AIM:The dental pulp is an immunologically active tissue that responds dynamically to cariogenic challenge. Peripheral pulp cells adjacent to dentine encounter bacterial stimuli earlier than cells located in the central pulp. To investigate signalling and immune interactions, this study profiled the transcriptomes of dentine-adherent cells (DACs) and central dental pulp cells (DPCs) cocultured with Streptococcus mutans. METHODOLOGY:Primary cultures of both DACs and DPCs were obtained from healthy third molars of three female and three male donors aged 13-16. Cells were cocultured with viable S. mutans (2 × 108 CFU/mL) for 6 h (n = 6). Controls included γ-inactivated bacteria and unexposed cells. RNA libraries (Illumina Stranded mRNA Prep) were sequenced on a NextSeq2000. Bioinformatic analysis included differential gene expression (DESeq2), gene set enrichment analysis (GSEA), and protein-protein interaction (PPI) network construction. Batch effects were corrected, and significantly regulated genes (|log2FC| > 1.5, padj < 0.05) were identified. Validation of DEGs was performed via reverse transcription quantitative polymerase chain reaction (RT-qPCR). RESULTS:RNA-Seq revealed a dynamic shift in the transcriptome of DACs and DPCs stimulated with S. mutans, while cells exposed to γ-inactivated or no bacteria did not. Although DACs and DPCs shared common DEGs (33 up, 8 down), several regulations were exclusive to DACs (22 up, 9 down) and DPCs (9 up, 25 down), highlighting a donor-independent functional specificity of the pulp subpopulations. Functional enrichment analysis revealed a strong and comparable activation of hypoxia-related pathways in both DPCs and DACs. However, DACs additionally showed enrichment in extracellular matrix organisation and cytokine signalling, while DPCs were characterised by intracellular stress responses and protein folding pathways. Additionally, protein-protein interaction analysis identified IL-6 as a key hub in DACs, while ANGPTL4 was central in DPCs. CONCLUSION:Following exposure to S. mutans, mechanically isolated DACs and DPCs displayed distinct transcriptomic profiles, indicating functional heterogeneity in the pulpal immune response. DACs engaged immunomodulatory pathways, while DPCs were marked by cellular stress responses, suggesting divergent contributions to tissue defence and homeostasis.

BeadChip array-based DNA methylation profiling has been recognized by the World Health Organization (WHO) as a key diagnostic tool for brain tumor classification. While its diagnostic utility has been well established, data on technical reproducibility, interlaboratory comparability, and data interpretation under real-world diagnostic conditions remain limited. Bridging this gap, we here report the results of an international proficiency test using the Infinium MethylationEPIC v2.0 platform and the corresponding Brain Tumor Classifier version 12.8. Tissue slides of eight FFPE brain tumor samples, covering a representative range of CNS tumor entities, were distributed among 24 laboratories in 10 different countries. Participants were asked to report methylation classes and copy number variation (CNV) profiles. Pre-array workflows were left to local procedures and results had to be submitted within 15 working days. Technical data reproducibility was high with a median pairwise beta-value correlation of 0.99 (range 0.93-1.0). In general, participating centers generated high-quality data, reflected by consistently low detection p-values (<0.01). Eighteen of the 24 participating centers (75%) successfully passed the test. Of the six centers that failed the test, two laboratories experienced technical issues that led to misclassification of individual cases and contributed to incorrect CNV reporting. Four additional centers showed substantial discrepancies in the interpretation of diagnostically highly relevant CNVs, whereas methylation classification was not impaired. While accurate DNA quantification proved to be an important pre-array step, the use of the DNA restoration kit had only minor influence on overall results. Taken together, our interlaboratory performance testing on EPIC v2.0 CNS tumor profiling confirms high reproducibility of tumor classification but reveals the need for harmonized CNV reporting.

