Maurizi A, Capulli M, Curle A, Patel R, Ucci A, Côrtes JA, Oxford H, Lamandé SR, Bateman JF, Rucci N, Teti A. Extra-skeletal manifestations in mice affected by Clcn7-dependent autosomal dominant osteopetrosis type 2 clinical and therapeutic implications. Bone Res. 2019 Jun 11;7:17. PMC free article

The electrochemistry of CIC7

The ClC7 2Cl/1H+ antiporter is coded for by the CLCN7 gene.  Mutations in just a single copy of this gene cause autosomal dominant osteopetrosis type 2 (ADO2) is a rare disease that makes bones dense but brittle.  CIC7 is found in acidic organelles that include lysosomes the ruffled border of osteoclasts.  The ruffled border protrudes into the resorption lacuna, representing the extracellular environment between the bone surface and the osteoclast membrane.

What does it mean when CIC7 is “anti-porting” Cland H+ in the opposite direction as this H+ pump is loading the lysosome? Part of this may be to maintain charge neutrality and another to keep things from getting too acidic as is shown in supplemental Figures 4b-c.  The authors used bone marrow mononuclear cells (BMMC) isolated from bone marrow of 2-month-old WT and ADO2 mice.

Lysosensor® is a fluorescent probe whose emission shifts from green (higher pH) to blue (lower pH).

In the first panel the Pubchem images of neutral red with and without the extra proton (gray square) have been superimposed on the lyosome. b. Lysosensor® probe fluorescence switched from blue (lower pH) in WT cells to green (higher pH) in ADO2 cells. Part of this original image was very confusing because the lysosomes could not be visualized in the wildtype cells. The image manipulation program Gimp2 was used to adjust the levels to see acidified lysosomes. c. Neutral Red dye uptake by lysosomeswas followed by a spectrophotometer in real timeThe lysosomes time for 10 minutes. The maximum absorbance of the Neutral Red dye shifts from 460 nm to 510 nm in acidic pH conditions.  A nuance of this probe is that it becomes trapped in lysosomes when it is protonated. 

The take home message

ADO2 lsyosomes can acidify, but not as much as the wildtype lsyosomes where CIC7 is there to maintain electroneutrality… even if it means antiporting some of the H+ out.

The mouse model

The Clcn7 G213R/WT (ADO2) mouse model has one wild type copy of CLCN7 and another copy with a single base pair mutation hat results in a neutral glycine at position 213 being replaced with an alkaline arginine.    This ADO2 mouse model mimics the human ADO2 phenotype.  The authors looked extra skeletal symptoms in eight human ADO2 patients with genetically confirmed disease.  Extra-skeletal symptoms tended to be observed in at most two of the eight patients with the exception of anemia which was observed in six of the eight patients.  The raises a therapeutic question of how does one compensate when half the usual CIC7 is not enough, perhaps because the patient expresses a lesser amount, or too much, of an accessory enzyme.  This post will follow the work of Maurizi and ask such questions.

1. Where is CIC7 expressed in normal, wildtype mice ?

The calvaria is one of the skull bones.  The femur of course is the long leg bone.  It is interesting to note that more CIC7 is expressed in the lung, spleen, and brown adipose tissue (BAT) but not white adipose tissue (WAT).  The main difference between brown and white adipose tissue is mitochondria in the BAT.  Also note the expression of CIC7 in the heart but not the skeletal muscle. the apparently pooled fast twitch glycolytic Ib fingers with slow twitch I and fast twitch Ia aerobic fibers that are rich in bitochondria.

Hassan Trichrome statin is used a lot in this study for the detection of collagen. Note that collagen is the matrix protein in bone.

Gene expression

Gene expression, or rather mRNA transcripts of the genes yet to be translated into proteins, were compared in lung, kidney, and muscle. ADO2 patients have skeletal muscle symptoms but expression is low in this mouse model.

