Biallelic NAA60 variants with impaired n-terminal acetylation capacity cause autosomal recessive primary familial brain calcifications.

Primary familial brain calcification (PFBC) is characterized by calcium deposition in the brain, causing progressive movement disorders, psychiatric symptoms, and cognitive decline. PFBC is a heterogeneous disorder currently linked to variants in six different genes, but most patients remain genetically undiagnosed. Here, we identify biallelic NAA60 variants in ten individuals from seven families with autosomal recessive PFBC. The NAA60 variants lead to loss-of-function with lack of protein N-terminal (Nt)-acetylation activity. We show that the phosphate importer SLC20A2 is a substrate of NAA60 in vitro. In cells, loss of NAA60 caused reduced surface levels of SLC20A2 and a reduction in extracellular phosphate uptake. This study establishes NAA60 as a causal gene for PFBC, provides a possible biochemical explanation of its disease-causing mechanisms and underscores NAA60-mediated Nt-acetylation of transmembrane proteins as a fundamental process for healthy neurobiological functioning.

Reticulon 2 deficiency results in an autosomal recessive distal motor neuropathy with lower limb spasticity.

Heterozygous RTN2 variants have been previously identified in a limited cohort of families affected by autosomal dominant spastic paraplegia (SPG12-OMIM:604805) with a variable age of onset. Nevertheless, the definitive validity of SPG12 remains to be confidently confirmed due to scarcity of supporting evidence. In our study, we identified and validated seven novel or ultra-rare homozygous loss-of-function RTN2 variants in 14 individuals from seven consanguineous families with distal hereditary motor neuropathy (dHMN) using exome, genome and Sanger sequencing coupled with deep-phenotyping. All affected individuals (seven males and seven females, aged 9-50 years) exhibited weakness in the distal upper and lower limbs, lower limb spasticity, hyperreflexia, with an onset in the first decade of life. Nerve conduction studies revealed axonal motor neuropathy with neurogenic changes in the electromyography. Despite a slowly progressive disease course, all patients remained ambulatory over a mean disease duration of 19.71 ± 13.70 years. Characterisation of C. elegans RTN2 homolog loss-of-function variants demonstrated morphological and behavioural differences compared to the parental strain. Treatment of the mutant with an endoplasmic/sarcoplasmic reticulum Ca2+ reuptake inhibitor (2,5-di-tert-butylhydroquinone) rescued key phenotypic differences, suggesting a potential therapeutic benefit for RTN2-disorder. Despite Reticulon-2 being an endoplasmic reticulum (ER)-resident membrane shaping protein, our analysis of patient fibroblast cells did not find significant alterations in ER structure or the response to ER stress. Our findings delineate a distinct form of autosomal recessive dHMN with pyramidal features associated with Reticulon-2 deficiency. This phenotype shares similarities with SIGMAR1-related dHMN, and Silver-like syndromes, providing valuable insights into the clinical spectrum and potential therapeutic strategies for RTN2-related dHMN.

Biallelic BORCS8 variants cause an infantile-onset neurodegenerative disorder with altered lysosome dynamics.

BLOC-One-Related Complex (BORC) is a multiprotein complex composed of eight subunits named BORCS1-8. BORC associates with the cytosolic face of lysosomes, where it sequentially recruits the small GTPase ARL8 and kinesin-1 and -3 microtubule motors to promote anterograde transport of lysosomes toward the peripheral cytoplasm in non-neuronal cells and the distal axon in neurons. The physiological and pathological importance of BORC in humans, however, remains to be determined. Here, we report the identification of compound heterozygous variants [missense c.85T > C (p.Ser29Pro) and frameshift c.71-75dupTGGCC (p.Asn26Trpfs*51)] and homozygous variants [missense c.196A > C (p.Thr66Pro) and c.124T > C (p.Ser42Pro)] in BORCS8 in five children with a severe early-infantile neurodegenerative disorder from three unrelated families. The children exhibit global developmental delay, severe-to-profound intellectual disability, hypotonia, limb spasticity, muscle wasting, dysmorphic facies, optic atrophy, leuko-axonopathy with hypomyelination, and neurodegenerative features with prevalent supratentorial involvement. Cellular studies using a heterologous transfection system show that the BORCS8 missense variants p.Ser29Pro, p.Ser42Pro and p.Thr66Pro are expressed at normal levels but exhibit reduced assembly with other BORC subunits and reduced ability to drive lysosome distribution toward the cell periphery. The BORCS8 frameshift variant p.Asn26Trpfs*51, on the other hand, is expressed at lower levels and is completely incapable of assembling with other BORC subunits and promoting lysosome distribution toward the cell periphery. Therefore, all the BORCS8 variants are partial or total loss-of-function alleles and are thus likely pathogenic. Knockout of the orthologous borcs8 in zebrafish causes decreased brain and eye size, neuromuscular anomalies and impaired locomotion, recapitulating some of the key traits of the human disease. These findings thus identify BORCS8 as a novel genetic locus for an early-infantile neurodegenerative disorder and highlight the critical importance of BORC and lysosome dynamics for the development and function of the central nervous system.

