The complexity of cellular functions necessitates intricate pathways that govern protein synthesis, transport, and degradation; yet the mechanisms governing the trafficking of key developmental proteins in vivo remain incompletely understood. N-Myc downstream-regulated genes (NDRGs) have recently emerged as stress-responsive proteins with roles in protein trafficking, but their functions during normal embryogenesis are largely unknown. Here, we identify N-Myc downstream-regulated gene 1b (Ndrg1b) as a novel regulator of N-cadherin (N-cad, cdh2) trafficking during zebrafish muscle development. We show that ndrg1b is broadly expressed during embryonic and larval stages and is required for proper skeletal muscle morphogenesis. Loss of Ndrg1b disrupts N-cad localization and impairs cell adhesion in vitro, highlighting a key role in maintaining tissue integrity. Mechanistically, Ndrg1b promotes the recycling of N-cad to the plasma membrane, identifying it as a novel component of the endocytic trafficking machinery in vivo. Given N-cad's central role in development and disease, these findings provide new insight into how its localization is fine-tuned during morphogenesis. This work also expands the functional repertoire of NDRGs beyond stress response, suggesting that Ndrg1b - and potentially other NDRG family members - may broadly regulate transmembrane protein trafficking in a context-dependent manner. Furthermore, these findings may shed greater light on the etiology of Charcot-Marie-Tooth Disease type 4D, which has been linked to mutations in NDRG1.

Charcot-Marie-Tooth neuropathy type 4D (CMT4D) is a rare genetic disorder of the peripheral nervous system caused by biallelic mutations in the N-Myc Downstream Regulated 1 gene (NDRG1). Patients present with an early onset demyelinating peripheral neuropathy causing severe distal muscle weakness and sensory loss, leading to loss of ambulation and progressive sensorineural hearing loss. The disorder was initially described in the Roma community due to a common founder mutation, and only a handful of disease-causing variants have been described in this gene so far. Here, we present genetic and clinical findings from a large Bulgarian cohort of demyelinating CMT patients harboring recurrent and novel variants in the NDRG1 gene. Notably, two splice-site variants are exclusive to Bulgarian Muslims and reside in ancestral haplotypes, suggesting a founder effect. Functional characterization of these novel variants implicates a loss-of-function mechanism due to shorter gene products. Our findings contribute to a deeper understanding of the genetic and clinical heterogeneity of CMT4D and highlight novel founder mutations in the ethnic minority of Bulgarian Muslims.

Charcot-Marie-Tooth disease, type 4D (CMT4D) is a progressive, autosomal recessive form of CMT, characterized by distal muscle weakness and atrophy, foot deformities, severe motor sensory neuropathy, and sensorineural hearing impairment. Mutations in NDRG1 gene cause neuropathy in humans, dogs, and rodents. Here, we describe clinical and genetic features of a 17-year-old male with wasting of hand muscle and foot and severe motor neuropathy. Whole exome sequencing was carried out on the patient and his unaffected parents. We identified a novel deletion of nine nucleotides (c.537 + 2_537 + 10del) on the splice donor site of intron 8 in NDRG1 gene. The Sanger sequencing confirmed the segregation of this mutation in autosomal recessive inheritance. Furthermore, transcript analysis confirmed a splice defect and reveals using of an alternate cryptic splice donor site on the downstream intronic region. It resulted in an insertion of 42 nucleotides to exon 8 of NDRG1. Translation of the resulting transcript sequence revealed an insertion of 14 amino acids in-frame to the existing NDRG1 protein. This insertion is predicted to disrupt an alpha helix which is involved in protein-protein interactions in homologous proteins. Our study expands the clinical and genetic spectrum of CMT4D. The splice defect we found in this patient reveals a novel splice isoform of NDRG1 as the potential cause for the neuropathy observed in this patient.

Mutations in the N-myc downstream-regulated gene 1 (NDRG1) can cause degenerative polyneuropathy in humans, dogs, and rodents. In humans, this motor and sensory neuropathy is known as Charcot-Marie-Tooth disease type 4D, and it is assumed that analogous canine diseases can be used as models for this disease. NDRG1 is also regarded as a metastasis-suppressor in several malignancies. The tissue distribution of NDRG1 has been described in humans and rodents, but this has not been studied in the dog. By immunolabeling and Western blotting, we present a detailed mapping of NDRG1 in dog tissues and primary canine Schwann cell cultures, with particular emphasis on peripheral nerves. High levels of phosphorylated NDRG1 appear in distinct subcellular localizations of the Schwann cells, suggesting signaling-driven rerouting of the protein. In a nerve from an Alaskan malamute homozygous for the disease-causing Gly98Val mutation in NDRG1, this signal was absent. Furthermore, NDRG1 is present in canine epithelial cells, predominantly in the cytosolic compartment, often with basolateral localization. Constitutive expression also occurs in mesenchymal cells, including developing spermatids that are transiently positive for NDRG1. In some cells, NDRG1 localize to centrosomes. Overall, canine NDRG1 shows a cell and context-dependent localization. Our data from peripheral nerves and primary Schwann cell cultures suggest that the subcellular localization of NDRG1 in Schwann cells is dynamically influenced by signaling events leading to reversible phosphorylation of the protein. We propose that disease-causing mutations in NDRG1 can disrupt signaling in myelinating Schwann cells, causing disturbance in myelin homeostasis and axonal-glial cross talk, thereby precipitating polyneuropathy.

Charcot-Marie-Tooth disease type 4D (CMT4D) is an autosomal-recessive demyelinating form of CMT characterized by a severe distal motor and sensory neuropathy. NDRG1 is the causative gene for CMT4D. To date, only four mutations in NDRG1 -c.442C>T (p.Arg148*), c.739delC (p.His247Thrfs*74), c.538-1G>A, and duplication of exons 6-8-have been described in CMT4D patients. Here, using targeted next-generation sequencing examination, we identified for the first time two homozygous missense variants in NDRG1, c.437T>C (p.Leu146Pro) and c.701G>A (p.Arg234Gln), in two Chinese CMT families with consanguineous histories. Further functional studies were performed to characterize the biological effects of these variants. Cell culture transfection studies showed that mutant NDRG1 carrying p.Leu146Pro, p.Arg148*, or p.Arg234Gln variant degraded faster than wild-type NDRG1, resulting in lower protein levels. Live cell confocal microscopy and coimmunoprecipitation analysis indicated that these variants did not disrupt the interaction between NDRG1 and Rab4a protein. However, NDRG1-knockdown cells expressing mutant NDRG1 displayed enlarged Rab4a-positive compartments. Moreover, mutant NDRG1 could not enhance the uptake of DiI-LDL or increase the fraction of low-density lipoprotein receptor on the cell surface. Taken together, our study described two missense mutations in NDRG1 and emphasized the important role of NDRG1 in intracellular protein trafficking.