Mutations in theMFN2gene encoding Mitofusin 2 lead to the development of Charcot–Marie–Tooth type 2A (CMT2A), a dominant axonal form of peripheral neuropathy. Mitofusin 2 is localized at both the outer membrane of mitochondria and the endoplasmic reticulum and is particularly enriched at specialized contact regions known as mitochondria-associated membranes (MAM). We observed that expression of MFN2R94Qinduces distal axonal degeneration in the absence of overt neuronal death. The presence of mutant protein leads to reduction in endoplasmic reticulum and mitochondria contacts in CMT2A patient-derived fibroblasts, in primary neurons and in vivo, in motoneurons of a mouse model of CMT2A. These changes are concomitant with endoplasmic reticulum stress, calcium handling defects, and changes in the geometry and axonal transport of mitochondria. Importantly, pharmacological treatments reinforcing endoplasmic reticulum–mitochondria cross-talk, or reducing endoplasmic reticulum stress, restore the mitochondria morphology and prevent axonal degeneration. These results highlight defects in MAM as a cellular mechanism contributing to CMT2A pathology mediated by mutated MFN2.

Much has been achieved in terms of understanding the complex clinical and genetic heterogeneity of Charcot-Marie-Tooth neuropathy (CMT). Since the identification of mutations in the first CMT associated gene, , the technological advancement in molecular genetics and gene technology has allowed scientists to generate diverse animal models expressing monogenetic mutations that closely resemble the CMT phenotype. Additionally, one can now culture patient-derived neurons in a dish using cellular reprogramming and differentiation techniques. Nevertheless, despite the fact that finding a disease-causing mutation offers a precise diagnosis, there is no cure for CMT at present. This review will shed light on the exciting advancement in CMT disease modelling, the breakthroughs, pitfalls, current challenges for scientists and key considerations to move the field forward towards successful therapies.

Mutations in the small heat shock protein B8 gene (HSPB8/HSP22) have been associated with distal hereditary motor neuropathy, Charcot–Marie–Tooth disease, and recently distal myopathy. It is so far not clear how mutant HSPB8 induces the neuronal and muscular phenotypes and if a common pathogenesis lies behind these diseases. Growing evidence points towards a role of HSPB8 in chaperone-associated autophagy, which has been shown to be a determinant for the clearance of poly-glutamine aggregates in neurodegenerative diseases but also for the maintenance of skeletal muscle myofibrils. To test this hypothesis and better dissect the pathomechanism of mutant HSPB8, we generated a new transgenic mouse model leading to the expression of the mutant protein (knock-in lines) or the loss-of-function (functional knock-out lines) of the endogenous protein Hspb8. While the homozygous knock-in mice developed motor deficits associated with degeneration of peripheral nerves and severe muscle atrophy corroborating patient data, homozygous knock-out mice had locomotor performances equivalent to those of wild-type animals. The distal skeletal muscles of the post-symptomatic homozygous knock-in displayed Z-disk disorganisation, granulofilamentous material accumulation along with Hspb8, αB-crystallin (HSPB5/CRYAB), and desmin aggregates. The presence of the aggregates correlated with reduced markers of effective autophagy. The sciatic nerve of the homozygous knock-in mice was characterized by low autophagy potential in pre-symptomatic and Hspb8 aggregates in post-symptomatic animals. On the other hand, the sciatic nerve of the homozygous knock-out mice presented a normal morphology and their distal muscle displayed accumulation of abnormal mitochondria but intact myofiber and Z-line organisation. Our data, therefore, suggest that toxic gain-of-function of mutant Hspb8 aggregates is a major contributor to the peripheral neuropathy and the myopathy. In addition, mutant Hspb8 induces impairments in autophagy that may aggravate the phenotype.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-017-1756-0) contains supplementary material, which is available to authorized users.

MACC1 (Metastasis Associated in Colon Cancer 1) is a key driver and prognostic biomarker for cancer progression and metastasis in a large variety of solid tumor types, particularly colorectal cancer (CRC). However, no MACC1 inhibitors have been identified yet. Therefore, we aimed to target MACC1 expression using a luciferase reporter-based high-throughput screening with the ChemBioNet library of more than 30,000 compounds. The small molecules lovastatin and rottlerin emerged as the most potent MACC1 transcriptional inhibitors. They remarkably inhibited MACC1 promoter activity and expression, resulting in reduced cell motility. Lovastatin impaired the binding of the transcription factors c-Jun and Sp1 to the MACC1 promoter, thereby inhibiting MACC1 transcription. Most importantly, in CRC-xenografted mice, lovastatin and rottlerin restricted MACC1 expression and liver metastasis. This is—to the best of our knowledge—the first identification of inhibitors restricting cancer progression and metastasis via the novel target MACC1. This drug repositioning might be of therapeutic value for CRC patients.

