RANBP17 Overexpression Restores Nucleocytoplasmic Transport and Ameliorates Neurodevelopment in Induced DYT1 Dystonia Motor Neurons.

DYT1 dystonia is a debilitating neurological movement disorder, and it represents the most frequent and severe form of hereditary primary dystonia. There is currently no cure for this disease due to its unclear pathogenesis. In our previous study utilizing patient-specific motor neurons (MNs), we identified distinct cellular deficits associated with the disease, including a deformed nucleus, disrupted neurodevelopment, and compromised nucleocytoplasmic transport (NCT) functions. However, the precise molecular mechanisms underlying these cellular impairments have remained elusive. In this study, we revealed the genome-wide changes in gene expression in DYT1 MNs through transcriptomic analysis. We found that those dysregulated genes are intricately involved in neurodevelopment and various biological processes. Interestingly, we identified that the expression level of RANBP17, a RAN-binding protein crucial for NCT regulation, exhibited a significant reduction in DYT1 MNs. By manipulating RANBP17 expression, we further demonstrated that RANBP17 plays an important role in facilitating the nuclear transport of both protein and transcript cargos in induced human neurons. Excitingly, the overexpression of RANBP17 emerged as a substantial mitigating factor, effectively restoring impaired NCT activity and rescuing neurodevelopmental deficits observed in DYT1 MNs. These findings shed light on the intricate molecular underpinnings of impaired NCT in DYT1 neurons and provide novel insights into the pathophysiology of DYT1 dystonia, potentially leading to the development of innovative treatment strategies.

Analysis of the GFP-labelled ß-dystroglycan interactome in HEK-293 transfected cells reveals novel intracellular networks.

Dystroglycan (DG) is a cell adhesion complex that is widely expressed in tissues. It is composed by two subunits, α-DG, a highly glycosylated protein that interacts with several extracellular matrix proteins, and transmembrane ß-DG whose, cytodomain binds to the actin cytoskeleton. Glycosylation of α-DG is crucial for functioning as a receptor for its multiple extracellular binding partners. Perturbation of α-DG glycosylation is the central event in the pathogenesis of severe pathologies such as muscular dystrophy and cancer. ß-DG acts as a scaffold for several cytoskeletal and nuclear proteins and very little is known about the fine regulation of some of these intracellular interactions and how they are perturbed in diseases. To start filling this gap by identifying uncharacterized intracellular networks preferentially associated with ß-DG, HEK-293 cells were transiently transfected with a plasmid carrying the ß-DG subunit with GFP fused at its C-terminus. With this strategy, we aimed at forcing ß-DG to occupy multiple intracellular locations instead of sitting tightly at its canonical plasma membrane milieu, where it is commonly found in association with α-DG. Immunoprecipitation by anti-GFP antibodies followed by shotgun proteomic analysis led to the identification of an interactome formed by 313 exclusive protein matches for ß-DG binding. A series of already known ß-DG interactors have been found, including ezrin and emerin, whilst significant new matches, which include potential novel ß-DG interactors and their related networks, were identified in diverse subcellular compartments, such as cytoskeleton, endoplasmic reticulum/Golgi, mitochondria, nuclear membrane and the nucleus itself. Of particular interest amongst the novel identified matches, Lamina-Associated Polypeptide-1B (LAP1B), an inner nuclear membrane protein, whose mutations are known to cause nuclear envelopathies characterized by muscular dystrophy, was found to interact with ß-DG in HEK-293 cells. This evidence was confirmed by immunoprecipitation, Western blotting and immunofluorescence experiments. We also found by immunofluorescence experiments that LAP1B looses its nuclear envelope localization in C2C12 DG-knock-out cells, suggesting that LAP1B requires ß-DG for a proper nuclear localization. These results expand the role of ß-DG as a nuclear scaffolding protein and provide novel evidence of a possible link between dystroglycanopathies and nuclear envelopathies displaying with muscular dystrophy.

Nuclear envelope budding: Getting large macromolecular complexes out of the nucleus.