Abstract Background Mucolipidosis type II (ML II) is a lysosomal storage disorder caused by deficiency of N-acetylglucosamine-1-phosphotransferase (GlcNAc-PT), which impairs the trafficking of lysosomal hydrolases. Of all ML II pathogenic variants, c.3503_3504del in GNPTAB exon 19 is the most prevalent, therefore constituting a compelling molecular target for the development of personalized therapeutic strategies. Here, we explored the feasibility of an innovative RNA based-therapeutic strategy using antisense oligonucleotides (ASOs) designed to induce exon 19 skipping in GNPTAB pre-mRNA. This approach was previously successfully tested at the mRNA level in fibroblasts from ML II patients, where it generated an in-frame mRNA. In the present study, our aim was to evaluate whether GNPTAB exon 19 skipping could increase GlcNAc-PT levels and consequently improve the cellular phenotype of ML II patients carrying this pathogenic variant. To address this, we designed a functional approach based on the overexpression of a GNPTAB construct carrying the exon 19 deletion enabling the indirect evaluation of the resulting protein´s functionality. Results Our first results demonstrated that in ML II fibroblasts, ASO treatment led to a modest increase in lysosomal hydrolase activity at 24 h and 48 h. Moreover, LAMP-1 expression remained elevated and comparable to untreated ML II cells, indicating that GlcNAc-PT activity was not restored. To further investigate the functional relevance of exon 19 skipping, overexpression studies were performed in HEK293T cells. Three constructs (pGNPTAB WT, pGNPTAB del_exon19 and pGNPTAB c.3503_3504del) were expressed. Both pGNPTAB WT and pGNPTAB del_exon19 constructs produced the α/β-precursor. However, only the WT construct generated the mature β-subunit, whereas the pGNPTAB c.3503_3504del construct showed no detectable expression. These findings indicate that exon 19 is essential for proper GlcNAc-PT processing and enzymatic activity. Conclusions Although ASO treatment corrected splicing at the mRNA level, it did not restore GlcNAc-PT activity in ML II patient cells. Nonetheless, our findings clarify the functional importance of exon 19 and demonstrate that overexpression of an exon-skipped construct provides a simple and effective strategy to indirectly assess protein functionality, supporting the prioritization of this kind of approach to test an exon-skipping ASO-based approach before advancing to studies in patient-derived cells.

Lysosomes are multifunctional organelles that play important roles in cellular recycling, signaling, and homeostasis, relying on precise trafficking and activation of lysosomal enzymes. While the Golgi apparatus plays a central role in lysosomal enzyme sorting, the mechanisms linking Golgi function to lysosomal activity remain incompletely understood. Here, we identify the Golgi-resident protein GRASP55, but not its paralog GRASP65, as necessary for lysosome function. Loss of GRASP55 expression leads to missorting and secretion of lysosomal enzymes, lysosomal dysfunction and bloating. GRASP55 deficiency also disrupts lysosomal mTORC1 signaling, reducing the phosphorylation of its lysosomal substrates TFEB/TFE3, while sparing its non-lysosomal targets. Mechanistically, GRASP55 binds and maintains the COPI adaptor GOLPH3 protein at the Golgi, thereby controlling the Golgi localization and stability of LYSET and GNPTAB that are required for mannose 6-phosphate (M6P) tagging of lysosomal enzymes. These findings reveal an essential role for GRASP55 in Golgi-lysosome communication and lysosomal enzyme trafficking and underscore the importance of Golgi-mediated protein sorting in lysosome function and lysosomal mTORC1 signaling.

