ADO2 is a debilitating genetic bone disease causing multiple fractures and other severe symptoms. A mouse model of ADO2, harbouring the heterozygous Clcn7G213R gene mutation, phenocopies the human syndrome. The Clc7 gene encodes the ClC7 dimeric 2Cl−/1H+ antiporter that is almost ubiquitously expressed, although the mutations hit especially the osteoclasts impairing bone resorption. By immunofluorescence, we observed that the mutant ClC7 was normally localized in the endoplasmic reticulum of monocytes and osteoclasts, significantly accumulated in the Golgi (+30-fold, P=0.01), which appeared enlarged and fragmented, and reduced in lysosomes (−0.52%, P=0.02). Lysosomes showed an impaired acidification (wildtype pH 3.63+0.19; mutant 5.27±0.68, P<0.001) improved by treatment with Clcn7G213R-specific siRNA (pH 4.64±0.69, P=0.03). The protein co-localised normally with OSTM1, suggesting no involvement of this pathway. In contrast, cells showed an increased expression of LC3, implying altered autophagy, typical of storage diseases. Since storage diseases may affect the brain, we subjected ADO2 mice to behavioural tests, observing increased anxiety (open field test, −48% time spent in centre; elevated plus maze test, −42% time spent in open arm; dark/light test, −46% time spent in lit compartment, P<0.05) and depression (forced swimming test, +1.4-fold time spent immobile, P<0.01). Conversely, memory, locomotion and aggressiveness were unaltered. Anxiety and depression worsened with age (+1.7, P<0.05), further supporting the hypothesis of a storage disease. Consistently, the Glo1 and Gad1 enzyme mRNAs, associated with anxiety/depression, but not the unrelated Gad2, Srr, Th and Dbh enzyme mRNAs, were increased in ADO2 brains (1.77±0.5, 1.23±0.07, respectively, P<0.05). Furthermore, β-amyloid aggregates accumulated in amygdala, cerebral cortex, hippocampus and thalamus, especially in aged (12 months old) ADO2 mice. Consistently, CLC7 protein accumulation was 15-fold increased in the Golgi of ADO2 primary neurons (P<0.05), which was 2.15-fold enlarged in hippocampus cryosections (P<0.02), confirming an aberrant localization of the mutant protein and the induction of a storage disease also in the neural cells.
14 - 17 May 2016
European Calcified Tissue Society