Transport of macromolecules from the nucleus to the cytoplasm is essential for nearly all cellular and developmental events, and when mis-regulated, is associated with diseases, tumor formation/growth, and cancer progression. Nuclear Envelope (NE)-budding is a newly appreciated nuclear export pathway for large macromolecular machineries, including those assembled to allow co-regulation of functionally related components, that bypasses canonical nuclear export through nuclear pores. In this pathway, large macromolecular complexes are enveloped by the inner nuclear membrane, transverse the perinuclear space, and then exit through the outer nuclear membrane to release its contents into the cytoplasm. NE-budding is a conserved process and shares many features with nuclear egress mechanisms used by herpesviruses. Despite its biological importance and clinical relevance, little is yet known about the regulatory and structural machineries that allow NE-budding to occur in any system. Here we summarize what is currently known or proposed for this intriguing nuclear export process.

The clinical and genetic spectrum of autosomal-recessive TOR1A-related disorders.

In the field of rare diseases, progress in molecular diagnostics led to the recognition that variants linked to autosomal-dominant neurodegenerative diseases of later onset can, in the context of biallelic inheritance, cause devastating neurodevelopmental disorders and infantile or childhood-onset neurodegeneration. TOR1A-associated arthrogryposis multiplex congenita 5 (AMC5) is a rare neurodevelopmental disorder arising from biallelic variants in TOR1A, a gene that in the heterozygous state is associated with torsion dystonia-1 (DYT1 or DYT-TOR1A), an early-onset dystonia with reduced penetrance. While 15 individuals with AMC5-TOR1A have been reported (less than 10 in detail), a systematic investigation of the full disease-associated spectrum has not been conducted. Here, we assess the clinical, radiological and molecular characteristics of 57 individuals from 40 families with biallelic variants in TOR1A. Median age at last follow-up was 3 years (0-24 years). Most individuals presented with severe congenital flexion contractures (95%) and variable developmental delay (79%). Motor symptoms were reported in 79% and included lower limb spasticity and pyramidal signs, as well as gait disturbances. Facial dysmorphism was an integral part of the phenotype, with key features being a broad/full nasal tip, narrowing of the forehead and full cheeks. Analysis of disease-associated manifestations delineated a phenotypic spectrum ranging from normal cognition and mild gait disturbance to congenital arthrogryposis, global developmental delay, intellectual disability, absent speech and inability to walk. In a subset, the presentation was consistent with foetal akinesia deformation sequence with severe intrauterine abnormalities. Survival was 71%, with higher mortality in males. Death occurred at a median age of 1.2 months (1 week-9 years), due to respiratory failure, cardiac arrest or sepsis. Analysis of brain MRI studies identified non-specific neuroimaging features, including a hypoplastic corpus callosum (72%), foci of signal abnormality in the subcortical and periventricular white matter (55%), diffuse white matter volume loss (45%), mega cisterna magna (36%) and arachnoid cysts (27%). The molecular spectrum included 22 distinct variants, defining a mutational hotspot in the C-terminal domain of the Torsin-1A protein. Genotype-phenotype analysis revealed an association of missense variants in the 3-helix bundle domain to an attenuated phenotype, while missense variants near the Walker A/B motif as well as biallelic truncating variants were linked to early death. In summary, this systematic cross-sectional analysis of a large cohort of individuals with biallelic TOR1A variants across a wide age-range delineates the clinical and genetic spectrum of TOR1A-related autosomal-recessive disease and highlights potential predictors for disease severity and survival.

Identical twins with progressive kyphoscoliosis and ophthalmoplegia.

The possible differential diagnoses for children presenting with kyphoscoliosis, skeletal deformities and ophthalmoplegia are diverse. We present 11-year-old identical twins with these symptoms, with interesting etiological concern for those practicing in the fields of neurology, pediatrics, spine surgery and related specialties. A new presentation for a rare genetic condition was the final diagnosis for our patients. In this movement disorder round we describe our approach to this clinical constellation and discuss clinical significance of this genetic condition.

Exploitation of Thermal Sensitivity and Hyperalgesia in a Mouse Model of Dystonia.