Lysosomes are membrane-enclosed organelles that play a crucial role in the degradation of intra- and extracellular substrates and the regulation of metabolic signaling. These functions are carried out by a variety of proteins, of which > 150 are currently known to be located in the lysosomal lumen or to be embedded in its membrane. These proteins are typically low abundant, necessitating organelle enrichment experiments to enable their investigation by unbiased mass spectrometry-based proteomics analyses. Enrichment strategies have been applied in a plethora of studies to gain a deeper understanding of lysosomal composition and relative changes of lysosomal proteome abundance in a variety of pathological conditions. Such experiments are restricted, however, to selected cell lines and tissues and do not allow a direct analysis of the lysosomal proteome from whole cell or tissue lysates. Furthermore, they do not provide absolute quantities. We developed a multiple reaction monitoring mass spectrometry-based assay for the highly sensitive and reproducible absolute quantification of 143 mouse lysosomal proteins from any type of sample following the QconCAT strategy. We applied our approach to the investigation of mouse embryonic fibroblast whole cell lysates and lysosome-enriched fractions, providing absolute copy numbers per cell/lysosome for lysosomal hydrolases and membrane proteins. These data reveal a dynamic range of more than three orders of magnitude in protein expression and strong differences in the subcellular distribution of individual lysosomal proteins. Furthermore, we applied our strategy to the investigation of four primary cell types (macrophages, lung fibroblasts, osteoblasts, and osteoclasts), demonstrating pathway-specific heterogeneity of individual lysosomal protein classes and indicating protein-specific post-transcriptional regulation of expression levels. ### Competing Interest Statement The authors have declared no competing interest.

Leukemias with ambiguous lineage comprise several loosely defined entities, often without a clear mechanistic basis. Here, we extensively profile the epigenome and transcriptome of a subgroup of such leukemias with CpG Island Methylator Phenotype. These leukemias exhibit comparable hybrid myeloid/lymphoid epigenetic landscapes, yet heterogeneous genetic alterations, suggesting they are defined by their shared epigenetic profile rather than common genetic lesions. Gene expression enrichment reveals similarity with early T-cell precursor acute lymphoblastic leukemia and a lymphoid progenitor cell of origin. In line with this, integration of differential DNA methylation and gene expression shows widespread silencing of myeloid transcription factors. Moreover, binding sites for hematopoietic transcription factors, including CEBPA, SPI1 and LEF1, are uniquely inaccessible in these leukemias. Hypermethylation also results in loss of CTCF binding, accompanied by changes in chromatin interactions involving key transcription factors. In conclusion, epigenetic dysregulation, and not genetic lesions, explains the mixed phenotype of this group of leukemias with ambiguous lineage. The data collected here constitute a useful and comprehensive epigenomic reference for subsequent studies of acute myeloid leukemias, T-cell acute lymphoblastic leukemias and mixed-phenotype leukemias.

Lysosomes are multifunctional organelles that play important roles in cellular recycling, signaling, and homeostasis, relying on precise trafficking and activation of lysosomal enzymes. While the Golgi apparatus plays a central role in lysosomal enzyme sorting, the mechanisms linking Golgi function to lysosomal activity remain incompletely understood. Here, we identify the Golgi-resident protein GRASP55, but not its paralog GRASP65, as a key regulator of lysosome function. More specifically, we demonstrate that loss of GRASP55 expression leads to missorting and secretion of lysosomal enzymes, lysosomal dysfunction and bloating. GRASP55 deficiency also disrupts lysosomal mTORC1 signaling, reducing the phosphorylation of its lysosomal substrates, TFEB and TFE3, while sparing its non-lysosomal targets. Mechanistically, GRASP55 interacts with GNPTAB, a critical enzyme required for mannose 6-phosphate (M6P) tagging of lysosomal enzymes, and is necessary for its correct trafficking and stability. These findings reveal an essential role for GRASP55 in Golgi-lysosome communication and lysosomal enzyme trafficking, and suggest that GRASP55/GORASP2 may act as a susceptibility gene for lysosomal storage disorder (LSD)-like conditions. Overall, this work underscores the importance of Golgi-mediated protein sorting in lysosome function and lysosomal mTORC1 signaling, and provides insights into the molecular basis of LSD-related pathologies. ### Competing Interest Statement The authors have declared no competing interest.

