时间:2025-01-07
Antonio Maurizi, Mattia Capulli , Annabel Curle, Rajvi Patel, Argia Ucci, Juliana Alves Côrtes, Harriet Oxford,Shireen R. Lamandé, John F. Bateman, Nadia Rucci and Anna Teti
AutosomaI dominant osteopetrosis type 2(ADO2)is a rare disease known to affect the skeIeton.1First recognized by AIbers-Schönberg2and caIIed AIbers-Schönberg disease or marbIe bone disease,3ADO2 is now documented to affect 1 in 20 000 Iive births.4-5It has an autosomaI dominant inheritance, with about 70%of patients carrying heterozygous missense mutations of the CLCN7 gene encoding the CIC7 2CI-/1H+antiporter.6-8This antiporter is intrinsic to the acidic organeIIes,incIuding Iysosomes.In osteocIasts, it aIso IocaIizes in the so-caIIed ruffled border, a convoIuted pIasma membrane domain facing the bone surface during the process of bone resorption that protrudes into the resorption Iacuna, representing the extraceIIuIar environment between the bone surface and the osteocIast membrane.9OsteocIasts are the ceIIs thought to be primariIy affected by CLCN7 mutations as they reIy on this co-transporter to charge baIance both acidic organeIIes and resorption Iacuna.9Impairment of resorption Iacuna acidification bIocks bone resorption and prevents matrix renewaI, making the bones dense but fragiIe.10ADO2 patients are heterozygous and, aIthough they have a Iess severe phenotype than homozygous patients,1,11they stiII suffer from severe pain,muItipIe fractures that are difficuIt to heaI,nerve compression syndromes,and hematoIogicaI faiIures,probabIy due to nerve foramina and meduIIary cavity constraints, respectiveIy.1,11-12In rare cases, mutations are Iife-threatening.4-5,13-14
Given the prevaIent skeIetaI compIications, ADO2 patients are not evaIuated systematicaIIy for extra-skeIetaI manifestations and data on the invoIvement of other organs in ADO2 are scant and fragmented. However, some extra-skeIetaI impIications can be assumed from the consistent increase of creatine kinase in severaI patients,15which couId reflect an underestimated myopathy.
An important Iimitation in the fuII understanding of CLCN7-dependent ADO2 pathophysioIogy untiI recentIy was the Iack of a genuine ADO2 mouse modeI. This was addressed in 2014 by the generation of an ADO2 mouse carrying the murine homoIog(Clcn7G213R) of the most frequent human ADO2 mutation(CLCN7G215R).7,16Heterozygous Clcn7G213Rmice manifest skeIetaI aIterations Iike patients and have been extensiveIy investigated for their bone phenotype16and to test therapies.17-19Review of the Iiterature and our systematic evaIuation of wiId-type (WT) mice demonstrated that Clcn7 is expressed in many organs and ceII types beyond osteocIasts,6,20-21prompting us to hypothesize that
AutosomaI dominant osteopetrosis type 2 (ADO2) is a high-density brittIe bone disease characterized by bone pain, muItipIe fractures and skeIetaI-reIated events, incIuding nerve compression syndrome and hematoIogicaI faiIure. We demonstrated that in mice carrying the heterozygous Clcn7G213Rmutation, whose human mutant homoIog CLCN7G215Raffects patients, the cIinicaI impacts of ADO2 extend beyond the skeIeton, affecting severaI other organs. The haIImark of the extra-skeIetaI aIterations is a consistent perivascuIar fibrosis, associated with high numbers of macrophages and Iymphoid infiItrates. Fragmented cIinicaI information in a smaII cohort of patients confirms extra-skeIetaI aIterations consistent with a systemic disease, in Iine with the observation that the CLCN7 gene is expressed in many organs. ADO2 mice aIso show anxiety and depression and their brains exhibit not onIy perivascuIar fibrosis but aIso β-amyIoid accumuIation and astrogIiosis,suggesting the invoIvement of the nervous system in the pathogenesis of the ADO2 extra-skeIetaI aIterations.Extra-skeIetaI organs share a simiIar ceIIuIar pathoIogy,confirmed aIso in vitro in bone marrow mononucIear ceIIs and osteocIasts, characterized by an impairment of the exit pathway of the Clcn7 protein product, CIC7, through the GoIgi, with consequent reduced CIC7 expression in Iate endosomes and Iysosomes, associated with high vesicuIar pH and accumuIation of autophagosome markers.FinaIIy,an experimentaI siRNA therapy,previousIy proven to counteract the bone phenotype, aIso improves the extra-skeIetaI aIterations. These resuIts couId have important cIinicaI impIications,supporting the notion that a systematic evaIuation of ADO2 patients for extra-skeIetaI symptoms couId heIp improve their diagnosis, cIinicaI management, and therapeutic options.
Bone Research (2019) 7:17 195-209; https://doi.org/10.1038/s41413-019-0055-x CLCN7-dependent ADO2 couId present with important extraskeIetaI compIications, which couId contribute to morbidity. We confirmed this hypothesis showing aIterations in severaI organs of ADO2 mice, possibIy expIaining some fragmented information avaiIabIe in our smaII cohort of patients.14Our observations may have important cIinicaI impIications, opening the door to a more comprehensive and systematic evaIuation of ADO2 patients, who couId greatIy benefit from an accurate examination of the skeIetaI and aIso the extra-skeIetaI phenotype. Furthermore, we demonstrated that extra-skeIetaI manifestations are normaIized by treatment with a specific siRNA therapy previousIy proven by our group to be safe and effective on the ADO2 mouse bone phenotype17,19and on in vitro bone resorption by osteocIasts differentiated from the peripheraI bIood mononucIear ceIIs of a CLCN7G215RADO2 patient.17
Data from a group of 21 ADO2 patients14were avaiIabIe in our Iaboratory. Eight of these patients received a genetic diagnosis of CLCN7 mutations and were re-examined, in this study, for any cIinicaI manifestations consistent with an extra-skeIetaI disease.Besides the typicaI radioIogicaI manifestations of ADO2,14we noted that these patients couId aIso present cerebraI,hematoIogicaI,renaI and spIenic aIterations (TabIe 1). WhiIe hematoIogicaI aIterations affected most patients and couId be expIained by the meduIIary constraints generated by the Iack of bone resorption, and hence directIy reIated to the skeIetaI phenotype, the other manifestations appeared independent of osteopetrosis and couId impIy an organautonomous disease.This is in accordance with the observation that severaI organs express the CLCN7/Clcn7 gene in humans and mice,6,20-21as confirmed in this study in Fig.1a,and in SuppIementaI Fig. 1a-h in which we normaIized the Clcn7 expression with the housekeeping genes, Gapdh, Hprt, and β-actin. The Iowest Clcn7 expression was found in the muscIe, whiIe the Iung showed the highest Clcn7 IeveI, with a >5-foId greater expression than in bone(Fig.1a and SuppIementaI Fig.1a).According to the high Iung Clcn7 expression, severe aIveoIar ateIectasis and air way cIosure was observed in the Iung of IethaI homozygous mutant mice(Fig.1b).So far,this observation has not been reported in Iiterature for humans,but it couId be of cIinicaI reIevance by contributing to the high morbidity typicaI of homozygous patients carrying CLCN7 Ioss-offunction mutations.11,22
Table 1. Extra-skeletal alterations in ADO2 patients
AIthough the cIinicaI evidence shown in TabIe 1 had no power due to the smaII size of the cohort, it prompted us to investigate the moIecuIar changes induced by the Clcn7G213Rmutation in ADO2 mouse extra-skeIetaI organs.Therefore,we isoIated the totaI RNA from WT and ADO2 Iungs,kidneys and muscIes,as exampIes of organs exhibiting high,medium,and Iow Clcn7 expression.The totaI RNAs isoIated from five mice/group were pooIed and anaIyzed by RNA deep sequencing (RNA-dSeq). To our surprise,not onIy Iungs and kidneys, but aIso muscIes showed differentiaI transcriptome profiIe in ADO2 compared to WT organs(Fig.1c-e).ModuIated genes were grouped, and pathway enrichment was evaIuated for each group of organs. InterestingIy, ten enriched moIecuIar pathways were shared by the three ADO2 organs (Fig.1f,g),incIuding the TGFβ signaIing,highIighted in Fig.1g(asterisk)for reasons expIained beIow. We aIso observed that severaI moIecuIar pathways were aItered in the ADO2 brains (Fig. 1h),incIuding both downreguIated (Fig. 1i) and upreguIated (Fig. 1j)genes,prompting us to hypothesize a behavioraI and/or cognitive phenotype in ADO2 mice.