Neuropathic pain is characterized by mechanical allodynia and thermal hyperalgesia to heat, and it affects some 20% of European population. Patients suffering from several neurologic diseases experience neuropathic pain, often finding no relief in therapy. Transgenic mice expressing the gene encoding the human mutant (hMT) or the human wild-type (hWT) torsin A represent a preclinical model of DYT1 dystonia which is the most common form of early-onset inherited dystonia. Baseline thermal sensitivity and hyperalgesia to heat have never been studied in models of dystonia. Therefore, the aim of this research has been to characterize thermal sensitivity in baseline conditions and hyperalgesia to heat after the induction of neuropathic pain through the spinal nerve ligation (SNL) model in mice overexpressing human wild-type and mutated torsin A in comparison to non-transgenic C57BL/6 mice. According to our results, the paw withdrawal latency time to heat in the Hargreaves' test is significantly lower in the hMT mice (Kruskal-Wallis test = 6.933; p = 0.0312*; hMT vs. hWT p = 0.0317*). On the other hand, no significant differences in SNL-induced thermal hyperalgesia was found among the three strains (Friedman test = 4.933; p = 0.1019). Future studies are needed to better understand the role of torsin A in sensory processing of heat stimuli.

Torsin and NEP1R1-CTDNEP1 phosphatase affect interphase nuclear pore complex insertion by lipid-dependent and lipid-independent mechanisms.

The interphase nuclear envelope (NE) is extensively remodeled during nuclear pore complex (NPC) insertion. How this remodeling occurs and why it requires Torsin ATPases, which also regulate lipid metabolism, remains poorly understood. Here, we show that Drosophila Torsin (dTorsin) affects lipid metabolism via the NEP1R1-CTDNEP1 phosphatase and the Lipin phosphatidic acid (PA) phosphatase. This includes that Torsins remove NEP1R1-CTDNEP1 from the NE in fly and mouse cells, leading to subsequent Lipin exclusion from the nucleus. NEP1R1-CTDNEP1 downregulation also restores nuclear pore membrane fusion in post-mitotic dTorsinKO fat body cells. However, dTorsin-associated nuclear pore defects do not correlate with lipidomic abnormalities and are not resolved by silencing of Lipin. Further testing confirmed that membrane fusion continues in cells with hyperactivated Lipin. It also led to the surprising finding that excessive PA metabolism inhibits recruitment of the inner ring complex Nup35 subunit, resulting in elongated channel-like structures in place of mature nuclear pores. We conclude that the NEP1R1-CTDNEP1 phosphatase affects interphase NPC biogenesis by lipid-dependent and lipid-independent mechanisms, explaining some of the pleiotropic effects of Torsins.

Structural Glycoprotein E2 of Classical Swine Fever Virus Critically Interacts with Host Protein Torsin-1A during the Virus Infectious Cycle.

The classical swine fever virus (CSFV) glycoprotein E2 is the major structural component of the virus particle. E2 is involved in several functions, such as virus adsorption to the cell, the elicitation of protective immune responses, and virus virulence in swine. Using a yeast two-hybrid system, we previously identified the swine host protein Torsin-1A, an ATPase protein residing in the endoplasmic reticulum and inner nucleus membrane of the cell, as a specific binding partner for E2. The interaction between Torsin-1A and E2 proteins was confirmed to occur in CSFV-infected swine cells using three independent methods: coimmunoprecipitation, confocal microscopy, and proximity ligation assay (PLA). Furthermore, the E2 residue critical to mediate the protein-protein interaction with Torsin-1A was identified by a reverse yeast two-hybrid assay using a randomly mutated E2 library. A recombinant CSFV E2 mutant protein with a Q316L substitution failed to bind swine Torsin-1A in the yeast two-hybrid model. In addition, a CSFV infectious clone harboring the E2 Q316L substitution, although expressing substantial levels of E2 protein, repetitively failed to produce virus progeny when the corresponding RNA was transfected into susceptible SK6 cells. Importantly, PLA analysis of the transfected cells demonstrated an abolishment of the interaction between E2 Q316L and Torsin-1A, indicating a critical role for that interaction during CSFV replication.IMPORTANCE Structural glycoprotein E2 is an important structural component of the CSFV particle. E2 is involved in several virus functions, particularly virus-host interactions. Here, we characterized the interaction between CSFV E2 and swine protein Torsin-1A during virus infection. The critical amino acid residue in E2 mediating the interaction with Torsin-1A was identified and the effect of disrupting the E2-Torsin-1A protein-protein interaction was studied using reverse genetics. It is shown that the amino acid substitution abrogating E2-Torsin-1A interaction constitutes a lethal mutation, demonstrating that this virus-host protein-protein interaction is a critical factor during CSFV replication. This highlights the potential importance of the E2-Torsin-1A protein-protein interaction during CSFV replication and provides a potential pathway toward blocking virus replication, an important step toward the potential development of novel virus countermeasures.

