Neurodegeneration Upon Dysfunction of Endosomal/Lysosomal CLC Chloride Transporters

The regulation of luminal ion concentrations is critical for the function of, and transport between intracellular organelles. The importance of the acidic pH in the compartments of the endosomal-lysosomal pathway has been well-known for decades. Besides the V-ATPase, which pumps protons into their lumen, a variety of ion transporters and channels is involved in the regulation of the organelles' complex ion homeostasis. Amongst these are the intracellular members of the CLC family, ClC-3 through ClC-7. They localize to distinct but overlapping compartments of the endosomal-lysosomal pathway, partially with tissue-specific expression. Functioning as 2Cl−/H+ exchangers, they can support the vesicular acidification and accumulate luminal Cl−. Mutations in the encoding genes in patients and mouse models underlie severe phenotypes including kidney stones with CLCN5 and osteopetrosis or hypopigmentation with CLCN7. Dysfunction of those intracellular CLCs that are expressed in neurons lead to neuronal defects. Loss of endosomal ClC-3, which heteromerizes with ClC-4, results in neurodegeneration. Mutations in ClC-4 are associated with epileptic encephalopathy and intellectual disability. Mice lacking the late endosomal ClC-6 develop a lysosomal storage disease with reduced pain sensitivity. Human gene variants have been associated with epilepsy, and a gain-of-function mutation causes early-onset neurodegeneration. Dysfunction of the lysosomal ClC-7 leads to a lysosomal storage disease and neurodegeneration in mice and humans. Reduced luminal chloride, as well as altered calcium regulation, has been associated with lysosomal storage diseases in general. This review discusses the properties of endosomal and lysosomal Cl−/H+ exchange by CLCs and how various alterations of ion transport by CLCs impact organellar ion homeostasis and function in neurodegenerative disorders

Impaired Autophagic Clearance with a Gain-of-Function Variant of the Lysosomal Cl−/H+ Exchanger ClC-7

ClC-7 is a ubiquitously expressed voltage-gated Cl−/H+ exchanger that critically contributes to lysosomal ion homeostasis. Together with its β-subunit Ostm1, ClC-7 localizes to lysosomes and to the ruffled border of osteoclasts, where it supports the acidification of the resorption lacuna. Loss of ClC-7 or Ostm1 leads to osteopetrosis accompanied by accumulation of storage material in lysosomes and neurodegeneration. Interestingly, not all osteopetrosis-causing CLCN7 mutations from patients are associated with a loss of ion transport. Some rather result in an acceleration of voltage-dependent ClC-7 activation. Recently, a gain-of-function variant, ClC-7Y715C, that yields larger ion currents upon heterologous expression, was identified in two patients with neurodegeneration, organomegaly and albinism. However, neither the patients nor a mouse model that carried the equivalent mutation developed osteopetrosis, although expression of ClC-7Y715C induced the formation of enlarged intracellular vacuoles. Here, we investigated how, in transfected cells with mutant ClC-7, the substitution of this tyrosine impinged on the morphology and function of lysosomes. Combinations of the tyrosine mutation with mutations that either uncouple Cl− from H+ counter-transport or strongly diminish overall ion currents were used to show that increased ClC-7 Cl−/H+ exchange activity is required for the formation of enlarged vacuoles by membrane fusion. Degradation of endocytosed material was reduced in these compartments and resulted in an accumulation of lysosomal storage material. In cells expressing the ClC-7 gain-of-function mutant, autophagic clearance was largely impaired, resulting in a build-up of autophagic material

Impaired Autophagic Clearance with a Gain-of-Function Variant of the Lysosomal Cl -/H + Exchanger ClC-7.

ClC-7 is a ubiquitously expressed voltage-gated Cl -/H + exchanger that critically contributes to lysosomal ion homeostasis. Together with its β-subunit Ostm1, ClC-7 localizes to lysosomes and to the ruffled border of osteoclasts, where it supports the acidification of the resorption lacuna. Loss of ClC-7 or Ostm1 leads to osteopetrosis accompanied by accumulation of storage material in lysosomes and neurodegeneration. Interestingly, not all osteopetrosis-causing CLCN7 mutations from patients are associated with a loss of ion transport. Some rather result in an acceleration of voltage-dependent ClC-7 activation. Recently, a gain-of-function variant, ClC-7 Y715C, that yields larger ion currents upon heterologous expression, was identified in two patients with neurodegeneration, organomegaly and albinism. However, neither the patients nor a mouse model that carried the equivalent mutation developed osteopetrosis, although expression of ClC-7 Y715C induced the formation of enlarged intracellular vacuoles. Here, we investigated how, in transfected cells with mutant ClC-7, the substitution of this tyrosine impinged on the morphology and function of lysosomes. Combinations of the tyrosine mutation with mutations that either uncouple Cl - from H + counter-transport or strongly diminish overall ion currents were used to show that increased ClC-7 Cl -/H + exchange activity is required for the formation of enlarged vacuoles by membrane fusion. Degradation of endocytosed material was reduced in these compartments and resulted in an accumulation of lysosomal storage material. In cells expressing the ClC-7 gain-of-function mutant, autophagic clearance was largely impaired, resulting in a build-up of autophagic material

Efficient generation of osteoclasts from human induced pluripotent stem cells and functional investigations of lethal CLCN7‐related osteopetrosis

Human induced pluripotent stem cells (hiPSCs) hold great potential for modeling human diseases and the development of innovative therapeutic approaches. Here, we report on a novel, simplified differentiation method for forming functional osteoclasts from hiPSCs. The three-step protocol starts with embryoid body formation, followed by hematopoietic specification, and finally osteoclast differentiation. We observed continuous production of monocyte-like cells over a period of up to 9 weeks, generating sufficient material for several osteoclast differentiations. The analysis of stage-specific gene and surface marker expression proved mesodermal priming, the presence of monocyte-like cells, and of terminally differentiated multinucleated osteoclasts, able to form resorption pits and trenches on bone and dentine in vitro. In comparison to peripheral blood mononuclear cell (PBMC)-derived osteoclasts hiPSC-derived osteoclasts were larger and contained a higher number of nuclei. Detailed functional studies on the resorption behavior of hiPSC-osteoclasts indicated a trend towards forming more trenches than pits and an increase in pseudoresorption. We used hiPSCs from an autosomal recessive osteopetrosis (ARO) patient (BIHi002-A, ARO hiPSCs) with compound heterozygous missense mutations p.(G292E) and p.(R403Q) in CLCN7, coding for the Cl-/H+-exchanger ClC-7, for functional investigations. The patient's leading clinical feature was a brain malformation due to defective neuronal migration. Mutant ClC-7 displayed residual expression and retained lysosomal co-localization with OSTM1, the gene coding for the osteopetrosis-associated transmembrane protein 1, but only ClC-7 harboring the mutation p.(R403Q) gave strongly reduced ion currents. An increased autophagic flux in spite of unchanged lysosomal pH was evident in undifferentiated ARO hiPSCs. ARO hiPSC-derived osteoclasts showed an increased size compared to hiPSCs of healthy donors. They were not able to resorb bone, underlining a loss-of-function effect of the mutations. In summary, we developed a highly reproducible, straightforward hiPSC-osteoclast differentiation protocol. We demonstrated that osteoclasts differentiated from ARO hiPSCs can be used as a disease model for ARO and potentially also other osteoclast-related diseases. (c) 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)