Spermine synthase, encoded by the SMS gene, is involved in polyamine metabolism, as it is required for the synthesis of spermine from its precursor molecule spermidine. Pathogenic variants of SMS are known to cause Snyder-Robinson syndrome (SRS), an X-linked recessive disorder causing various symptoms, including intellectual disability, muscular hypotonia, infertility, but also skeletal abnormalities, such as facial dysmorphisms and osteoporosis. Since the impact of a murine SMS deficiency has so far only been analyzed in Gy mice, where a large genomic deletion also includes the neighboring Phex gene, there is only limited knowledge about the potential role of SMS in bone cell regulation. In the present manuscript, we describe 2 patients carrying distinct SMS variants, both diagnosed with osteoporosis. Whereas the first patient displayed all characteristic hallmarks of SRS, the second patient was initially diagnosed, based on laboratory findings, as a case of adult-onset hypophosphatasia. To study the impact of SMS inactivation on bone remodeling, we took advantage of a newly developed mouse model carrying a pathogenic SMS variant (p.G56S). Compared to their wildtype littermates, 12-wk-old male SMSG56S/0 mice displayed reduced trabecular bone mass and cortical thickness, as assessed by μCT analysis of the femur. This phenotype was histologically confirmed by the analysis of spine and tibia sections, where we also observed a moderate enrichment of non-mineralized osteoid in SMSG56S/0 mice. Cellular and dynamic histomorphometry further identified a reduced bone formation rate as a main cause of the low bone mass phenotype. Likewise, primary bone marrow cells from SMSG56S/0 mice displayed reduced capacity to form a mineralized matrix ex vivo, thereby suggesting a cell-autonomous mechanism. Taken together, our data identify SMS as an enzyme with physiological relevance for osteoblast activity, thereby demonstrating an important role of polyamine metabolism in the control of bone remodeling.

Although differentiation and activity of bone-forming osteoblasts are regulated at various levels, there is ample evidence that components of the Wnt signaling pathway play a dominant role in bone mass acquisition. Moreover, a neutralizing antibody blocking the Wnt signaling inhibitor sclerostin has recently been established as a novel osteoanabolic treatment option for osteoporotic patients. Importantly, however, the molecular mechanisms explaining the osteogenic influence of Wnt molecules are still not fully clarified. In this issue of EMBO Reports, You and co-workers show that O-GlcNAcylation is required for the osteoanabolic effect of Wnt stimulation, in vitro and in vivo (You et al, 2024). The molecular mechanisms explaining the osteogenic influence of Wnt molecules are still not fully clarified. A study in this issue shows that O-GlcNAcylation is required for the osteoanabolic effects of Wnt stimulation.

Little is known about the mechanistic significance of the ubiquitin proteasome system (UPS) in a kidney autoimmune environment. In membranous nephropathy (MN), autoantibodies target podocytes of the glomerular filter resulting in proteinuria. Converging biochemical, structural, mouse pathomechanistic, and clinical information we report that the deubiquitinase Ubiquitin C-terminal hydrolase L1 (UCH-L1) is induced by oxidative stress in podocytes and is directly involved in proteasome substrate accumulation. Mechanistically, this toxic gain-of-function is mediated by non-functional UCH-L1, which interacts with and thereby impairs proteasomes. In experimental MN, UCH-L1 becomes non-functional and MN patients with poor outcome exhibit autoantibodies with preferential reactivity to non-functional UCH-L1. Podocyte-specific deletion of UCH-L1 protects from experimental MN, whereas overexpression of non-functional UCH-L1 impairs podocyte proteostasis and drives injury in mice. In conclusion, the UPS is pathomechanistically linked to podocyte disease by aberrant proteasomal interactions of non-functional UCH-L1.