Effect of Gabapentin in a Neuropathic Pain Model in Mice Overexpressing Human Wild-Type or Human Mutated Torsin A.

BACKGROUND: DYT1 dystonia is the most common form of early-onset inherited dystonia, which is caused by mutation of torsin A (TA) belonging to the "ATPases associated with a variety of cellular activities" (AAA + ATPase). Dystonia is often accompanied by pain, and neuropathic pain can be associated to peripherally induced movement disorder and dystonia. However, no evidence exists on the effect of gabapentin in mice subjected to neuropathic pain model overexpressing human normal or mutated TA. METHODS: Mice subjected to L5 spinal nerve ligation (SNL) develop mechanical allodynia and upregulation of the α2δ-1 L-type calcium channel subunit, forming a validated experimental model of neuropathic pain. Under these experimental conditions, TA is expressed in dorsal horn neurons and astrocytes and colocalizes with α2δ-1. Similar to this subunit, TA is overexpressed in dorsal horn 7 days after SNL. This model has been used to investigate (1) basal mechanical sensitivity; (2) neuropathic pain phases; and (3) the effect of gabapentin, an α2δ-1 ligand used against neuropathic pain, in non-transgenic (NT) C57BL/6 mice and in mice overexpressing human wild-type (hWT) or mutant (hMT) TA. RESULTS: In comparison to non-transgenic mice, the threshold for mechanical sensitivity in hWT or hMT does not differ (Kruskal-Wallis test = 1.478; p = 0.4777, although, in the latter animals, neuropathic pain recovery phase is delayed. Interestingly, gabapentin (100 mg/Kg) reduces allodynia at its peak (occurring between post-operative day 7 and day 10) but not in the phase of recovery. CONCLUSIONS: These data lend support to the investigation on the role of TA in the molecular machinery engaged during neuropathic pain.

Function of Torsin AAA+ ATPases in Pseudorabies Virus Nuclear Egress.

Newly assembled herpesvirus nucleocapsids traverse the intact nuclear envelope by a vesicle-mediated nucleo-cytoplasmic transport for final virion maturation in the cytoplasm. For this, they bud at the inner nuclear membrane resulting in primary enveloped particles in the perinuclear space (PNS) followed by fusion of the primary envelope with the outer nuclear membrane (ONM). While the conserved viral nuclear egress complex orchestrates the first steps, effectors of fusion of the primary virion envelope with the ONM are still mostly enigmatic but might include cellular proteins like SUN2 or ESCRT-III components. Here, we analyzed the influence of the only known AAA+ ATPases located in the endoplasmic reticulum and the PNS, the Torsins (Tor), on nuclear egress of the alphaherpesvirus pseudorabies virus. For this overexpression of wild type and mutant proteins as well as CRISPR/Cas9 genome editing was applied. Neither single overexpression nor gene knockout (KO) of TorA or TorB had a significant impact. However, TorA/B double KO cells showed decreased viral titers at early time points of infection and an accumulation of primary virions in the PNS pointing to a delay in capsid release during nuclear egress.

Fantastic nuclear envelope herniations and where to find them.

Morphological abnormalities of the bounding membranes of the nucleus have long been associated with human diseases from cancer to premature aging to neurodegeneration. Studies over the past few decades support that there are both cell intrinsic and extrinsic factors (e.g. mechanical force) that can lead to nuclear envelope 'herniations', a broad catch-all term that reveals little about the underlying molecular mechanisms that contribute to these morphological defects. While there are many genetic perturbations that could ultimately change nuclear shape, here, we focus on a subset of nuclear envelope herniations that likely arise as a consequence of disrupting physiological nuclear membrane remodeling pathways required to maintain nuclear envelope homeostasis. For example, stalling of the interphase nuclear pore complex (NPC) biogenesis pathway and/or triggering of NPC quality control mechanisms can lead to herniations in budding yeast, which are remarkably similar to those observed in human disease models of early-onset dystonia. By also examining the provenance of nuclear envelope herniations associated with emerging nuclear autophagy and nuclear egress pathways, we will provide a framework to help understand the molecular pathways that contribute to nuclear deformation.