Bi-allelic loss-of-function variants in WBP4, encoding a spliceosome protein, result in a variable neurodevelopmental syndrome.

Over two dozen spliceosome proteins are involved in human diseases, also referred to as spliceosomopathies. WW domain-binding protein 4 (WBP4) is part of the early spliceosomal complex and has not been previously associated with human pathologies in the Online Mendelian Inheritance in Man (OMIM) database. Through GeneMatcher, we identified ten individuals from eight families with a severe neurodevelopmental syndrome featuring variable manifestations. Clinical manifestations included hypotonia, global developmental delay, severe intellectual disability, brain abnormalities, musculoskeletal, and gastrointestinal abnormalities. Genetic analysis revealed five different homozygous loss-of-function variants in WBP4. Immunoblotting on fibroblasts from two affected individuals with different genetic variants demonstrated a complete loss of protein, and RNA sequencing analysis uncovered shared abnormal splicing patterns, including in genes associated with abnormalities of the nervous system, potentially underlying the phenotypes of the probands. We conclude that bi-allelic variants in WBP4 cause a developmental disorder with variable presentations, adding to the growing list of human spliceosomopathies.

Evaluating the association of biallelic OGDHL variants with significant phenotypic heterogeneity.

BACKGROUND: Biallelic variants in OGDHL, encoding part of the α-ketoglutarate dehydrogenase complex, have been associated with highly heterogeneous neurological and neurodevelopmental disorders. However, the validity of this association remains to be confirmed. A second OGDHL patient cohort was recruited to carefully assess the gene-disease relationship. METHODS: Using an unbiased genotype-first approach, we screened large, multiethnic aggregated sequencing datasets worldwide for biallelic OGDHL variants. We used CRISPR/Cas9 to generate zebrafish knockouts of ogdhl, ogdh paralogs, and dhtkd1 to investigate functional relationships and impact during development. Functional complementation with patient variant transcripts was conducted to systematically assess protein functionality as a readout for pathogenicity. RESULTS: A cohort of 14 individuals from 12 unrelated families exhibited highly variable clinical phenotypes, with the majority of them presenting at least one additional variant, potentially accounting for a blended phenotype and complicating phenotypic understanding. We also uncovered extreme clinical heterogeneity and high allele frequencies, occasionally incompatible with a fully penetrant recessive disorder. Human cDNA of previously described and new variants were tested in an ogdhl zebrafish knockout model, adding functional evidence for variant reclassification. We disclosed evidence of hypomorphic alleles as well as a loss-of-function variant without deleterious effects in zebrafish variant testing also showing discordant familial segregation, challenging the relationship of OGDHL as a conventional Mendelian gene. Going further, we uncovered evidence for a complex compensatory relationship among OGDH, OGDHL, and DHTKD1 isoenzymes that are associated with neurodevelopmental disorders and exhibit complex transcriptional compensation patterns with partial functional redundancy. CONCLUSIONS: Based on the results of genetic, clinical, and functional studies, we formed three hypotheses in which to frame observations: biallelic OGDHL variants lead to a highly variable monogenic disorder, variants in OGDHL are following a complex pattern of inheritance, or they may not be causative at all. Our study further highlights the continuing challenges of assessing the validity of reported disease-gene associations and effects of variants identified in these genes. This is particularly more complicated in making genetic diagnoses based on identification of variants in genes presenting a highly heterogenous phenotype such as "OGDHL-related disorders".

Lunapark deficiency leads to an autosomal recessive neurodevelopmental phenotype with a degenerative course, epilepsy and distinct brain anomalies.