Missense variants in the MBTPS2 gene, located on the X chromosome, have been associated with an X-linked recessive form of osteogenesis imperfecta (X-OI), an inherited bone dysplasia characterized by multiple and recurrent bone fractures, short stature, and various skeletal deformities in affected individuals. The role of site-2 protease, encoded by MBTPS2, and the molecular pathomechanism underlying the disease are to date elusive. This study is the first to report on the generation of two Mbtps2 mouse models, a knock-in mouse carrying one of the disease-causative MBTPS2 variants (N455S) and a Mbtps2 knock-out (ko) mouse. Because both loss-of-function variants lead to embryonic lethality in hemizygous male mutant mice, we performed a comprehensive skeletal analysis of heterozygous Mbtps2+/N455S and Mbtps2+/ko female mice. Both models displayed osteochondral abnormalities such as thinned subchondral bone, altered subchondral osteocyte interconnectivity as well as thickened articular cartilage with chondrocyte clustering, altogether resembling an early osteoarthritis (OA) phenotype. However, distant from the joints, no alterations in the bone mass and turnover could be detected in either of the mutant mice. Based on our findings we conclude that MBTPS2 haploinsufficiency results in early OA-like alterations in the articular cartilage and underlying subchondral bone, which likely precede the development of typical OI phenotype in bone. Our study provides first evidence for a potential role of site-2 protease for maintaining homeostasis of both bone and cartilage.

3D (organoid) models in cancer research are considered the novel gold standard for in vitro experiments. Nevertheless, a variety of different protocols exits and the models are often used uncritically in lack of further validation. In this study, we aimed to characterize a scaffold-based (MatrigelTM) 3D glioma cell culture approach. We compared the 3D technique to conventional 2D culturing in its efficacy of generating models and in respect to its exact modeling properties. Models were generated from 62 patients with CNS tumors, most of them with glioblastomas, IDH-wildtype (GBM, n = 43). Pairs of 11 GBM models (2D and 3D) together with corresponding patient material were further characterized using bulk and single-cell RNA sequencing as well as 850k methylation analyses. Functional aspects of 3D tumor organoids were analyzed using a coculture system (Incucyte® SX5 live cell imager) with tumor infiltrating lymphocytes. Compared to conventional 2D culture the MatrigelTM-based 3D protocol was not superior in terms of the successful generation of in vitro models. However, NGS-based bulk and single-cell sequencing of matched pairs of 2D and 3D models showed an altered transcription of key immune regulatory genes in the 3D models, indicating the reoccurrence of an immunosuppressive phenotype. Changes included the presentation of different HLA surface molecules as well as cellular stressors. In the coculturing approach, lymphocyte-mediated cell killing appeared less effective in clearing 3D models than their 2D counterparts. IFN-γ release as well as live cell staining and proliferation analysis pointed towards an elevated resistance of 3D models. In conclusion, we found that the Scaffold-based (MatrigelTM) 3D culture technique depicts clinically highly important aspects of tumor-immune cell interactions, with the potential to explore immunotherapeutic approaches in an easily accessible in vitro system.

Conventional 2D cultures are commonly used in cancer research though they come with limitations such as the lack of microenvironment or reduced cell heterogeneity. In this study, we investigated in what respect a scaffold-based (Matrigel™) 3D culture technique can ameliorate the limitations of 2D cultures. NGS-based bulk and single-cell sequencing of matched pairs of 2D and 3D models showed an altered transcription of key immune regulatory genes in around 36% of 3D models, indicating the reoccurrence of an immune suppressive phenotype. Changes included the presentation of different HLA surface molecules as well as cellular stressors. We also investigated the 3D tumor organoids in a co-culture setting with tumor-infiltrating lymphocytes (TILs). Of note, lymphocyte-mediated cell killing appeared less effective in clearing 3D models than their 2D counterparts. IFN-γ release, as well as live cell staining and proliferation analysis, pointed toward an elevated resistance of 3D models. In conclusion, we found that the scaffold-based (Matrigel™) 3D culture technique affects the transcriptional profile in a subset of GBM models. Thus, these models allow for depicting clinically relevant aspects of tumor-immune interaction, with the potential to explore immunotherapeutic approaches in an easily accessible in vitro system.