Volcano Plots in 1c-e can be a bit tricky to understand. All red dots above the p=0.05 can be ignored as their changes are not significant. Dots to the left of the vertical represent increased transcript. Dots to the left, decreased transcripts. The higher the dot on the graph, the more significant the change in the transcript. Panel 1d is a zen diagram. Transcripts were grouped into pathways in which the proteins they code for belong to. Ten pathways that were changed in ADO2 mice were common to these three diverse tissue types. The pathways are shown in 1h. Then Côrtes and coauthors moved on to the brain.

I’m not honestly sure what these graphs mean. I’ve seen the blue and red grids used for gene expression profiles. In this case, each row would be an individual animal. The white lines are new to me and not explained in the text. The take home message of this study is that G213R mutation changes gene transcription similarly in very different tissues.

2. Honing in on fibrosis related genes

The gene enrichment software revealed an increase in fibrosis related genes in the ADO2 mice.  Note that this is the genes…. Not just protein levels because of dysfunctional lysosome acidification.  The authors performed histological analysis to further define this observation.  Masson’s trichrome staining revealed more perivascular collagen in the ADO2 lungs as well as more expression of the mutant CIC7 protein.  More macrophages were present in ADO2 lungs (Fig. 2e). The increased Clcn7 expression in lungs was observed.  The authors were focused on the role of the tumor growth factor TFGβ signaling p known to be involved in fibrosis and found that collagen associated proteins were also upregulated: αSMA and Grem2, and collagen I and III (Fig. 2f). The involvement of TGFβ was confirmed by histological evidence of the TGFβ downstream downstream transcription factor, pSmad2/3.  Similar results were observed in the kidneys and muscle.

Figure 3  How does being ADO2 affect transcripts in phagocytic cells?

There are genes in pathways that are up regulated in the ADO2 mice that just cannot be explained by the proteolytic function of lysosomes.  Some, like long-term depression and neuroactive ling-receptor interaction pathways could explain the neurological symptoms.  Moving on to resident macrophage of  lungs, kidneys, and muscles. NES normalized enrichment score. Images are representative, and data are the mean ± S.D. of three mice per organ. 

Figure 3 of the Côrtes publication examines this question in peripheral blood mononuclear cells from WT and SADO2 mice.  A macrophage cell line was also genetically manipulated to resemble the PMN of the mice.  Only the graphs showing colocalization of markers are shown in this post.  Cartoons showing what these proteins do have been added for lay readers.


(Fig. 3b, Supplemental Fig. 5), and no difference in

the expression of the ER stress proteins, GRp94, Bip1, and ERp57

  • Calnexin, ER marker
  • Lamp1, lysosome marker
  • TGN46, trans-Golgi marker
  • EEA early endosome marker
  • γ-adaptin clathrin-coated vesicle marker,  paranuclear, suggesting an impaired vesicular transit through the Golgi stacks. Lysotracker® accumulated in the γ-adaptin-positive clathrin-coated vesicles of ADO2 cells, suggesting that this compartment exhibits a lower pH compared to WT cells, (not shown in this post). This result indirectly suggests that the mutant ClC7 keeps the chloride conductance capacity and that the pH in the γ-adaptin-positive clathrin-coated vesicles is likely to be
  • EEA2 early endosome marker ADO2 cells and incubated them with neutral red, a probe that is uptaken by these organelles and retained inside their membrane upon protonation. The results showed that the neutral red content in ADO2 lysosomes was significantly lower than in WT lysosomes
  • M6PR, mannose-6P Receptor was normal suggesting that ClC7 trafficking impairment did not involve its sorting receptor. M5PR targets lysosomal enzymes to the lysosome.

Supplemental data demonstrated that the ClC7 β-subunit Ostm1 did not perfectly track CIC7

Clcn7G213R mutation results in impairment of ClC7

  1. Golgi exit
  2. vesicular trafficking
  3. lysosomal acidification
  4. altered autophagy

in ADO2 cells. Interestingly, similar alterations were observed in ADO2 lungs, kidneys and muscles as exemplified by their increase of γ-adaptin .  No quantitation data were shown for LC3a; however, the immunocytochemistry images were unusually clear and suitable for those not trained in pathology. 

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