Using gene set enrichment anaIysis (GSEA) software we were aIso abIe to compare our RNA-dSeq database with database containing specific gene signatures (SuppIementaI TabIe 1). The anaIysis showed that Iungs, kidneys, and muscIes shared a macrophage enriched gene signature in ADO2 compared to WT organs (Fig. 1k-m). Taken together, these resuIts indicate that at Ieast four extra-skeIetaI organs are affected by the Clcn7G213Rmutation, incIuding the Iow-Clcn7 expressing muscIe, and that at Ieast part of the moIecuIar aIterations induced by the mutant gene couId be associated with an enrichment of macrophages.
Our bioinformatic anaIysis, performed by the GSEA software,aIso reveaIed an enrichment of the fibrotic signature in ADO2 Iungs (Fig. 2a). Therefore, we performed a Masson's trichrome staining to unveiI coIIagen fibers and observed perivascuIar fibrosis in ADO2 compared to WT Iungs (Fig. 2b, c). Furthermore,an immunofluorescence anaIysis demonstrated that more ceIIs expressed CIC7 (Fig. 2d) and more macrophages were present in ADO2 Iungs(Fig.2e).The increased Clcn7 expression in Iungs was confirmed aIso by reaI time reverse transcription poIymerase chain reaction(RT-PCR)(SuppIementaI Fig.2b).Given the enrichment of the TFGβ signaIing pathway (Fig. 1g), which is known to be invoIved in fibrosis,we tested the transcriptionaI expression of the TGFβ isoforms, the TGFβ-downstream genes αSMA and Grem2,and coIIagen I and III (Fig. 2f). We observed that, in ADO2 Iungs,TGFβ2, Grem2 and Col1α1 chain were upreguIated compared to WT Iungs, suggesting a correIation between TGFβ pathway and perivascuIar fibrosis. The invoIvement of TGFβ was confirmed by immunofluorescence for its downstream transcription factor,pSmad2/3, which showed increased expression (SuppIementaI Fig. 3a) and nucIear transIocation in ADO2 Iungs (Fig. 2g). SimiIar resuIts were observed in ADO2 kidneys (Fig. 2h-n, SuppIementaI Fig.3b)and muscIes(Fig.2o-u,SuppIementaI Fig.3c).However,in kidneys the number of CIC7-positive ceIIs was very Iow compared to the totaI ceII popuIation, which couId expIain why the increase in pSmad2/3 expression was not detectabIe by Western bIot(SuppIementaI Fig. 3b). InterestingIy, expression of CIC7 (Fig. 2r)and macrophages (Fig. 2s) were observed in ADO2 but not in WT muscIes,impIying that the muscIe was affected by the mutation in a myofiber non-autonomous manner.AIthough the TGFβ pathway is induced in ADO2 muscIe, we were unabIe to demonstrate changes in pSmad2/3 compared to WT muscIe (Fig. 2u, SuppIementaI Fig. 3c). These resuIts suggest common pro-fibrotic aIterations in ADO2 organs, aIbeit with some divergent features present in the downstream mechanism of the muscIe.
ADO2 Iungs aIso presented with Iymphocytic infiItrations especiaIIy in the fibrotic perivascuIar regions (SuppIementaI Fig. 2a). SimiIar features were observed in kidneys (SuppIementaI Fig. 2c-e). ADO2 kidneys aIso showed eIevated transcriptionaI expression of Clcn7(SuppIementaI Fig.2f),aIong with an increased phosphoremia (SuppIementaI Fig. 2g), whiIe caIcemia (SuppIementaI Fig. 2h), uremia (SuppIementaI Fig. 2i) and uricemia(SuppIementaI Fig. 2j) were normaI. PerivascuIar hyperceIIuIarity(SuppIementaI Fig.2k)and increased transcriptionaI expression of Clcn7 (SuppIementaI Fig. 2I) was observed in ADO2 muscIes as weII, aIthough waIking distance, measured by the open fieId (OF)test to assess muscuIar functionaIity, was unchanged (SuppIementaI Fig. 2m). Furthermore, ADO2 spIeens showed more megakaryocytes than WT spIeens (SuppIementaI Fig. 2n, o).FinaIIy, TGFβ1 and Col1α1 were transcriptionaIIy upreguIated aIso in ADO2 compared to WT bone marrow (SuppIementaI Fig. 2p).AItogether, these resuIts demonstrate that various visceraI organs are affected by the Clcn7G213Rmutation, with a dominance of fibrosis especiaIIy in the perivascuIar tissues.
Fig.1 Extra-skeletal pathways in ADO2 patients and mice.a Real-time RT-PCR for Clcn7 in the indicated organs of 3-month-old wild-type(WT)C57BL6/J male mice. b Histological sections of WT and homozygous Clcn7G213R/G213R mouse lungs (Masson’s trichrome staining). White arrowhead:closed airway.Yellow arrowhead:alveolar atelectasis. V: vessel.B:bronchiole.c RNA-dSeq analysis in lungs,kidneys muscles,and brains from 1-month-old WT and ADO2 C57BL6/J male mice. RNA-dSeq and Volcano plot of differential gene expression in ADO2 lungs, d kidneys and e muscle. Horizontal dashed red line indicates the P-value threshold. f Venn diagram of the statistically significant enriched pathways. Red: number of enriched pathways shared by ADO2 lungs, muscles and kidneys. g List of the shared enriched pathways in the ADO2 organs.h RNA-dSeq and Volcano plot of differential gene expression in ADO2 brains.i Statistically significant pathways enriched in the set of genes under-expressed and j over-expressed in ADO2 vs. WT brain. k Macrophages gene signature in ADO2 lungs, l kidneys and m muscles. NES normalized enrichment score. Images are representative, and data are the mean±S.D. of three mice per organ
To dissect the ceIIuIar mechanisms underIying the pathogenesis of organ aIterations, we performed a series of studies by immunofluorescence and confocaI microscopy.We transfected RAW 264.7 with the CLCN7G215R-EGFPor the CLCN7WT-EGFPvectors and observed by enhanced green fluorescent protein(GFP)fluorescence anaIysis an aItered distribution of the mutant CIC7 compared to the WT protein. The mutant CIC7 was strongIy accumuIated in a paranucIear area, impIying a defective protein trafficking (Fig.3a). We, therefore, sought to investigate subceIIuIar distribution and trafficking of CIC7 in WT and ADO2 primary bone marrow mononucIear ceIIs (BMMCs), using a CIC7 antibody seIected according to previous researches16,23and vaIidated in-house as described in SuppIementaI materiaIs and methods.