The mutation responsible for torsion dystonia type 1 shows the ability to stimulate intracellular aggregation of mutant huntingtin.

OBJECTIVE: Introduction: Torsion dystonia type 1 is the most common form of early-onset primary dystonia. Previous reports have suggested that torsin 1A, a protein mutated in this disease, might function as a chaperone that prevents the toxic aggregation of misfolded polypeptides. The aim of the study: The aim of this study was to verify the chaperone function of torsin 1A by investigating its ability to prevent the aggregation of huntingtin model peptides. PATIENTS AND METHODS: Materials and methods: N-terminal mutant huntingtin fragments of different length were co-expressed in neuronal HT-22 and non-neuronal HeLa cells with either the wild-type or mutant (ΔE302/303) torsin 1A protein. The transfected cells were immunostained and analyzed for the presence of huntingtin aggregates using fluorescence microscopy. RESULTS: Results: The immunofluorescence analysis of huntingtin subcellular distribution within the transfected cells showed no significant difference between the huntingtin aggregation levels in cells co-expressing the wild-type torsin 1A and in control cells co-transfected with an empty vector. Instead, it was the increased level of huntingtin aggregation in the presence of the torsion dystonia-causing ΔE302/303 mutant that reached statistical significance in both neuronal and non-neuronal cells. CONCLUSION: Conclusions: Either torsin 1A does not function as a chaperone protein or huntingtin is not an efficient substrate for such a hypothetical chaperone activity. However, the ability of mutant torsin 1A to stimulate the accumulation of aggregation-prone polypeptides might constitute an important source of ΔE302/303 pathogenicity and thus a potential target for future therapy.

Site-specific Proteolysis Mobilizes TorsinA from the Membrane of the Endoplasmic Reticulum (ER) in Response to ER Stress and B Cell Stimulation.

Torsin ATPases are the only representatives of the AAA+ ATPase family that reside in the lumen of the endoplasmic reticulum (ER) and nuclear envelope. Two of these, TorsinA and TorsinB, are anchored to the ER membrane by virtue of an N-terminal hydrophobic domain. Here we demonstrate that the imposition of ER stress leads to a proteolytic cleavage event that selectively removes the hydrophobic domain from the AAA+ domain of TorsinA, which retains catalytic activity. Both the pharmacological inhibition profile and the identified cleavage site between two juxtaposed cysteine residues are distinct from those of presently known proteases, suggesting that a hitherto uncharacterized, membrane-associated protease accounts for TorsinA processing. This processing occurs not only in stress-exposed cell lines but also in primary cells from distinct organisms including stimulated B cells, indicating that Torsin conversion in response to physiologically relevant stimuli is an evolutionarily conserved process. By establishing 5-nitroisatin as a cell-permeable inhibitor for Torsin processing, we provide the methodological framework for interfering with Torsin processing in a wide range of primary cells without the need for genetic manipulation.

Genetic mutations strengthen functional association of LAP1 with DYT1 dystonia and muscular dystrophy.

Lamina-associated polypeptide 1 (LAP1) is a ubiquitously expressed integral protein of the inner nuclear membrane. It interacts physically with lamins, torsinA, emerin and protein phosphatase 1; potentially providing a pivotal mechanism for transducing signals across the inner nuclear membrane. In neurons a functional protein complex is formed, comprising LAP1 and torsinA and in skeletal muscle LAP1 and emerin likewise form a protein complex. Several isoforms of LAP1 have been reported across species. However, in humans only two isoforms have been described, LAP1B and LAP1C. The latter has only recently been reported, but its physiological function and mode of action are not clear. The first TOR1AIP1 (gene encoding LAP1) mutation identified is a single nucleotide deletion resulting in a frameshift and a putative truncated LAP1B protein (Turkish mutation). This has deleterious effects associated with a specific form of muscular dystrophy. A second point mutation, affecting both human LAP1 isoforms, was also recently described. This mutation involves the replacement of a single glutamic acid to alanine at position 482 (Moroccan Mutation), thereby causing severe dystonia, cerebellar atrophy and cardiomyopathy. This review focuses on the recently described human LAP1 isoform (LAP1C), the two recently reported LAP1 mutations and post-translational LAP1 modifications. The latter play an important role in regulating this protein. These scientific contributions strengthen the role of LAP1 in DYT1 dystonia and muscular dystrophy.