HSPB1 (heat shock protein family B [small] member 1) is a ubiquitously expressed molecular chaperone. Most mutations in HSPB1 cause axonal Charcot-Marie-Tooth neuropathy and/or distal hereditary motor neuropathy. In this study we show that mutations in HSPB1 lead to impairment of macroautophagic/ autophagic flux. In HSPB1 knockout cells, we demonstrate that HSPB1 is necessary for autophagosome formation, which was rescued upon re-expression of HSPB1. Employing a label-free LC-MS/MS analysis on the various HSPB1 variants (wild type and mutants), we identified autophagy-specific interactors. We reveal that the wild-type HSPB1 protein binds to the autophagy receptor SQSTM1/p62 and that the PB1 domain of SQSTM1 is essential for this interaction. Mutations in HSPB1 lead to a decrease in the formation of SQSTM1/p62 bodies, and subsequent impairment of phagophore formation, suggesting a regulatory role for HSPB1 in autophagy via interaction with SQSTM1. Remarkably, autophagy deficits could also be confirmed in patient-derived motor neurons thereby indicating that the impairment of autophagy might be one of the pathomechanisms by which mutations in HSPB1 lead to peripheral neuropathy.

MACC1, Metastasis associated in colon cancer 1, is a newly identified prognostic biomarker for colorectal cancer metastasis and patient survival, when determined in the primary tumor or patient blood. MACC1 induces cell motility and proliferation in cell culture and metastasis in mouse models. MACC1 acts as a transcriptional regulator of the receptor tyrosine kinase gene Met via binding to its promoter. However, no information about the promoter of the MACC1 gene and its transcriptional regulation has been reported so far. Here we report the identification of the MACC1 promoter using a promoter luciferase construct that directs transcription of MACC1. To gain insights into the essential domains within this promoter region, we constructed 5' truncated deletion constructs. Our results show that the region from -426 to -18 constitutes the core promoter and harbors functional motifs for the binding of AP-1, Sp1, and C/EBP transcription factors as validated by site directed mutagenesis study. Using electrophoretic mobility shift assay and chromatin immunoprecipitation assay, we demonstrated the physical interaction of these transcription factors to a minimal essential MACC1 core promoter sequence. Knock down of these transcription factors using RNAi strategy reduced MACC1 expression (P < 0.001), and resulted in decrease of cell migration (P < 0.01) which could be specifically rescued by ectopic overexpression of MACC1. In human colorectal tumors, expression levels of c-Jun and Sp1 correlated significantly to MACC1 (P = 0.0007 and P = 0.02, respectively). Importantly, levels of c-Jun and Sp1 also showed significant correlation to development of metachronous metastases (P = 0.01 and P = 0.001, respectively). This is the first study identifying the MACC1 promoter and its transcriptional regulation by AP-1 and Sp1. Knowledge of the transcriptional regulation of the MACC1 gene will implicate in enhanced understanding of its role in cancer progression and metastasis.

Purpose: We have previously identified the gene MACC1 as a strong prognostic biomarker for colorectal cancer metastasis and patient survival. Here, we report for the first time the generation of transgenic mouse models for MACC1.Experimental Design: We generated mice with transgenic overexpression of MACC1 in the intestine driven by the villin promoter (vil-MACC1) and crossed them with Apc Min mice (vil-MACC1/Apc Min ). Results: vil-MACC1/ApcMin mice significantly increased the total number of tumors (P ¼ 0.0056). This was particularly apparent in large tumors (!3-mm diameter; P ¼ 0.0024). A detailed histopathologic analysis of these lesions demonstrated that the tumors from the vil-MACC1/Apc Min mice had a more invasive phenotype and, consequently, showed a significantly reduced survival time than Apc Min mice (P ¼ 0.03). Molecular analysis revealed an increased Wnt and pluripotency signaling in the tumors of vil-MACC1/Apc Min mice. Specifically, we observed a prominent upregulation of the pluripotency markers Oct4 and Nanog in these tumors compared with Apc Min controls. Finally, we could also validate that Oct4 and Nanog are regulated by MACC1 in vitro and strongly correlate with MACC1 levels in a cohort of 60 tumors of colorectal cancer patients (r ¼ 0.7005 and r ¼ 0.6808, respectively; P > 0.0001 and P > 0.0002, respectively).Conclusions: We provide proof of principle that MACC1-induced tumor progression in colorectal cancer acts, at least in part, via the newly discovered MACC1/Nanog/Oct4 axis. These findings might have important implications for the design of novel therapeutic intervention strategies to restrict tumor progression.

Colorectal cancer is one of the most common cancers worldwide and one of the leading causes of cancer-related death in the Western world. Tumor progression towards metastasis affects a large number of patients with colorectal cancer and seriously affects their clinical outcome. Therefore, considerable effort has been made towards the development of therapeutic strategies that can decrease or prevent colorectal cancer metastasis. Standard treatment of metastatic colorectal cancer with chemotherapy has been improved in the last 10 years by the addition of new targeted agents. The currently used antibodies bevacizumab, cetuximab and panitumumab target the VEGF and EGFR signaling pathways, which are crucial for tumor progression and metastasis. These antibodies have shown relevant efficacy in both first- and second-line treatment of metastatic colorectal cancer. Additionally, other signaling pathways, including the Wnt and HGF/Met pathways, have a well-established role in colorectal cancer progression and metastasis and constitute, therefore, promising targets for new therapeutic approaches. Several new drugs targeting these pathways, including different antibodies and small-molecule tyrosine kinase inhibitors, are currently being developed and tested in clinical trials. In this review, we summarize the new developments in this field, focusing on the inhibitors that show more promising results for use in colorectal cancer patients.