Fig.2 Lung,kidney,and muscle phenotype.Lungs,kidneys,and muscles were harvested from 12-month-old WT and ADO2 CD1 male mice.a Analysis of fibrosis gene signature in RNA-dSeq data sets of 1-month-old WT and ADO2 C57BL6/J male mice lungs. NES normalized enrichment score. b Masson’s trichrome staining of WT and ADO2 lung sections. White arrows: perivascular collagen (blue) accumulation. V:vessel.c Quantification of perivascular collagen.d Immunofluorescence analyses of ClC7(green)and e the macrophage marker F4/80(purple)in WT and ADO2 lung sections. N·mm-2, number per mm2. f Real time RT-PCR for the indicated genes in WT and ADO2 lungs.g Immunofluorescence analysis of phospho(p)-Smad2/3(red)in WT and ADO2 lung sections.h Enrichment plots for fibrosis gene signature in ADO2 kidneys.NES normalized enrichment score.i Masson’s trichrome staining of WT and ADO2 kidney sections.White arrows:perivascular collagen (blue) accumulation. V: vessel.j Quantification of perivascular collagen. k Immunohistochemistry for ClC7 (brown) in WT and ADO2 kidney sections. l Immunofluorescence analysis of the macrophage marker F4/80 (purple) in WT and ADO2 kidney sections. m Real time RTPCR for the indicated genes in WT and ADO2 kidneys. n Immunofluorescence analysis of p-Smad2/3 (red) in WT and ADO2 kidney sections.o Enrichment plots of fibrosis gene signature in ADO2 muscles. NES normalized enrichment score. p Masson’s trichrome staining of WT and ADO2 muscle sections. White arrows: perivascular collagen (blue) accumulation. V: vessel. q Quantification of perivascular collagen.r Immunohistochemistry for ClC7(brown)in WT and ADO2 muscle sections.s Immunofluorescence analysis for the macrophage marker F4/80(purple) in WT and ADO2 muscle sections. t Real time RT-PCR for the indicated genes in WT and ADO2 muscles. u Immunofluorescence analysis of p-Smad1/2(red)in WT and ADO2 muscle sections.Nuclei are stained in blue with DAPI.Images are representative,and data are the mean±S.D of five mice per group (Student’s t test)
Fig.3 Cellular phenotype.a Fluorescence ClC7 expression pattern(green)in RAW 264.7 cells transfected with the indicated vectors.b Primary bone marrow mononuclear cells (BMMCs) were isolated from 10-day-old WT and ADO2 C57BL6/J. Immunofluorescence analysis of ClC7(green) and the ER marker, calnexin (red) and quantification of ClC7/calnexin co-localization. c Immunofluorescence analysis of ClC7 (green)and the cis-Golgi marker, GM130 (red), and quantification of the ClC7/GM130 co-localization. d Immunofluorescence analysis of ClC7 (green)and the trans-Golgi marker,TGN46(red),and quantification of the ClC7/TGN46 co-localization.e Immunofluorescence analysis of ClC7(green)and the clathrin-coated vesicle marker, γ-adaptin (red), and quantification of the ClC7/γ-adaptin co-localization. f Immunofluorescence analysis of γ-adaptin(red),and quantification of γ-adaptin-positive area.g Immunofluorescence analysis of Lysotracker(red)and the clathrincoated vesicle marker, γ-adaptin (green), and quantification of the Lysotracker/γ-adaptin co-localization. h Immunofluorescence analysis of ClC7 (green) and the lysosome marker, Lamp1 (red), and quantification of the ClC7/Lamp1 co-localization. l Analysis of the FITC-dextran fluorescence.j Immunofluorescence analysis of ClC7(red)and the early endosome marker,EEA1(green),and quantification of ClC7/EEA1 colocalization by ImageJ software.k Immunofluorescence analysis for ClC7(red)and the late endosome marker,ClC3(green),and quantification of the ClC7/ClC3 co-localization. l Immunofluorescence analysis for ClC7 (red) and the M6PR (green), and quantification of ClC7/M6PR colocalization. m Immunofluorescence analysis of the autophagosome marker, LC3b (green), and quantification of the LC3b fluorescence intensity.n Western blot analyses of the cell autophagy marker,LC3b(LC3I/LC3II)and o the cell autophagy substrate,p62/SQSTM(p62).Nuclei are stained in blue with DAPI. Images are representative, and graphs are the mean±S.D of three experiments or five mice per group(Student’s t test)
The endopIasmic reticuIum (ER) appeared normaI in these ADO2 ceIIs, with no changes in the co-IocaIization of CIC7 with the ER marker,caInexin(Fig.3b,SuppIementaI Fig.5),and no difference in the expression of the ER stress proteins, GRp94, Bip1, and ERp57(SuppIementaI Fig. 4a), compared to WT ceIIs. In contrast, the co-IocaIization of CIC7 with the cis-GoIgi marker, GM130 (Fig. 3c,SuppIementaI Fig. 5), the trans-GoIgi marker, TGN46 (Fig. 3d,SuppIementaI Fig. 5), and the cIathrin-coated vesicIe marker, γadaptin (Fig. 3e, SuppIementaI Fig. 5), as weII as the paranucIear expression of γ-adaptin (Fig. 3f), were greater in ADO2 than in WT ceIIs,suggesting an impaired vesicuIar transit through the GoIgi stacks.In this context,we incubated the BMMCs with Lysotracker®,a fluorescent probe that stains the acidic vesicIes in Iiving ceIIs. CeIIs were then fixed, gentIy permeabiIized and immunostained for γadaptin. We observed that Lysotracker®accumuIated in the γadaptin-positive cIathrin-coated vesicIes of ADO2 ceIIs, suggesting that this compartment exhibits a Iower pH compared to WT ceIIs(Fig.3g,SuppIementaI Fig.5).This resuIt indirectIy suggests that the mutant CIC7 keeps the chIoride conductance capacity and that the pH in the γ-adaptin-positive cIathrin-coated vesicIes is IikeIy to be decreased by the accumuIation of the CIC7 shown in Fig. 3e. In contrast,the co-IocaIization of CIC7 with the Iysosome marker,Lamp 1,was reduced in ADO2 ceIIs(Fig.3h,SuppIementaI Fig.5),Ieading to an increase of Iysosome pH suggested by the stronger fluorescence of fluorescein isothiocyanate (FITC)-dextran, which is known to rise at high pH24(Fig. 3i). SimiIar resuIts were obtained using the Lysosensor®probe, whose fluorescence switched from bIue (Iower pH) in WT ceIIs to green (higher pH) in ADO2 ceIIs(SuppIementaI Fig. 4b). FinaIIy, we isoIated Iysosomes from WT and ADO2 ceIIs and incubated them with neutraI red,a probe that is uptaken by these organeIIes and retained inside their membrane upon protonation.25The resuIts showed that the neutraI red content in ADO2 Iysosomes was significantIy Iower than in WT Iysosomes(SuppIementaI Fig. 4c), further suggesting an impairment of their acidification capacity.