LNPK encodes a conserved membrane protein that stabilizes the junctions of the tubular endoplasmic reticulum network playing crucial roles in diverse biological functions. Recently, homozygous variants in LNPK were shown to cause a neurodevelopmental disorder (OMIM#618090) in four patients displaying developmental delay, epilepsy and nonspecific brain malformations including corpus callosum hypoplasia and variable impairment of cerebellum. We sought to delineate the molecular and phenotypic spectrum of LNPK-related disorder. Exome or genome sequencing was carried out in 11 families. Thorough clinical and neuroradiological evaluation was performed for all the affected individuals, including review of previously reported patients. We identified 12 distinct homozygous loss-of-function variants in 16 individuals presenting with moderate to profound developmental delay, cognitive impairment, regression, refractory epilepsy and a recognizable neuroimaging pattern consisting of corpus callosum hypoplasia and signal alterations of the forceps minor ('ear-of-the-lynx' sign), variably associated with substantia nigra signal alterations, mild brain atrophy, short midbrain and cerebellar hypoplasia/atrophy. In summary, we define the core phenotype of LNPK-related disorder and expand the list of neurological disorders presenting with the 'ear-of-the-lynx' sign suggesting a possible common underlying mechanism related to endoplasmic reticulum-phagy dysfunction.

Biallelic loss of function variants in WBP4, encoding a spliceosome protein, result in a variable neurodevelopmental delay syndrome.

Over two dozen spliceosome proteins are involved in human diseases, also referred to as spliceosomopathies. WBP4 (WW Domain Binding Protein 4) is part of the early spliceosomal complex, and was not described before in the context of human pathologies. Ascertained through GeneMatcher we identified eleven patients from eight families, with a severe neurodevelopmental syndrome with variable manifestations. Clinical manifestations included hypotonia, global developmental delay, severe intellectual disability, brain abnormalities, musculoskeletal and gastrointestinal abnormalities. Genetic analysis revealed overall five different homozygous loss-of-function variants in WBP4. Immunoblotting on fibroblasts from two affected individuals with different genetic variants demonstrated complete loss of protein, and RNA sequencing analysis uncovered shared abnormal splicing patterns, including enrichment for abnormalities of the nervous system and musculoskeletal system genes, suggesting that the overlapping differentially spliced genes are related to the common phenotypes of the probands. We conclude that biallelic variants in WBP4 cause a spliceosomopathy. Further functional studies are called for better understanding of the mechanism of pathogenicity.

Gain and loss of function variants in EZH1 disrupt neurogenesis and cause dominant and recessive neurodevelopmental disorders.

Genetic variants in chromatin regulators are frequently found in neurodevelopmental disorders, but their effect in disease etiology is rarely determined. Here, we uncover and functionally define pathogenic variants in the chromatin modifier EZH1 as the cause of dominant and recessive neurodevelopmental disorders in 19 individuals. EZH1 encodes one of the two alternative histone H3 lysine 27 methyltransferases of the PRC2 complex. Unlike the other PRC2 subunits, which are involved in cancers and developmental syndromes, the implication of EZH1 in human development and disease is largely unknown. Using cellular and biochemical studies, we demonstrate that recessive variants impair EZH1 expression causing loss of function effects, while dominant variants are missense mutations that affect evolutionarily conserved aminoacids, likely impacting EZH1 structure or function. Accordingly, we found increased methyltransferase activity leading to gain of function of two EZH1 missense variants. Furthermore, we show that EZH1 is necessary and sufficient for differentiation of neural progenitor cells in the developing chick embryo neural tube. Finally, using human pluripotent stem cell-derived neural cultures and forebrain organoids, we demonstrate that EZH1 variants perturb cortical neuron differentiation. Overall, our work reveals a critical role of EZH1 in neurogenesis regulation and provides molecular diagnosis for previously undefined neurodevelopmental disorders.

The clinical and genetic spectrum of autosomal-recessive TOR1A-related disorders.

