A weakened interface in the P182L variant of HSP27 associated with severe Charcot-Marie-Tooth neuropathy causes aberrant binding to interacting proteins.

HSP27 is a human molecular chaperone that forms large, dynamic oligomers and functions in many aspects of cellular homeostasis. Mutations in HSP27 cause Charcot-Marie-Tooth (CMT) disease, the most common inherited disorder of the peripheral nervous system. A particularly severe form of CMT disease is triggered by the P182L mutation in the highly conserved IxI/V motif of the disordered C-terminal region, which interacts weakly with the structured core domain of HSP27. Here, we observed that the P182L mutation disrupts the chaperone activity and significantly increases the size of HSP27 oligomers formed in vivo, including in motor neurons differentiated from CMT patient-derived stem cells. Using NMR spectroscopy, we determined that the P182L mutation decreases the affinity of the HSP27 IxI/V motif for its own core domain, leaving this binding site more accessible for other IxI/V-containing proteins. We identified multiple IxI/V-bearing proteins that bind with higher affinity to the P182L variant due to the increased availability of the IxI/V-binding site. Our results provide a mechanistic basis for the impact of the P182L mutation on HSP27 and suggest that the IxI/V motif plays an important, regulatory role in modulating protein-protein interactions.

Downregulation of PMP22 ameliorates myelin defects in iPSC-derived human organoid cultures of CMT1A.

Charcot-Marie-Tooth disease is the most common inherited disorder of the PNS. CMT1A accounts for 40-50% of all cases and is caused by a duplication of the PMP22 gene on chromosome 17, leading to dysmyelination in the PNS. Patient-derived models to study such myelination defects are lacking as the in vitro generation of human myelinating Schwann cells has proved to be particularly challenging. Here, we present an induced pluripotent stem cell-derived organoid culture, containing various cell types of the PNS, including myelinating human Schwann cells, which mimics the human PNS. Single-cell analysis confirmed the PNS-like cellular composition and provides insight into the developmental trajectory. We used this organoid model to study disease signatures of CMT1A, revealing early ultrastructural myelin alterations, including increased myelin periodic line distance and hypermyelination of small axons. Furthermore, we observed the presence of onion-bulb-like formations in a later developmental stage. These hallmarks were not present in the CMT1A-corrected isogenic line or in a CMT2A iPSC line, supporting the notion that these alterations are specific to CMT1A. Downregulation of PMP22 expression using short-hairpin RNAs or a combinatorial drug consisting of baclofen, naltrexone hydrochloride and D-sorbitol was able to ameliorate the myelin defects in CMT1A-organoids. In summary, this self-organizing organoid model can capture biologically meaningful features of the disease and capture the physiological complexity, forms an excellent model for studying demyelinating diseases and supports the therapeutic approach of reducing PMP22 expression.

Genetic landscape of congenital insensitivity to pain and hereditary sensory and autonomic neuropathies.

Congenital insensitivity to pain (CIP) and hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders exclusively or predominantly affecting the sensory and autonomic neurons. Due to the rarity of the diseases and findings based mainly on single case reports or small case series, knowledge about these disorders is limited. Here, we describe the molecular workup of a large international cohort of CIP/HSAN patients including patients from normally under-represented countries. We identify 80 previously unreported pathogenic or likely pathogenic variants in a total of 73 families in the >20 known CIP/HSAN-associated genes. The data expand the spectrum of disease-relevant alterations in CIP/HSAN, including novel variants in previously rarely recognized entities such as ATL3-, FLVCR1- and NGF-associated neuropathies and previously under-recognized mutation types such as larger deletions. In silico predictions, heterologous expression studies, segregation analyses and metabolic tests helped to overcome limitations of current variant classification schemes that often fail to categorize a variant as disease-related or benign. The study sheds light on the genetic causes and disease-relevant changes within individual genes in CIP/HSAN. This is becoming increasingly important with emerging clinical trials investigating subtype or gene-specific treatment strategies.

Induced pluripotent stem cell-derived motor neurons of CMT type 2 patients reveal progressive mitochondrial dysfunction.