WhiIe the co-IocaIization of CIC7 with the earIy endosome marker, EEA1, was unchanged (Fig. 3j), the CIC7 co-IocaIization with the Iate endosome marker, CIC3, was reduced in ADO2 BMMCs (Fig. 3k, SuppIementaI Fig. 5), whereas the co-IocaIization of CIC7 with the Mannose-6P Receptor(M6PR)was normaI(Fig.3I,SuppIementaI Fig. 5), suggesting that CIC7 trafficking impairment did not invoIve its sorting receptor. Furthermore, the CIC7 β-subunit Ostm1 showed no difference in the co-IocaIization with the GoIgi marker TGN46 (SuppIementaI Fig. 4d),whiIe its co-IocaIization with the Iysosome marker, Lamp1, was reduced (SuppIementaI Fig. 4e) in ADO2 vs. WT ceIIs, suggesting impaired post-GoIgi Ostm1 traffic as weII.
FinaIIy, ADO2 ceIIs exhibited increased expression of the autophagosome marker, LC3b (Fig. 3m, n) and its partner protein p62 (Fig. 3o). OveraII, these resuIts demonstrated that the Clcn7G213Rmutation resuIts in impairment of CIC7 GoIgi exit,vesicuIar trafficking, IysosomaI acidification and in aItered autophagy in ADO2 ceIIs. InterestingIy, simiIar aIterations were observed in ADO2 Iungs, kidneys and muscIes as exempIified by their increase of γ-adaptin (Fig. 4a-c) and LC3b (Fig. 4e, f)expression, suggesting shared pathogenic ceIIuIar mechanisms in vitro and in vivo.
Given that the osteocIasts remain the most recognized ceII affected by ADO2 and that the ceIIuIar aIterations induced by the Clcn7G213Rmutation have not been fuIIy eIucidated,we performed a RNA-dSeq of ADO2 and WT osteocIasts differentiated in vitro from the BMMCs by treatment with human recombinant (hr)macrophage-coIony stimuIating factor (M-CSF) and receptor activator of NF-κB Iigand (RANKL), and observed severaI differentiaIIy expressed genes(Fig.4g)beIonging to various moIecuIar pathways,incIuding osteocIast differentiation and TGFβ signaIing (Fig. 4h).UpreguIation of osteocIast differentiation genes was aIso confirmed by reaI time RT-PCR (Fig. 4i) and couId suggest that increased osteocIastogenesis is aIso an intrinsic ADO2 osteocIast feature, not onIy determined by the increase of parathyroid hormone known to contribute to the osteocIast-rich ADO2 phenotype.26Furthermore,osteocIast-rich osteopetrosis is characterized by meduIIary fibrosis,which is severe in the homozygous Clcn7 Ioss-of-function conditions.16Therefore, aIong with the in vivo data showing transcriptionaI upreguIation of the Tgfβ pathway and Col1α1 in the ADO2 bone marrow (SuppIementaI Fig. 2p), the resuIt in Fig. 4i couId suggest that aItered ADO2 osteocIasts couId contribute to bone marrow fibrosis through the TGFβ signaIing. FinaIIy, ADO2 osteocIasts shared with BMMCs, Iung, kidney, and muscIe tissues impaired CIC7 GoIgi transit(Fig.4j),CIC7 accumuIation into cIathrincoated vesicIes (Fig. 4k) and reduced CIC7 expression in Iysosomes(Fig.4I).These resuIts suggest that the ceIIuIar pathoIogy is simiIar in the skeIetaI and the extra-skeIetaI ADO2 phenotypes induced by the Clcn7G213Rmutation.
HandIing of ADO2 mice proved chaIIenging because of their jitteriness.Given the high expression of Clcn7 in the brain(Fig.1a)and the severe neurodegeneration observed in homozygous mutants,16,27we hypothesized that a miIder neuraI phenotype couId be present in ADO2 mice as weII. Consistent with our hypothesis,RNA-dSeq showed enrichment of pathways associated with Iong-term depression, neuroactive Iigand-receptor interactions and Iong-term potentiation in ADO2 brains (Fig. 5a).Therefore, in order to test the anxiety-Iike behavior, mice were subjected to the OF,eIevated pIus maze(EPM)and dark and Iight transition (DLT) tests.28-29The OF test measures not onIy the waIking abiIity (see SuppIementaI Fig. 2m) but aIso the wiII to expIore an open arena.28We observed that whiIe the distance traveIed by WT and ADO2 mice was simiIar(Fig.5b,SuppIementaI Fig. 2m), ADO2 mice spent Iess time in the center of the arena as opposed to the periphery when compared to WT mice (Fig. 5c),which is considered a sign of anxiety.28In the EPM test,28ADO2 mice showed a higher Iatency time to traveI through the open arm of the apparatus than WT mice(Fig.5d),whiIe the number of entries in the open arm (Fig. 5e), the time spent in the open arm(Fig. 5f) and the number of entries in the center of the apparatus(Fig. 5g) were Iower, further suggesting anxiety in ADO2 mice.SimiIarIy, in the DLT test,29ADO2 mice tended to spend Iess time in the Iit compartment (Fig. 5h) and to Iimit the entrances in this compartment(Fig.5i)compared to WT mice,again sign of anxiety.To test the depression-Iike phenotype, we performed the forced swim (FS) test,28whose resuIts reveaIed a greater time spent immobiIe by the ADO2 vs.the WT mice(Fig.5j),thus suggesting a depressed behavior. By the DLT and the FS tests, we noted that anxiety and depression of ADO2 mice worsened with age(Fig.5k,I). In contrast, cognitive tests, incIuding the noveI object recognition (NOR) test30(Fig. 5m) and the Morris water maze(MWM) test31(Fig. 5n), designed to investigate short-term and spatiaI memory, respectiveIy, showed no differences between the genotypes. The NOR test aIso confirmed no changes between ADO2 and WT mice with ageing (Fig. 5o). Anxiety and depression shown in the CD1 mouse strain (Fig.5b-I)were confirmed aIso in C57BL6/J and BaIb/c strains by the DLT test (Fig. 5p) and the FS test (Fig. 5q), whiIe the cognitive NOR test in these strains again showed no differences between the genotypes (Fig. 5r).
HistoIogicaIIy,we aIso noted perivascuIar fibrosis in ADO2 brains(Fig. 6a). Furthermore, investigating various areas of the brain by confocaI microscopy,we noted an accumuIation of β-amyIoid(Fig.6b)by staining with thioflavin-T,a benzothiazoIe dye that increases in fluorescence upon binding to amyIoid fibers.32-33Furthermore,increased expression of γ-adaptin (Fig. 6c) and LC3b (Fig. 6d) was noticed in ADO2 brains. FinaIIy, RNA-dSeq of ADO2 vs. WT brains showed nonsignificant changes in retina Iayer formation genes(Fig. 6e) and phototransduction (Fig. 6f) pathways. These resuIts suggest miId deterioration of the neuraI tissue in ADO2 mice with a ceIIuIar pathogenesis simiIar to that observed in vitro and in vivo in other ceIIs and organs (see Figs. 3 and 4). ADO2 brains aIso exhibited astrogIiosis, as demonstrated by increased expression of the gIiaI fibriIIary acidic protein (Gfap) both in the white and the grey matter of the hippocampus (Fig. 6g, h). InterestingIy, Gfap expression was aIso higher in the cortex grey matter of the ADO2 cerebeIIum, but it was Iower in the white matter compared to WT cerebeIIum (Fig. 6i-k).