In the field of rare diseases, progress in molecular diagnostics led to the recognition that variants linked to autosomal-dominant neurodegenerative diseases of later onset can, in the context of biallelic inheritance, cause devastating neurodevelopmental disorders and infantile or childhood-onset neurodegeneration. TOR1A-associated arthrogryposis multiplex congenita 5 (AMC5) is a rare neurodevelopmental disorder arising from biallelic variants in TOR1A, a gene that in the heterozygous state is associated with torsion dystonia-1 (DYT1 or DYT-TOR1A), an early-onset dystonia with reduced penetrance. While 15 individuals with AMC5-TOR1A have been reported (less than 10 in detail), a systematic investigation of the full disease-associated spectrum has not been conducted. Here, we assess the clinical, radiological and molecular characteristics of 57 individuals from 40 families with biallelic variants in TOR1A. Median age at last follow-up was 3 years (0-24 years). Most individuals presented with severe congenital flexion contractures (95%) and variable developmental delay (79%). Motor symptoms were reported in 79% and included lower limb spasticity and pyramidal signs, as well as gait disturbances. Facial dysmorphism was an integral part of the phenotype, with key features being a broad/full nasal tip, narrowing of the forehead and full cheeks. Analysis of disease-associated manifestations delineated a phenotypic spectrum ranging from normal cognition and mild gait disturbance to congenital arthrogryposis, global developmental delay, intellectual disability, absent speech and inability to walk. In a subset, the presentation was consistent with foetal akinesia deformation sequence with severe intrauterine abnormalities. Survival was 71%, with higher mortality in males. Death occurred at a median age of 1.2 months (1 week-9 years), due to respiratory failure, cardiac arrest or sepsis. Analysis of brain MRI studies identified non-specific neuroimaging features, including a hypoplastic corpus callosum (72%), foci of signal abnormality in the subcortical and periventricular white matter (55%), diffuse white matter volume loss (45%), mega cisterna magna (36%) and arachnoid cysts (27%). The molecular spectrum included 22 distinct variants, defining a mutational hotspot in the C-terminal domain of the Torsin-1A protein. Genotype-phenotype analysis revealed an association of missense variants in the 3-helix bundle domain to an attenuated phenotype, while missense variants near the Walker A/B motif as well as biallelic truncating variants were linked to early death. In summary, this systematic cross-sectional analysis of a large cohort of individuals with biallelic TOR1A variants across a wide age-range delineates the clinical and genetic spectrum of TOR1A-related autosomal-recessive disease and highlights potential predictors for disease severity and survival.

Biallelic loss of LDB3 leads to a lethal pediatric dilated cardiomyopathy.

Autosomal dominant variants in LDB3 (also known as ZASP), encoding the PDZ-LIM domain-binding factor, have been linked to a late onset phenotype of cardiomyopathy and myofibrillar myopathy in humans. However, despite knockout mice displaying a much more severe phenotype with premature death, bi-allelic variants in LDB3 have not yet been reported. Here we identify biallelic loss-of-function variants in five unrelated cardiomyopathy families by next-generation sequencing. In the first family, we identified compound heterozygous LOF variants in LDB3 in a fetus with bilateral talipes and mild left cardiac ventricular enlargement. Ultra-structural examination revealed highly irregular Z-disc formation, and RNA analysis demonstrated little/no expression of LDB3 protein with a functional C-terminal LIM domain in muscle tissue from the affected fetus. In a second family, a homozygous LDB3 nonsense variant was identified in a young girl with severe early-onset dilated cardiomyopathy with left ventricular non-compaction; the same homozygous nonsense variant was identified in a third unrelated female infant with dilated cardiomyopathy. We further identified homozygous LDB3 frameshift variants in two unrelated probands diagnosed with cardiomegaly and severely reduced left ventricular ejection fraction. Our findings demonstrate that recessive LDB3 variants can lead to an early-onset severe human phenotype of cardiomyopathy and myopathy, reminiscent of the knockout mouse phenotype, and supporting a loss of function mechanism.

A homozygous MED11 C-terminal variant causes a lethal neurodegenerative disease.