We previously reported on the first neuropathological round robin trials operated together with Quality in Pathology (QuIP) GmbH in 2018 and 2019 in Germany, i.e., the trials on IDH mutational testing and MGMT promoter methylation analysis [1]. For 2020 and 2021, the spectrum of round robin trials has been expanded to cover the most commonly used assays in neuropathological institutions. In addition to IDH mutation and MGMT promoter methylation testing, there is a long tradition for 1p/19q codeletion testing relevant in the context of the diagnosis of oligodendroglioma. With the 5th edition of the World Health Organization (WHO) classification of the central nervous system tumors, additional molecu lar markers came into focus: TERT promoter mutation is often assessed as a molecular diagnostic criterion for IDH-wildtype glioblastoma. Moreover, several molecular diagnostic markers have been introduced for pediatric brain tumors. Here, trials on KIAA1549::BRAF fusions (common in pilocytic astrocytomas) and H3-3A mutations (in diffuse midline gliomas, H3-K27-altered and diffuse hemispheric gliomas, H3-G34 -mutant) were most desired by the neuropathological community. In this update, we report on these novel round robin trials. In summary, success rates in all four trials ranged from 75 to 96%, arguing for an overall high quality level in the field of molecular neuropathological diagnostics.

Mucolipidosis type II (MLII), an ultra-rare lysosomal storage disorder, manifests as a fatal multi-systemic disease. Mental inhibition and progressive neurodegeneration are commonly reported disease manifestations. Nevertheless, longitudinal data on neurocognitive testing and neuroimaging lack in current literature. This study aimed to provide details on central nervous system manifestations in MLII. All MLII patients with at least one standardized developmental assessment performed between 2005 and 2022 were included by retrospective chart review. A multiple mixed linear regression model was applied. Eleven patients with a median age of 34.0 months (range 1.6-159.6) underwent 32 neurocognitive and 28 adaptive behaviour assessments as well as 14 brain magnetic resonance imagings. The scales used were mainly BSID-III (42%) and VABS-II (47%). Neurocognitive testing (per patient: mean 2.9, standard deviation (SD) 2.0) performed over 0-52.1 months (median 12.1) revealed profound impairment with a mean developmental quotient of 36.7% (SD 20.4) at last assessment. The patients showed sustained development; on average, they gained 0.28 age-equivalent score points per month (confidence interval 0.17-0.38). Apart from common (63%) cervical spinal stenosis, neuroimaging revealed unspecific, non-progressive abnormalities (i.e., mild brain atrophy, white matter lesions). In summary, MLII is associated with profound developmental impairment, but not with neurodegeneration and neurocognitive decline.

Die neuropathologische Diagnostik von Hirntumoren hat in den vergangenen Jahren eine rasante Entwicklung erfahren. Während vor wenigen Jahren Hirntumordiagnosen noch rein histologisch und immunhistochemisch gestellt wurden, ergab sich bereits mit der 2016er-WHO-Klassifikation ein Paradigmenwechsel hin zum erstmaligen Einbezug von molekularer Information in die Tumorklassifikation. Dieser Trend hat sich in der aktuellen 2021er-WHO-Klassifikation weiter verstärkt und zum Neuzuschnitt zahlreicher bekannter Tumorentitäten geführt. Die zusätzliche Einführung ganz neuer Tumorentitäten ist nicht zuletzt auf die Technik der DNA-Methylierungsarrays und die Entwicklung des Heidelberger Hirntumor-Classifiers zurückzuführen. Zusätzlich werden die Tumorklassifikationen immer komplexer. So sind insbesondere bei den pädiatrischen Hirntumoren Tumorsubtypen definiert, die sich nicht mehr allein mit molekularen Einzelassays, sondern nur noch unter Einsatz von Methylierungsarrays oder von Next Generation Sequencing (NGS) voneinander abgrenzen lassen. Zwischen diesen Tumorsubtypen bestehen teils gravierende prognostische Unterschiede, sodass der klinische Wert des erhöhten diagnostischen Aufwandes nicht von der Hand zu weisen ist. Auch zur Therapieprädiktion steigt die Bedeutung der molekular-neuropathologischen Diagnostik. Die qualitätskontrollierte Durchführung von wissenschaftlich begleiteten Molekularen Neuroonkologischen Tumorboards stellt hier einen vielversprechenden Ansatzpunkt zur Schaffung innovativer Therapiepfade dar.