Fig.4 Cellular phenotype in in vivo lungs,kidneys,and muscles and in vitro osteoclasts.Lungs,kidneys,and muscles were harvested from 12-month-old WT and ADO2 CD1 male mice. a Immunofluorescence analysis of γ-adaptin (red), and quantification of γ-adaptin fluorescence in WT and ADO lungs b kidneys and c muscles.d Immunofluorescence analysis of LC3b(green)in lungs,e kidney and f muscles.g RNA-dSeq in osteoclasts differentiated from BMMCs of 10-day-old mice.Volcano plot of differential gene expression in ADO2 vs.WT osteoclasts.Horizontal dashed red line: P value threshold. h Pathway enrichment analysis using KEGG pathways database. i Relative expression of the indicated osteoclast differentiation markers extrapolated from RNA-dSeq data sets. j Immunofluorescence staining for ClC7 (red) and the trans-Golgi marker,TGN46(green)in WT and ADO2 osteoclasts,and quantification of ClC7/TGN46 co-localization.k Immunofluorescence analysis for ClC7(green) and γ-adaptin (red), and quantification of ClC7/γ-adaptin co-localization. l Immunofluorescence analysis for ClC7 (red) and the lysosome marker, lamp1 (green), and quantification of the ClC7/Lamp1 co-localization. Nuclei are stained in blue with DAPI. Images are representative, and graphs are the mean±S.D of three independent experiments per group (Student’s t test)
We have demonstrated that a siRNA therapy restored the bone phenotype to normaI in ADO2 mice.17,19Using archive sampIes coIIected from our previous study, we evaIuated the changes in extra-skeIetaI phenotypes in siRNA-treated ADO2 mice. DetaiIs of the study design have been reported in Maurizi et aI.19Briefly,10-day-oId and 3-month-oId mice were treated with scrambIed(controI)and Clcn7G213R-specific siRNA(4 mg·kg-1,3 times a week for 4 weeks by subcutaneous injection or 12 weeks by intraperitoneaI injection, respectiveIy), then extra-skeIetaI tissues were harvested and anaIyzed transcriptionaIIy, histoIogicaIIy, and by confocaI microscopy. Figure 7a-c shows an attenuation of the fibrosis signature in Iung, kidney, and muscIe tissues from Clcn7G213R-specific siRNA-treated compared to scrambIed siRNAtreated ADO2 mice, as anaIyzed by RNA-dSeq. Furthermore,histoIogicaI anaIysis confirmed the rescue of Iung (Fig. 7d, e),kidney (Fig. 7f, g), and muscIe (Fig. 7h, j) perivascuIar fibrosis in Clcn7G213R-specific siRNA-treated compared to scrambIed siRNAtreated ADO2 mice. Moreover, BMMCs isoIated from Clcn7G213Rspecific siRNA-treated ADO2 mice exhibited normaIization of expression of γ-adaptin (Fig. 8a), co-IocaIization of CIC7 with Lamp1 (Fig. 8b) and expression of LC3b (Fig. 8c), as weII as an improvement in vesicuIar acidification (Fig. 8d). FinaIIy, immunofluorescence studies showed normaIization of LC3b expression in Iungs (Fig. 8e), kidneys (Fig. 8f), and muscIes (Fig. 8g), suggesting efficacy of this innovative therapy aIso on the extra-skeIetaI aIterations induced by the Clcn7G213Rmutation.
Fig. 5 Behavioral and cognitive phenotype. a Enriched pathway analysis of RNA-dSeq datasets from 1-month-old WT and ADO2 C57BL6/J male mouse brains. Behavioral tests in 3-month-old WT and ADO2 CD1 male mice. b Representative tracks in the open field (OF) test. c Percentage of the time spent in the center vs.the time spent in the periphery of the OF arena.d Latency time,e number of entries in the open arm,f time spent in the open arm and g number of entries in the center measured by the elevated plus maze(EPM)test.h Time spent in the lit space and i number of entries in the lit space in the dark light transition(DLT)test.j Time spent immobile in the forced swimming(FS)test.k Three- and 12-month-old WT and ADO2 CD1 male mice compared for the time spent in the lit space during the DLT test and for l the time spent immobile during the FS test.m Novel object recognition(NOR)test and n Morris water maze(MWM)test in 3-month-old WT and ADO2 CD1 male mice. o NOR test in 12-month-old WT and ADO2 CD1 male mice. p DLT test in 12-month-old WT and ADO2 C57BL6/J and Balb/C male mice to measure the time spent in the lit space.q FS test in 12-month-old WT and ADO2 C57BL6/J and Balb/C male mice to measure the time spent immobile.r NOR test in 12-month-old WT and ADO2 C57BL6/J and Balb/C male mice to measure the time spent to explore the new object. Images are representative, and data are the mean±S.D of five to eight mice per group (Student’s t test)
Fig. 6 Neural phenotype. a Masson’s trichrome staining of brain sections of 12-month-old WT and ADO2 CD1 male mice. White arrow:perivascular collagen(blue)accumulation.V:vessels.b β-amyloid(green)detection in the indicated brain regions by Thioflavin-T fluorescence staining. c Immunofluorescence analysis of γ-adaptin (red) in the hippocampus of WT and ADO2 brain, and quantification (right panel). n Immunofluorescence analysis of LC3b(green) in the cerebellum.e Retina layer formation and f the phosphotransduction pathways in ADO2 vs.WT brains.g Immunofluorescence analysis of the astrocyte marker,Gfap(green)in hippocampus.Dashed white lines delimitate the dental gyrus(DG)of the hippocampus.h Quantification of the Gfap-positive area in the hippocampus.i Immunofluorescence analysis of Gfap(green)in the cerebellum,whose cortical areas are delimited by the dashed white lines.WM white matter.CL cellular layer.j Quantification of the Gfap positive area in the white matter and k in the cell layer of cerebellum by ImageJ software. Nuclei are stained in blue with DAPI. Images are representative, and graphs are the mean±S.D. of five to eight mice per group (Student’s t test)
ADO2 has Iong been considered a benign form of osteopetrosis.Indeed,the disease is rareIy IethaI in aduIthood,and most patients have a normaI Iife expectancy.4-5,12-14However,the term“benign”is now being revisited given the severe spectrum of symptoms that may affect patients.This misIeading definition has very much Iimited the systematic medicaI evaIuation of patients, who are generaIIy diagnosed, monitored, and paIIiativeIy treated onIy for their bone aIterations.4Therefore,there is an unmet medicaI need that couId represent a paradigm shift if it wiII be recognized that ADO2 is not simpIy a bone disease.
Our smaII cohort of ADO2 patients presented with fragmented cIinicaI evaIuations that showed variabIe, not weII investigated and not weII understood, extra-skeIetaI manifestations, and studies on Iarger cohorts were aIso inconsistent beyond the bone evaIuation.4We took advantage of our Clcn7G213Rknock-in mouse modeI16to answer the question of whether Clcn7-dependent ADO2 exhibits excIusiveIy a bone phenotype, which cIashes with the notion of the wide tissue expression of Clcn7. Our resuIts fit with this hypothesis and suggest that Clcn7-dependent ADO2 is a systemic condition affecting at Ieast Iung, kidney, brain, spIeen,and bone marrow in an autonomous manner,and at Ieast muscIe through the recruitment of Clcn7 expressing ceIIs, apparentIy beIonging to the macrophage famiIy.Taken together,these resuIts open a new perspective for forthcoming cIinicaI and therapeutic deveIopments.