PURPOSE: The mediator (MED) multisubunit-complex modulates the activity of the transcriptional machinery, and genetic defects in different MED subunits (17, 20, 27) have been implicated in neurologic diseases. In this study, we identified a recurrent homozygous variant in MED11 (c.325C>T; p.Arg109Ter) in 7 affected individuals from 5 unrelated families. METHODS: To investigate the genetic cause of the disease, exome or genome sequencing were performed in 5 unrelated families identified via different research networks and Matchmaker Exchange. Deep clinical and brain imaging evaluations were performed by clinical pediatric neurologists and neuroradiologists. The functional effect of the candidate variant on both MED11 RNA and protein was assessed using reverse transcriptase polymerase chain reaction and western blotting using fibroblast cell lines derived from 1 affected individual and controls and through computational approaches. Knockouts in zebrafish were generated using clustered regularly interspaced short palindromic repeats/Cas9. RESULTS: The disease was characterized by microcephaly, profound neurodevelopmental impairment, exaggerated startle response, myoclonic seizures, progressive widespread neurodegeneration, and premature death. Functional studies on patient-derived fibroblasts did not show a loss of protein function but rather disruption of the C-terminal of MED11, likely impairing binding to other MED subunits. A zebrafish knockout model recapitulates key clinical phenotypes. CONCLUSION: Loss of the C-terminal of MED subunit 11 may affect its binding efficiency to other MED subunits, thus implicating the MED-complex stability in brain development and neurodegeneration.

Biallelic Variants in the Ectonucleotidase ENTPD1 Cause a Complex Neurodevelopmental Disorder with Intellectual Disability, Distinct White Matter Abnormalities, and Spastic Paraplegia.

OBJECTIVE: Human genomics established that pathogenic variation in diverse genes can underlie a single disorder. For example, hereditary spastic paraplegia is associated with >80 genes, with frequently only few affected individuals described for each gene. Herein, we characterize a large cohort of individuals with biallelic variation in ENTPD1, a gene previously linked to spastic paraplegia 64 (Mendelian Inheritance in Man # 615683). METHODS: Individuals with biallelic ENTPD1 variants were recruited worldwide. Deep phenotyping and molecular characterization were performed. RESULTS: A total of 27 individuals from 17 unrelated families were studied; additional phenotypic information was collected from published cases. Twelve novel pathogenic ENTPD1 variants are described (NM 001776.6): c.398_399delinsAA; p.(Gly133Glu), c.540del; p.(Thr181Leufs*18), c.640del; p.(Gly216Glufs*75), c.185 T > G; p.(Leu62*), c.1531 T > C; p.(*511Glnext*100), c.967C > T; p.(Gln323*), c.414-2_414-1del, and c.146 A > G; p.(Tyr49Cys) including 4 recurrent variants c.1109 T > A; p.(Leu370*), c.574-6_574-3del, c.770_771del; p.(Gly257Glufs*18), and c.1041del; p.(Ile348Phefs*19). Shared disease traits include childhood onset, progressive spastic paraplegia, intellectual disability (ID), dysarthria, and white matter abnormalities. In vitro assays demonstrate that ENTPD1 expression and function are impaired and that c.574-6_574-3del causes exon skipping. Global metabolomics demonstrate ENTPD1 deficiency leads to impaired nucleotide, lipid, and energy metabolism. INTERPRETATION: The ENTPD1 locus trait consists of childhood disease onset, ID, progressive spastic paraparesis, dysarthria, dysmorphisms, and white matter abnormalities, with some individuals showing neurocognitive regression. Investigation of an allelic series of ENTPD1 (1) expands previously described features of ENTPD1-related neurological disease, (2) highlights the importance of genotype-driven deep phenotyping, (3) documents the need for global collaborative efforts to characterize rare autosomal recessive disease traits, and (4) provides insights into disease trait neurobiology. ANN NEUROL 2022;92:304-321.

Altered voltage-dependence of slowly activating chloride-proton antiport by late endosomal ClC-6 explains distinct neurological disorders.

ClC-6 is an intracellularly localised member of the CLC family of chloride transport proteins. It presumably functions in the endolysosomal compartment as a chloride-proton antiporter, despite a paucity of biophysical studies in direct support. Observations of lysosomal storage disease, as well as neurodegenerative disorders, emerge with its disruption by knockout or mutation, respectively. An incomplete understanding of wild-type ClC-6 function obscures clear mechanistic insight into disease aetiology. Here, high-resolution recording protocols that incorporate extreme voltage pulses permit detailed biophysical measurement and analysis of transient capacitive, as well as ionic transport currents. This approach reveals that wild-type ClC-6 activation and transport require depolarisation to voltages beyond 140 mV. Mutant Y553C associated with early-onset neurodegeneration exerts gain-of-function by shifting the half-maximal voltage for activation to less depolarised voltages. Moreover, we show that the E267A proton glutamate mutant conserves transport currents, albeit reduced. Lastly, the positive shift in activation voltage shown by V580M, a mutant identified in a patient with late-onset lysosomal storage disease, can explain loss-of-function leading to disease. KEY POINTS: Ionic composition and pH within intracellular compartments, such as endolysosomes, rely on the activity of chloride/proton transporters including ClC-6. Distinct CLCN6 mutations were previously found in individuals with neurodegenerative disease, and also putatively associated with neuronal ceroid lipofuscinosis. Limited knowledge of wild-type ClC-6 transport function impedes understanding of mechanisms underlying these conditions. We resolved transient and transport currents that permit measurement of voltage- and pH-dependences, as well as kinetics, for wild-type and disease-associated mutant ClC-6s. These findings define wild-type ClC-6 function robustly, and reveal how alterations of the slow activation gating of the transporter cause different kinds of neurological diseases.

