The cytoskeleton mediates fundamental cellular processes by organizing inter-organelle interactions. Pathogenic variants of inverted formin 2 (INF2) CAAX isoform, an actin assembly factor that is predominantly expressed in the endoplasmic reticulum (ER), are linked to focal segmental glomerulosclerosis (FSGS) and Charcot–Marie–Tooth (CMT) neuropathy. To investigate how pathogenic INF2 variants alter ER integrity, we used high-resolution live imaging of HeLa cells. Cells expressing wild-type (WT) INF2 showed a predominant tubular ER with perinuclear clustering. Cells expressing INF2 FSGS variants that cause mild and intermediate disease induced more sheet-like ER, a pattern similar to that seen for cells expressing WT-INF2 that were treated with actin and microtubule (MT) inhibitors. Dual CMT-FSGS INF2 variants led to more severe ER dysmorphism, with a diffuse, fragmented ER and coarse INF2 aggregates. Proper organization of both F-actin and MT was needed to modulate the tubule vs. sheet conformation balance, while MT arrays regulated spatial expansion of tubular ER in the cell periphery. Pathogenic INF2 variants also induced mitochondria fragmentation and dysregulated mitochondria distribution. Such mitochondrial abnormalities were more prominent for cells expressing CMT-FSGS compared to those with FSGS variants, indicating that the severity of the dysfunction is linked to the degree of cytoskeletal disorganization. Our observations suggest that pathogenic INF2 variants disrupt ER continuity by altering interactions between the ER and the cytoskeleton that in turn impairs inter-organelle communication, especially at ER–mitochondria contact sites. ER continuity defects may be a common disease mechanism involved in both peripheral neuropathy and glomerulopathy.

Background: Charcot–Marie–Tooth (CMT) disease is an inherited peripheral neuropathy primarily involving motor and sensory neurons. Mutations in INF2, an actin assembly factor, cause two diseases: peripheral neuropathy CMT-DIE (MIM614455) and/or focal segmental glomerulosclerosis (FSGS). These two phenotypes arise from the progressive degeneration affecting podocytes and Schwann cells. In general, nerve enlargement has been reported in 25% of the demyelinating CMT subtype (CMT1), while little is known about the CMT-DIE caused by INF2 variants. Methods: To characterize the peripheral nerve phenotype of INF2-related CMT, we studied the clinical course, imaging, histology, and germline genetic variants in two unrelated CMT-DIE patients. Results: Patient 1 (INF2 p.Gly73Asp) and patient 2 (p.Val108Asp) first noticed walking difficulties at 10 to 12 years old. Both of them were electrophysiologically diagnosed with demyelinating neuropathy. In patient 2, the sural nerve biopsy revealed an onion bulb formation. Both patients developed nephrotic syndrome almost simultaneously with CMT and progressed into renal failure at the age of 16 to 17 years. Around the age of 30 years, both patients manifested multiple hypertrophy of the trunk, plexus, and root in the cervical, brachial, lumbosacral nerves, and cauda equina. The histology of the cervical mass in patient 2 revealed Schwannoma. Exome analysis showed that patient 2 harbors a germline LZTR1 p.Arg68Gly variant, while patient 1 has no schwannomatosis-related mutations. Conclusions: Peripheral neuropathy caused by INF2 variants may lead to the development of multifocal hypertrophy with age, likely due to the initial demyelination and subsequent Schwann cell proliferation. Schwannoma could co-occur when the tissues attain additional hits in schwannomatosis-related genes (e.g., LZTR1).

The endoplasmic reticulum (ER) is a single dynamic, and continuous network, which mediates a variety of biological processes. The ER spreads throughout the cytoplasm as an interconnected network mainly consisting of flat (sheets) or reticular (tubules) networks. Such ER integrity is regulated by actin-microtubule interaction. INF2 is an actin assembly factor that exclusively located in ER and is mutated in hereditary form of glomerulopathy (focal segmental glomerulosclerosis, FSGS) and peripheral neuropathy (Charcot-Marie Tooth, CMT-DIE, MIM 614455). It remains unclear how INF2 variants could affect ER morphology. High-resolution, live-imaging of HeLa cells revealed that pathogenic INF2-CAAX variants disrupt peripheral ER complexity, generating focal clustering of polygonal tubules and preferential sheet-like appearance. G73D (causing CMT+FSGS) induced more remarkable alterations than T161N, N202S and R218W (leading to FSGS). Both actin and microtubule inhibitors shifted the ER balance towards sheet predominance and focal compaction of ER tubules, suggesting the role of cytoskeleton in shaping tubular ER networks. INF2 variants induced mitochondria fragmentation with peripheral mis-distribution. The mitochondrial alterations correlated with the degree of cytoskeletal disorganization, leading to defective respiratory function. Moreover, lysosomal trafficking was restricted by INF2 variants in the cell cortex. These organelle-cytoskeletal interactions were more remarkably impaired by CMT+FSGS variant than in FSGS variants. Our observations underscore that INF2 variants disrupt ER integrity by disorganizing cytoskeletons, which leads to defective mitochondria function and vesicle trafficking in INF2 disorders. INF2 CMT+FSGS variants impair ER-organelles interaction more prominent than FSGS variants, suggesting the existence of specific mediators for CMT+FSGS variants.