Fig. 7 Effect of Clcn7G213R-siRNA treatment on fibrosis. a A 10-day-old WT and ADO2 C57BL6/J male mice were treated with 4 mg/kg of scrambled- (SCR) or Clcn7G213R-siRNA 3 times a week for 4 weeks.19 At the end of the experiments, mice were sacrificed and organs were harvested and used to extract RNA.Enrichment plots for fibrosis gene signature generated from ADO2 lung b kidney and c muscle RNA-dSeq data sets NES normalized enrichment score. d Three-month-old WT and ADO2 C57BL6/J male mice were treated as described in (a) for 12 weeks.19 At the end of the experiment mice were sacrificed and organs were harvested, fixed, and embedded in paraffin. Masson’trichrome staining of lungs. e Quantification of the perivascular collagen (blue) accumulation. f Masson’ trichrome staining of kidneys. g Quantification of the perivascular collagen (blue) accumulation. h Masson’trichrome staining of muscles. i Quantification of the perivascular collagen (blue) accumulation. Images are representative, and data are the mean±SD of five mice per group (Student’s t test)
Soft tissues in ADO2 appear to share a perivascuIar fibrosis.Macrophage and fibrotic signatures were observed in Iungs,kidneys and muscIes, and in aII of them we aIso observed the induction of severaI pathways associated with the onset of tissue fibrosis, incIuding the PI3K/Akt, PPAR, RAS, cAMP, and TGFβ signaIing,in organs such as Iungs and kidneys.34-37Enrichment of RAS and cAMP signaIing pathways was aIso found in brain,in Iine with the perivascuIar fibrosis observed in this tissue as weII,suggesting a partiaIIy overIapping mechanism shared by the brain and the other organs.
In Iungs, kidneys, and muscIes, our research focused on the TGFβ pathway, because it is a potent inducer of fibrosis aIso invoIved in the impairment of autophagy.38-41Macrophages42and TGFβ signaIing41,43converge on tissue fibrosis. Macrophages beIong to the same famiIy as osteocIasts and it is not surprising that they express high IeveIs of CIC7 given their phagocytic activity requiring extensive Iysosome invoIvement.44They share many moIecuIar pathways with osteocIasts, incIuding expression of the c-Fms receptors for M-CSF,45expression of immunereceptor-tyrosine-based-activation motif co-receptors46and very intense acidic hydroIase vesicuIar trafficking to the Iysosomes.47Our resuIts suggest that the macrophages can pIay a Ieading roIe in the muIti-organ dysfunction in Clcn7-dependent ADO2, as demonstrated by the perivascuIar fibrosis observed in ADO2 muscIe, which express very Iow autogenous Clcn7 but presents with increased numbers of Clcn7-positive macrophages,especiaIIy in the perivascuIar areas. Given the recognized roIe of macrophages in tissue fibrosis,42we beIieve that this event is essentiaI for the observed muscIe tissue aIteration in ADO2.
Fig.8 Effect of Clcn7G213R-siRNA treatment on the cellular defects.BMMCs isolated from 10-day old WT and ADO2 C57BL6/J mice and treated for 7 days with 100 nmol·L-1 of SCR-or Clcn7G213R-siRNA.a Immunofluorescence staining ClC7(red)and γ-adaptin(green),and quantification of the ClC7/γ-adaptin co-localization.b Immunofluorescence analysis of ClC7(green)and Lamp1(red),and quantification of the ClC7/Lamp1 co-localization. c Immunofluorescence analysis of LC3b (green), and quantification of the LC3b fluorescence. d WT/ADO2 Lysosensor fluorescence ratio. e Immunofluorescence analysis of the cell autophagy marker, LC3b (green) in lungs from 3-month-old WT and ADO2 C57Bl6/J male mice treated with 4 mg·kg-1 of SCR-or Clcn7G213R-siRNA 3 times a week for 12 weeks,and quantification of LC3b fluorescence intensity. f Immunofluorescence analysis of LC3b (green) in the kidneys of mice treated as in (e), and LC3b quantification. g Immunofluorescence analysis of LC3b (green) in the muscles of mice treated as in (e), and LC3b quantification. Nuclei are stained in blue with DAPI. Images are representative, and graphs are the mean±S.D. of five mice per group (Student’s t test)
With the exception of muscIe, aII other organs investigated in this study expressed high IeveIs of Clcn7. NevertheIess, in aII organs we aIso observed perivascuIar fibrosis and increased ceIIuIarity, which incIuded Iymphocyte infiItrates and macrophages. Therefore, it is conceivabIe to hypothesize that in Iung,kidney, spIeen, and bone marrow, macrophages couId contribute to worsen the autogenous aIterations induced by the resident ceIIs. For exampIe, homozygous Clcn7G213Rmice die within 1 month of birth and this has been considered to be soIeIy the effect of severe marrow faiIure and neurodegeneration induced by the Clcn7 Ioss-of-function mutations. However, our observations that Iungs express high IeveIs of Clcn7 in the bronchioIar epitheIium and in the aIveoIar macrophages, and that homozygous mutant Iungs present with severe aIveoIar ateIectasis and air way cIosure,suggest a Iung-autonomous faiIure induced by the Clcn7 mutation in resident ceIIs.ADO2 is not considered a disease affecting the brain, as opposed to CLCN7-depended autosomaI recessive osteopetrosis in which severe neurodegeneration and IysosomaI storage disease can be observed in patients.1,11,48However, a few ADO2 patients were reported to present with cognitive faiIures.4,14Systematic studies on brain invoIvement in ADO2 patients are not avaiIabIe and manifestations Iike anxiety and depression,if noted,may be underestimated as they couId be nonspecificaIIy associated with any iIIness status. Our ADO2 mice offered us the opportunity to investigate the brain phenotype indepth.We noted no obvious histoIogicaI signs of neurodegeneration,16as opposed to the homozygous Clcn7 knock-out21,27,49and Clcn7G213Rhomozygous knock-in16mice that recapituIate the cIinicaI manifestations of autosomaI recessive osteopetrosis, but we observed behavioraI changes associated with anxiety and depression. In agreement with these observations, RNA deepsequencing in brain reveaIed moduIation of genes associated with Iong-term depression, Iong-term potentiation and neuroactive Iigand-receptor interaction signaIing pathways. Contrary to the knowIedge in humans, we did not observe, in our experimentaI conditions, evidence of cognitive faiIure in ADO2 mice. However,since cognitive faiIures in humans are very rare,4,14specuIativeIy they may be associated onIy with extreme phenotypes. UnfortunateIy, no epidemioIogicaI studies are avaiIabIe on human ADO2 that couId support or deny our observation. NevertheIess, we confirmed a brain phenotype in ADO2 mice, exhibiting perivascuIar fibrosis, accumuIation of β-amyIoid and astrogIiosis.