The Role of the Lysosomal Cl-/H+ Antiporter ClC-7 in Osteopetrosis and Neurodegeneration.

CLC proteins comprise Cl- channels and anion/H+ antiporters involved in several fundamental physiological processes. ClC-7 is a lysosomal Cl-/H+ antiporter that together with its beta subunit Ostm1 has a critical role in the ionic homeostasis of lysosomes and of the osteoclasts' resorption lacuna, although the specific underlying mechanism has so far remained elusive. Mutations in ClC-7 cause osteopetrosis, but also a form of lysosomal storage disease and neurodegeneration. Interestingly, both loss-of- and gain-of-function mutations of ClC-7 can be pathogenic, but the mechanistic implications of this finding are still unclear. This review will focus on the recent advances in our understanding of the biophysical properties of ClC-7 and of its role in human diseases with a focus on osteopetrosis and neurodegeneration.

TRPM2 Oxidation Activates Two Distinct Potassium Channels in Melanoma Cells through Intracellular Calcium Increase.

Tumor microenvironments are often characterized by an increase in oxidative stress levels. We studied the response to oxidative stimulation in human primary (IGR39) or metastatic (IGR37) cell lines obtained from the same patient, performing patch-clamp recordings, intracellular calcium ([Ca2+]i) imaging, and RT-qPCR gene expression analysis. In IGR39 cells, chloramine-T (Chl-T) activated large K+ currents (KROS) that were partially sensitive to tetraethylammonium (TEA). A large fraction of KROS was inhibited by paxilline-a specific inhibitor of large-conductance Ca2+-activated BK channels. The TEA-insensitive component was inhibited by senicapoc-a specific inhibitor of the Ca2+-activated KCa3.1 channel. Both BK and KCa3.1 activation were mediated by an increase in [Ca2+]i induced by Chl-T. Both KROS and [Ca2+]i increase were inhibited by ACA and clotrimazole-two different inhibitors of the calcium-permeable TRPM2 channel. Surprisingly, IGR37 cells did not exhibit current increase upon the application of Chl-T. Expression analysis confirmed that the genes encoding BK, KCa3.1, and TRPM2 are much more expressed in IGR39 than in IGR37. The potassium currents and [Ca2+]i increase observed in response to the oxidizing agent strongly suggest that these three molecular entities play a major role in the progression of melanoma. Pharmacological targeting of either of these ion channels could be a new strategy to reduce the metastatic potential of melanoma cells, and could complement classical radio- or chemotherapeutic treatments.

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. © 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).

Large transient capacitive currents in wild-type lysosomal Cl-/H+ antiporter ClC-7 and residual transport activity in the proton glutamate mutant E312A.

ClC-7 is a lysosomal 2 Cl-/1 H+ antiporter of the CLC protein family, which comprises Cl- channels and other Cl-/H+ antiporters. Mutations in ClC-7 and its associated ß subunit Ostm1 lead to osteopetrosis and lysosomal storage disease in humans and mice. Previous studies on other mammalian CLC transporters showed that mutations of a conserved, intracellularly located glutamate residue, the so-called proton glutamate, abolish steady-state transport activity but increase transient capacitive currents associated with partial reactions of the transport cycle. In contrast, we observed large, transient capacitive currents for the wild-type ClC-7, which depend on external pH and internal, but not external, Cl-. Very similar transient currents were observed for the E312A mutant of the proton glutamate. Interestingly, and unlike in other mammalian CLC transporters investigated so far, the E312A mutation strongly reduces, but does not abolish, stationary transport currents, potentially explaining the intermediate phenotype observed in the E312A mouse line.