The site-1 and site-2 proteases (S1P and S2P) were identified over 20 years ago, and the functions of both have been addressed in numerous studies ever since. Whereas S1P processes a set of substrates independently of S2P, the latter acts in concert with S1P in a mechanism, called regulated intramembrane proteolysis, that controls lipid metabolism and response to unfolded proteins. This review summarizes the molecular roles that S1P and S2P jointly play in these processes. As S1P and S2P deficiencies mainly affect connective tissues, yet with varying phenotypes, we discuss the segregated functions of S1P and S2P in terms of cell homeostasis and maintenance of the connective tissues. In addition, we provide experimental data that point at S2P, but not S1P, as a critical regulator of cell adaptation to proteotoxicity or lipid imbalance. Therefore, we hypothesize that S2P can also function independently of S1P activity.

Purpose Mucolipidosis (ML) II, MLIII alpha/beta, and MLIII gamma are rare autosomal recessive lysosomal storage disorders. Data on the natural course of the diseases are scarce. These data are important for counseling, therapies development, and improvement of outcome. The aim of this study is to gain knowledge on the natural history of ML by obtaining data on survival, symptom onset, presenting symptoms, diagnosis, and pathogenic variants associated with the MLII or MLIII phenotype. Methods A systematic review on all published MLII and MLIII cases between 1968 and August 2019 was performed. Results Three hundred one articles provided data on 843 patients. Median age at diagnosis: 0.7 for MLII and 9.0 years for MLIII. Median survival: 5.0 for MLII and 62.0 years for MLIIIII. Median age of death: 1.8 for MLII and 33.0 years for MLIII. Most frequent causes of death in all ML were pulmonary and/or cardiac complications. Pathogenic variants were described in 388 patients (GNPTAB: 571, GNPTG 179). Conclusion This review provides unique insights into the natural history of MLII and MLIII, with a clear genotype-phenotype correlation with the most frequent pathogenic variant c.3503_3504del in MLII and in MLIII alpha/beta c.22A>G for GNPTAB. All pathogenic GNPTG variants resulted in MLIII gamma.

Severe skeletal alterations are common symptoms in patients with mucolipidosis type II (MLII), a rare lysosomal storage disorder of childhood. We have previously reported that progressive bone loss in a mouse model for MLII is caused by an increased number of bone-resorbing osteoclasts, which is accompanied by elevated expression of the cytokine interleukin-6 (IL-6) in the bone microenvironment. In the present study we addressed the question, if pharmacological blockade of IL-6 can prevent the low bone mass phenotype of MLII mice. Since the cellular IL-6 response can be mediated by either the membrane-bound (classic signaling) or the soluble IL-6 receptor (trans-signaling), we first performed cell culture assays and found that both pathways can increase osteoclastogenesis. We then crossed MLII mice with transgenic mice expressing the recombinant soluble fusion protein sgp130Fc, which represents a natural inhibitor of IL-6 trans-signaling. By undecalcified histology and bone-specific histomorphometry we found that high circulating sgp130Fc levels do not affect skeletal growth or remodeling in wild-type mice. Most importantly, blockade of IL-6 trans-signaling did neither reduce osteoclastogenesis, nor increase bone mass in MLII mice. Therefore, our data clearly demonstrate that the bone phenotype of MLII mice cannot be corrected by blocking the IL-6 trans-signaling.