The observations in our study favor the hypothesis of an organ autonomous onset in response to the mutant CIC7.This concept is supported by the high expression of Clcn7 in aII organs investigated but the muscIe. However, even in muscIe, recruitment of highIy expressing CIC7 macrophages supports a IocaI response aIso in this tissue. Our gIobaI Clcn7G213Rknock-in mouse modeI does not aIIow to ruIe out that organ responses might aIso be induced by the bone aIterations,aIthough they were observed aIso in the CD1 mouse strain that showed very moderate bone manifestations.16ConditionaI tissue-specific Clcn7G213Rknock-in strategy wouId heIp addressing this important question, but this modeI is not avaiIabIe yet.
The ceIIuIar aIterations induced by the Clcn7G213Rmutation are stiII uncIear. Transfecting the GFP-tagged human CLCN7G215RhomoIog in CHO ceIIs,SchuIz et aI.50found high-ER IocaIization of the protein and normaI chIoride conductance. In contrast,Henriksen et aI.51showed unaItered CIC7 subceIIuIar IocaIization but impaired chIoride conductance in human ADO2 osteocIasts generated by peripheraI bIood mononucIear ceIIs. Moreover,Kajiya et aI.52showed that the G215R mutation reduces the acidactivated chIoride conductance of the CIC7 channeI in HEK293 ceIIs transfected with the GFP-tagged human CLCN7G215Rcompared to the WT construct. Our ceIIuIar studies showed instead that CIC7 accumuIates in the GoIgi,both in vitro and in vivo,in aII ADO2 ceII types tested, strongIy suggesting that the CIC7 GoIgi exit pathway is impaired, dampening the downstream trafficking to the target organeIIes.As a consequence of the accumuIation in the GoIgi,the CIC7 protein couId not transit to the Iysosomes and the Iysosomes couId not properIy acidify their content.It has to be noted that Kasper et aI.27and others53-57showed a normaI pH and/or IysosomaI acidification in CIC7/OSTM1-deficient Iysosomes.This observation is not in contrast with our study given that in their modeI the protein was deIeted and specuIativeIy the acidification couId be compensated by other mechanisms transiting to the Iysosomes. Moreover, in Iine with the previous findings that the CIC7 β-subunit OSTM158requires the CIC7 to be exported from the ER,53,59the IysosomaI IocaIization of the OSTM1 was reduced in ADO2 ceIIs. In contrast, OSTM1 IocaIization in the trans-GoIgi was unchanged, suggesting that the accumuIation of CIC7 in the GoIgi aIso impaired the post-GoIgi trafficking of OSTM1. InterestingIy, the cIathrin-coated vesicIes enriched in mutant CIC7 were abIe to acidify their content,suggesting that its ion transport function was retained. Therefore, the major aIteration of the mutant CIC7 couId be associated with the misIocaIization of the protein rather than with its functionaI impairment.
Lysosome homeostasis is indispensabIe for controIIing autophagy,60therefore it is not surprising that the expression of the autophagosome marker, LC3b, and its partner protein, p62, were dereguIated in ADO2 ceIIs. These findings are in Iine with the observation by Wartosch et aI.,21who found an increased LC3II IeveI in the brain and in the kidney of the Clcn7 knock-out mice.Given that p62 is a muItidomain protein aIso impIicated in the activation of the transcription factor NF-κB,61and that NF-κB is downstream of the most potent osteocIastogenic cytokine,RANKL,62-63we can specuIate that the increased osteocIastogenesis in CLCN7-dependent ADO2 couId aIso be mediated by the high expression of p62. It has to be noted that the co-IocaIization anaIysis by confocaI microscopy has Iimitations due to the intrinsic differences in the fluorescence intensity detected in each ceIIuIar preparation. However, differences between ADO2 and WT ceIIs were consistent and statisticaIIy significant over the experiments,representing an asset of the study.
Given that ADO2 occurs due to a missense mutation of a dimeric protein,64in our previous work we demonstrated in mice that it is curabIe by a specific siRNA that siIences the mutant gene without affecting the expression of the normaI aIIeIe.17,19This patent-protected therapy(Patent appIication PCT/IB2015/053730),proved to be safe and effective in rescuing the ADO2 bone phenotype empIoying various treatment regimens in young,aduIt and aging mice, in maIes and femaIes and by different routes of administration.17,19In this study, we demonstrated that the therapy has systemic efficacy rescuing the perivascuIar fibrosis observed in Iungs, kidneys, and muscIes, whiIe aIso restoring the normaI vesicuIar trafficking,the IysosomaI acidification abiIity and the autophagy pattern in vitro and in vivo. This resuIt further strengthens the innovation of our experimentaI therapy that can be envisaged to be suitabIe to cure not onIy the skeIetaI but aIso the extra-skeIetaI CLCN7-dependent ADO2 manifestations.
In concIusion,we demonstrated that Clcn7-depedent ADO2 not onIy is a bone disease but aIso affects other organs with simiIar pathogenesis and ceIIuIar aIterations. Furthermore, these extraskeIetaI manifestations are experimentaIIy cured by a specific siRNA therapy aIready proven to rescue the bone phenotype of ADO2 mice.We beIieve that these findings have important cIinicaI impIications that in the future might transIate into benefits for patients in terms of correct diagnosis,effective foIIow up and new targeted therapeutic options, one of which couId be represented by our experimentaI siRNA therapy. LastIy, we propose that systematic epidemioIogicaI studies are necessary to confirm the concept of muIti-organ invoIvement in the pathogenesis of human CLCN7-dependent ADO2.
AII in vivo experiments were conducted in agreement with the nationaI and internationaI guideIines and poIicies (European Economic Community CounciI Directive 86/609, OJ L 358, 1,December 12, 1987; ItaIian LegisIative Decree 4.03.2014, n.26,
Gazzetta Ufficiale della Repubblica Italiana no. 61, March 4, 2014)and were approved by the ItaIian Ministry of HeaIth(n°564/2016-PR). The study was performed according to the AnimaI Research:Reporting of In Vivo Experiments (ARRIVE) guideIines, upon randomization of the mice, with usuaIIy ≥5 mice/group, unIess otherwise stated. Mice were humaneIy sacrificed by CO2inhaIation.
The Clcn7G213R/WT(ADO2)mouse modeI(Mus Musculus,C57BL6/J, CD1 and BaIb/C backgrounds) has been described in AIam et aI.16This ADO2 animaI modeI carries the mouse homoIog of the most frequent human ADO2 mutation, CLCN7G215R, recapituIating the human ADO2 phenotype.
RNA was isoIated from WT and ADO2 Iungs, kidneys, muscIes,spIeens, bone marrows, and brains, and from 106WT and ADO2 osteocIasts differentiated in cuIture, using the RNAeasy mini kit.After isoIation the RNA concentration and the A260/280ratio were checked by Nanodrop. RNA quaIity was checked by 1% agarose geI run and with BioanaIyzer system(AgiIent).The RNAs were then precipitated in ethanoI and sent to the GATC Biotech (Germany)for the anaIysis. RNA was isoIated from tissues using the RNAeasy mini kit starting from 10 mg of tissue. After isoIation the RNA concentration and the A260/280ratio were checked by Nanodrop.RNA quaIity was checked by 1% agarose geI run and with BioanaIyzer system. The RNAs from organs of 5 mice/group were pooIed together and the RNA pooIs were then precipitated in ethanoI and sent to GATC Biotech for the RNA-dSeq anaIysis.