Axonal Charcot-Marie-Tooth neuropathies (CMT type 2) are caused by inherited mutations in various genes functioning in different pathways. The types of genes and multiplicity of mutations reflect the clinical and genetic heterogeneity in CMT2 disease, which complicates its diagnosis and has inhibited the development of therapies. Here, we used CMT2 patient-derived pluripotent stem cells (iPSCs) to identify common hallmarks of axonal degeneration shared by different CMT2 subtypes. We compared the cellular phenotypes of neurons differentiated from CMT2 patient iPSCs with those from healthy controls and a CRISPR/Cas9-corrected isogenic line. Our results demonstrated neurite network alterations along with extracellular electrophysiological abnormalities in the differentiated motor neurons. Progressive deficits in mitochondrial and lysosomal trafficking, as well as in mitochondrial morphology, were observed in all CMT2 patient lines. Differentiation of the same CMT2 iPSC lines into peripheral sensory neurons only gave rise to cellular phenotypes in subtypes with sensory involvement, supporting the notion that some gene mutations predominantly affect motor neurons. We revealed a common mitochondrial dysfunction in CMT2-derived motor neurons, supported by alterations in the expression pattern and oxidative phosphorylation, which could be recapitulated in the sciatic nerve tissue of a symptomatic mouse model. Inhibition of a dual leucine zipper kinase could partially ameliorate the mitochondrial disease phenotypes in CMT2 subtypes. Altogether, our data reveal shared cellular phenotypes across different CMT2 subtypes and suggests that targeting such common pathomechanisms could allow the development of a uniform treatment for CMT2.

Harmony in chaos: understanding cancer through the lenses of developmental biology.

When we think about cancer, the link to development might not immediately spring to mind. Yet, many foundational concepts in cancer biology trace their roots back to developmental processes. Several defining traits of cancer were indeed initially observed and studied within developing embryos. As our comprehension of embryonic mechanisms deepens, it not only illuminates how and why cancer cells hijack these processes but also spearheads the emergence of innovative technologies for modeling and comprehending tumor biology. Among these technologies are stem cell-based models, made feasible through our grasp of fundamental mechanisms related to embryonic development. The intersection between cancer and stem cell research is evolving into a tangible synergy that extends beyond the concepts of cancer stem cells and cell-of-origin, offering novel tools to unravel the mechanisms of cancer initiation and progression.

Advances and challenges in modeling inherited peripheral neuropathies using iPSCs.

Inherited peripheral neuropathies (IPNs) are a group of diseases associated with mutations in various genes with fundamental roles in the development and function of peripheral nerves. Over the past 10 years, significant advances in identifying molecular disease mechanisms underlying axonal and myelin degeneration, acquired from cellular biology studies and transgenic fly and rodent models, have facilitated the development of promising treatment strategies. However, no clinical treatment has emerged to date. This lack of treatment highlights the urgent need for more biologically and clinically relevant models recapitulating IPNs. For both neurodevelopmental and neurodegenerative diseases, patient-specific induced pluripotent stem cells (iPSCs) are a particularly powerful platform for disease modeling and preclinical studies. In this review, we provide an update on different in vitro human cellular IPN models, including traditional two-dimensional monoculture iPSC derivatives, and recent advances in more complex human iPSC-based systems using microfluidic chips, organoids, and assembloids.

Genetic pain loss disorders.

Genetic pain loss includes congenital insensitivity to pain (CIP), hereditary sensory neuropathies and, if autonomic nerves are involved, hereditary sensory and autonomic neuropathy (HSAN). This heterogeneous group of disorders highlights the essential role of nociception in protecting against tissue damage. Patients with genetic pain loss have recurrent injuries, burns and poorly healing wounds as disease hallmarks. CIP and HSAN are caused by pathogenic genetic variants in >20 genes that lead to developmental defects, neurodegeneration or altered neuronal excitability of peripheral damage-sensing neurons. These genetic variants lead to hyperactivity of sodium channels, disturbed haem metabolism, altered clathrin-mediated transport and impaired gene regulatory mechanisms affecting epigenetic marks, long non-coding RNAs and repetitive elements. Therapies for pain loss disorders are mainly symptomatic but the first targeted therapies are being tested. Conversely, chronic pain remains one of the greatest unresolved medical challenges, and the genes and mechanisms associated with pain loss offer new targets for analgesics. Given the progress that has been made, the coming years are promising both in terms of targeted treatments for pain loss disorders and the development of innovative pain medicines based on knowledge of these genetic diseases.

Defects in Axonal Transport in Inherited Neuropathies.

Axonal transport is a highly complex process essential for sustaining proper neuronal functioning. Disturbances can result in an altered neuronal homeostasis, aggregation of cargoes, and ultimately a dying-back degeneration of neurons. The impact of dysfunction in axonal transport is shown by genetic defects in key proteins causing a broad spectrum of neurodegenerative diseases, including inherited peripheral neuropathies. In this review, we provide an overview of the cytoskeletal components, molecular motors and adaptor proteins involved in axonal transport mechanisms and their implication in neuronal functioning. In addition, we discuss the involvement of axonal transport dysfunction in neurodegenerative diseases with a particular focus on inherited peripheral neuropathies. Lastly, we address some recent scientific advances most notably in therapeutic strategies employed in the area of axonal transport, patient-derived iPSC models, in vivo animal models, antisense-oligonucleotide treatments, and novel chemical compounds.