New Insights into the Mechanism of NO3- Selectivity in the Human Kidney Chloride Channel ClC-Ka and the CLC Protein Family.

BACKGROUND: The mechanism of anion selectivity in the human kidney chloride channels ClC-Ka and ClC-Kb is unknown. However, it has been thought to be very similar to that of other channels and antiporters of the CLC protein family, and to rely on anions interacting with a conserved Ser residue (Sercen) at the center of three anion binding sites in the permeation pathway Scen. In both CLC channels and antiporters, mutations of Sercen alter the anion selectivity. Structurally, the side chain of Sercen of CLC channels and antiporters typically projects into the pore and coordinates the anion bound at Scen. METHODS: To investigate the role of several residues in anion selectivity of ClC-Ka, we created mutations that resulted in amino acid substitutions in these residues. We also used electrophysiologic techniques to assess the properties of the mutants. RESULTS: Mutations in ClC-Ka that change Sercen to Gly, Pro, or Thr have only minor effects on anion selectivity, whereas the mutations in residues Y425A, F519A, and Y520A increase the NO3-/Cl- permeability ratio, with Y425A having a particularly strong effect. CONCLUSION: s ClC-Ka's mechanism of anion selectivity is largely independent of Sercen, and it is therefore unique in the CLC protein family. We identified the residue Y425 in ClC-Ka-and the corresponding residue (A417) in the chloride channel ClC-0-as residues that contribute to NO3- discrimination in these channels. This work provides important and timely insight into the relationship between structure and function for the kidney chloride channels ClC-Ka and ClC-Kb, and for CLC proteins in general.

The 2018 correlative microscopy techniques roadmap.

Developments in microscopy have been instrumental to progress in the life sciences, and many new techniques have been introduced and led to new discoveries throughout the last century. A wide and diverse range of methodologies is now available, including electron microscopy, atomic force microscopy, magnetic resonance imaging, small-angle x-ray scattering and multiple super-resolution fluorescence techniques, and each of these methods provides valuable read-outs to meet the demands set by the samples under study. Yet, the investigation of cell development requires a multi-parametric approach to address both the structure and spatio-temporal organization of organelles, and also the transduction of chemical signals and forces involved in cell-cell interactions. Although the microscopy technologies for observing each of these characteristics are well developed, none of them can offer read-out of all characteristics simultaneously, which limits the information content of a measurement. For example, while electron microscopy is able to disclose the structural layout of cells and the macromolecular arrangement of proteins, it cannot directly follow dynamics in living cells. The latter can be achieved with fluorescence microscopy which, however, requires labelling and lacks spatial resolution. A remedy is to combine and correlate different readouts from the same specimen, which opens new avenues to understand structure-function relations in biomedical research. At the same time, such correlative approaches pose new challenges concerning sample preparation, instrument stability, region of interest retrieval, and data analysis. Because the field of correlative microscopy is relatively young, the capabilities of the various approaches have yet to be fully explored, and uncertainties remain when considering the best choice of strategy and workflow for the correlative experiment. With this in mind, the Journal of Physics D: Applied Physics presents a special roadmap on the correlative microscopy techniques, giving a comprehensive overview from various leading scientists in this field, via a collection of multiple short viewpoints.

A tale of two CLCs: biophysical insights toward understanding ClC-5 and ClC-7 function in endosomes and lysosomes.

The CLC protein family comprises both Cl(-) channels and H(+) -coupled anion transporters. The understanding of the critical role of CLC proteins in a number of physiological functions has greatly contributed to a revision of the classical paradigm that attributed to Cl(-) ions only a marginal role in human physiology. The endosomal ClC-5 and the lysosomal ClC-7 are the best characterized human CLC transporters. Their dysfunction causes Dent's disease and osteopetrosis, respectively. It had been originally proposed that they would provide a Cl(-) shunt conductance allowing efficient acidification of intracellular compartments. However, this model seems to conflict with the transport properties of these proteins and with recent physiological evidence. Currently, there is no consensus on their specific physiological role. CLC proteins present also a number of peculiar biophysical properties, such as the dimeric architecture, the co-existence of intrinsically different thermodynamic modes of transport based on similar structural principles, and the gating mechanism recently emerging for the transporters, just to name a few. This review focuses on the biophysical properties and physiological roles of ClC-5 and ClC-7.