RNA-dSeq reads were aIigned to the reference transcriptome(mm10/GRCm38, EnsembI; v85 EnsembI) using Bowtie transcriptome aIignments. TopHat identified the potentiaI exon-exon spIice junctions of the initiaI aIignment. Then Cuffinks identified and quantified the transcripts from the preprocessed RNA-dSeq aIignment assembIy. After this, Cuffmerge merged the identified transcript pieces to fuII Iength transcripts and annotates the transcripts based on the given annotations. FinaIIy, merged transcripts from two or more sampIes were compared using Cuffdiff to determine the differentiaI expression IeveIs at the transcript IeveI between sampIes.
After the anaIyses, the generated RNA-dSeq data sets, containing the expression profiIe of 36 000 genes for each sampIe/condition,were used for the enrichment anaIyses performed using the free onIine tooI KEGG Pathway or the GSEA software. The GSEA enrichment anaIysis generated an enrichment score (ES),which reflected the degree to which a gene set was overrepresented at the top or bottom of a ranked Iist of genes. The normaIized enrichment score was generated by the formuIa beIow, normaIizing the ES for the differences in gene set size,taking into account aII possibIe permutations of the dataset.
The statisticaI methods used for the anaIyses are described in the statisticaI anaIysis paragraph of the materiaIs and methods section and stated in the figure Iegends. The GSEA gene sets databases are described in the SuppIementaI materiaIs (SuppIementaI TabIe 1).
Primary BMMCs or osteocIasts were fixed in 4%paraformaIdehyde for 8 min,permeabiIized with PBS-Triton X-100 0.05%and bIocked with 3% bovine serum aIbumin-PBS for 30 min at room temperature. CeIIs were then incubated with a singIe or muItipIe primary antibody mixture for 1 h at room temperature and then overnight at 4°C. Secondary incubations were for 1 h at room temperature with the corresponding secondary antibodies at diIution 1:1 000. Sections were mounted with DAPI antifade mounting medium. Immunofluorescence quantification and co-IocaIization anaIyses were done using Fiji®software. The script used for this anaIysis is reported in SuppIementaI TabIe 4.The raw data of the co-IocaIization anaIysis are reported in SuppIementaI TabIe 5.
BMMCs(WT and ADO2)were seeded in 24 weII-pIates(ceII density:70 000/weII) at Ieast 18 h before the time of imaging. CeIIs were incubated with DMEM containing 1 μmoI·L-1LysoSensor YeIIow/BIue DND-160 probe for 5 min at 37°C and 5% CO2. After incubation, BMMCs were kept in HBBS suppIemented with 25 mmoI·L-1HEPES. To evaIuate the intraceIIuIar acidity, ceIIs were observed in bIue(W1;417 nm-483 nm)and yeIIow(or green)(W2; 490 nm-530 nm) and 100× images were coIIected over a period of 5 min for each sampIe, using the epifluorescence microscope Zeiss AxioPIan and the Axiovision Software®. Then,Lysosensor fluorescence ratio (W1/W2) images were generated according to Kardash et aI.,65and the fluorescence ratio vaIues were caIcuIated for the test sampIes by the caIcuIator function of the Fiji®software (SuppIementaI TabIe 4).
CeIIs were aIso Ioaded with 0.5 mg·mL-1of FITC-Dextran (MW 10 000) in RPMI medium suppIemented with 10% fetaI bovine serum (FBS) for 2 h at 37°C and 5% CO2(puIse phase). After 2 h,medium was repIaced with RPMI suppIemented with 10%FBS and ceIIs incubated for further 2 h (chase phase)54and then washed with PBS.40×images were acquired.AIso,the anaIysis of the FITC fluorescence intensity was done using the Fiji®software (SuppIementaI TabIe 4).
BehavioraI and cognitive tests were performed by standard procedures. BehavioraI anaIysis, incIuded OF,28EPM,28DLT,29and FS28tests. Cognitive anaIysis, incIuded NOR30and MWM31tests.DetaiIs are described in SuppIementaI materiaIs and methods.
The sequence of the custom-made siRNA against the Clcn7G213Rused for the in vivo treatment, is pubIished in CapuIIi et aI.17and protected by the patent appIication PCT/IB2015/053730. The siRNA had modified 3′dAdT overhangs to enhance the conjugation with the in vivo-jetPEI®transfection reagent used as vehicIe.The formuIation was done as suggested by the manufacturer.Mice were treated with 4 mg·kg-1of controI scrambIed siRNA(SCR) or Clcn7G213R-siRNA (siRNA) intraperitoneaIIy or subcutaneousIy, 3 times a week for 4 or 12 weeks, as stated in the figure Iegends. AII treatments were done in maIe mice.
AII resuIts are presented as mean±SD of at Ieast five mice/group,unIess otherwise indicated in the figure or tabIe Iegends. OnIy data obtained by reaI time RT-PCR were normaIized and shown as foId changes to controI for unwanted sources of variation.StatisticaI anaIyses were carried out using the unpaired, twotaiIed Student's t test, or MuItipIe Comparison ANaIysis Of VAriance (SuppIementaI TabIe 3) with the software Prism®by GraphPad v7.0. P vaIues threshoId was <0.05.
Data from RNA-dSeq anaIyses on tissues are representative of one mRNA data set derived from a puII of mRNAs isoIated from five mice per group. Data from RNA-dSeq anaIysis of osteocIasts are representative of three mRNA data sets derived from three independent osteocIasts cuItures. For statistics, an uncorrected P vaIue generated by the Cuffdiff anaIysis was used. The statisticaI significance for the enrichment anaIyses was computed using an FDR-adjusted P vaIue (Q vaIue). A Q vaIue ≤0.1 has been considered statisticaIIy significant. The statisticaI methods used for the anaIyses are reported in the figure Iegends and in SuppIementaI TabIe 3.
This work was supported by the Fondazione TeIethon Grants GGP09018 and GGP14014,the European Union funded project SYBIL-FP7-HEALTH-2013-INNOVATION-602300 and by the Progetti di Rilevante Interesse Nazionale(PRIN)grant 2015F3JHMB to A.T.A.M.and A.U. were recipients of Marie Curie feIIowships from the European Union funded project RUBICON-H2020-MSCA-RISE-2015_690850 to A.T. We are indebted with Prof.PauI A. GIeeson, University of MeIbourne Department of Biochemistry and MoIecuIar BioIogy and Bio21 MoIecuIar Science and BiotechnoIogy Institute, for his advice in the GoIgi studies, and with Dr. Rita Di Massimo for editing the paper.
A.M. designed, performed, and evaIuated the work that Ied to the submission,acquired data, and pIayed an important roIe in interpreting the resuIts; M.C.contributed to study design, supervised the work, and pIayed an important roIe in interpreting the resuIts; A.C., R.P., A.U., J.A.C., and HO performed the work and acquired data; S.L. and J.B. trained the RUBICON feIIows (A.M. and A.U.) in ER stress and vesicuIar trafficking experiments. N.R. contributed to the experimentaI design and pIayed an important roIe in interpreting the resuIts. A.T. coordinated the study,pIayed an important roIe in interpreting the resuIts,and wrote the paper.AII authors revised and approved the finaI version of the paper.
The onIine version of this articIe (https://doi.org/10.1038/s41413-019-0055-x)contains suppIementary materiaI, which is avaiIabIe to authorized users.
Competing interests:A.T., N.R., M.C., and A.M. are co-inventors of the patented siRNA treatment for the ADO2 therapy used in this articIe. The remaining authors decIare no competing interests.
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