula: Neutral Red uptake= ∆A/min(510) -∆A/min(460)

Behavioral and cognitive tests
For the behavioral tests, male mice of both genotypes (WT and ADO2) were kept in the testing room the day before the tests, at standard laboratory condition. All the tests were done between the 10 a.m. and 4 p.m. and mice were transported in their home cages. The apparatus used for the tests were cleaned with 50% ethanol before and at the end of each session or test. The tests were conducted blindly, and the mouse genotype was checked at the end of the test.
Open Field (OF) test 3 . This test was used to assess anxiety-like behavior and locomotor activity. Mice were put in the center of a 50x50 cm plastic box and their activity was recorded for 30 minutes using a video system. After the test, the videos were analyzed using the video tracking software Kinovea (version 0.8.15) to measure the total walking distance and the time spent in the center and in the periphery of the OF arena by the mice. 3 . This test was used to assess anxiety-like behavior. The EPM apparatus was made of plastic and consisted of four arms, two enclosed and two open, each measuring 30 cm in length and elevated 50 cm from the floor. Each mouse was placed in the center of the maze and allowed to explore the apparatus for 5 minutes. During the test the number of entries and the time spent in the open arms were measured by Stopwatch+ software (Version 1.5.1). The time spent in the center of the apparatus and the latency were recorded as well. 4 . This test was used to assess anxiety-like behavior. It was performed using a 30x60 cm plastic box divided in two compartments: the lit and the dark compartments. Mice were put individually into the dark compartment facing away from the doorway to the light compartment. The number of entries and the time spent in the lit compartment were recorded for 5 minutes using Stopwatch+ software (Version 1.5.1). 3 . This test was used to assess depression. During this test mice were placed individually into glass cylinders containing 10 cm deep water warmed to 25°C. Animals were tested for 6 minutes and the time spent immobile recorded with Stopwatch+ software (Version 1.5.1). Mice were considered immobile when they made no attempts to escape, except for the movements necessary to keep their heads above the water.

Forced Swimming (FS) test
Novel Object Recognition (NOR) test 5 . This test was used to assess the short-term memory. A plastic box was divided into two spaces with same dimension (30x20 cm each). This configuration of the apparatus allows two mice to run simultaneously avoiding the contact during the test. To test the short-term memory of the mice, a black plastic object (object 1) and a clear plastic funnel (object 2) were used. The test was divided into 3 sessions run on three different days. During day 1, mice were placed into the box without the objects for 5 minutes; on day 2 mice were exposed to two similar objects (object 1) for 10 minutes to allow them to familiarize with the object. Finally, on day 3, one of the old objects (object 1) was replaced with the new one (object 2), letting the mice to explore the novel object for 15 minutes. All the sessions were recorded, and the time spent to explore the novel and the old objects was calculated using the Stopwatch+ software (Version 1.5.1). 6 . This test was used to assess spatial memory. The maze had a diameter of 150 cm and contained warmed water (22-23°C) made opaque with non-toxic white paint. The pool was equipped with distal visual cues, including geometric figures attached to the walls, which were used by the mice for the spatial orientation. Mice were assessed across repeated trials (4 trials/day for 10 days). During these trials, a 15 cm round platform was hidden 1 cm beneath the surface of the water at a fixed position.

Morris Water Maze (MWM) test
Each daily trial consisted of four swimming trials starting randomly from each of four starting positions. The duration of each trial was 2 minutes and mice that failed to find the platform within this time were guided to the platform. Mice had to remain on the platform for 15 seconds before they were returned to their cages.
The MWM test were done on 10 subsequent days. The time spent by the mice to find the hidden platform was recorded using Stopwatch+ software (Version 1.5.1).

Validation of antibodies
The antibodies against ClC7, ClC3 and OSTM1, for which there was poor information on specificity, were validated in this study as follows: ClC7 by Western blot in WT and ADO2 cells (Supplemental Figure 4f) and by immunofluorescence in scrambled-siRNA and Clcn7-siRNA treated primary WT BBMCs (Supplemental Figure 4g); ClC3 and OSTM1 antibodies were validated by immunofluorescence in scrambled-siRNA or Clcn3-siRNA and Ostm1-siRNA treated primary WT BBMCs (Supplemental Figure 4g).