Access of torsinA to the inner nuclear membrane is activity dependent and regulated in the endoplasmic reticulum.

TorsinA (also known as torsin-1A) is a membrane-embedded AAA+ ATPase that has an important role in the nuclear envelope lumen. However, most torsinA is localized in the peripheral endoplasmic reticulum (ER) lumen where it has a slow mobility that is incompatible with free equilibration between ER subdomains. We now find that nuclear-envelope-localized torsinA is present on the inner nuclear membrane (INM) and ask how torsinA reaches this subdomain. The ER system contains two transmembrane proteins, LAP1 and LULL1 (also known as TOR1AIP1 and TOR1AIP2, respectively), that reversibly co-assemble with and activate torsinA. Whereas LAP1 localizes on the INM, we show that LULL1 is in the peripheral ER and does not enter the INM. Paradoxically, interaction between torsinA and LULL1 in the ER targets torsinA to the INM. Native gel electrophoresis reveals torsinA oligomeric complexes that are destabilized by LULL1. Mutations in torsinA or LULL1 that inhibit ATPase activity reduce the access of torsinA to the INM. Furthermore, although LULL1 binds torsinA in the ER lumen, its effect on torsinA localization requires cytosolic-domain-mediated oligomerization. These data suggest that LULL1 oligomerizes to engage and transiently disassemble torsinA oligomers, and is thereby positioned to transduce cytoplasmic signals to the INM through torsinA.

The mechanism of Torsin ATPase activation.

Torsins are membrane-associated ATPases whose activity is dependent on two activating cofactors, lamina-associated polypeptide 1 (LAP1) and luminal domain-like LAP1 (LULL1). The mechanism by which these cofactors regulate Torsin activity has so far remained elusive. In this study, we identify a conserved domain in these activators that is predicted to adopt a fold resembling an AAA+ (ATPase associated with a variety of cellular activities) domain. Within these domains, a strictly conserved Arg residue present in both activating cofactors, but notably missing in Torsins, aligns with a key catalytic Arg found in AAA+ proteins. We demonstrate that cofactors and Torsins associate to form heterooligomeric assemblies with a defined Torsin-activator interface. In this arrangement, the highly conserved Arg residue present in either cofactor comes into close proximity with the nucleotide bound in the neighboring Torsin subunit. Because this invariant Arg is strictly required to stimulate Torsin ATPase activity but is dispensable for Torsin binding, we propose that LAP1 and LULL1 regulate Torsin ATPase activity through an active site complementation mechanism.

How lamina-associated polypeptide 1 (LAP1) activates Torsin.

Lamina-associated polypeptide 1 (LAP1) resides at the nuclear envelope and interacts with Torsins, poorly understood endoplasmic reticulum (ER)-localized AAA+ ATPases, through a conserved, perinuclear domain. We determined the crystal structure of the perinuclear domain of human LAP1. LAP1 possesses an atypical AAA+ fold. While LAP1 lacks canonical nucleotide binding motifs, its strictly conserved arginine 563 is positioned exactly where the arginine finger of canonical AAA+ ATPases is found. Based on modeling and electron microscopic analysis, we propose that LAP1 targets Torsin to the nuclear envelope by forming an alternating, heterohexameric (LAP1-Torsin)3 ring, in which LAP1 acts as the Torsin activator. The experimental data show that mutation of arginine 563 in LAP1 reduces its ability to stimulate TorsinA ATPase hydrolysis. This knowledge may help scientists understand the etiology of DYT1 primary dystonia, a movement disorder caused by a single glutamate deletion in TorsinA.

Arresting a Torsin ATPase reshapes the endoplasmic reticulum.

Torsins are membrane-tethered AAA+ ATPases residing in the nuclear envelope (NE) and endoplasmic reticulum (ER). Here, we show that the induction of a conditional, dominant-negative TorsinB variant provokes a profound reorganization of the endomembrane system into foci containing double membrane structures that are derived from the ER. These double-membrane sinusoidal structures are formed by compressing the ER lumen to a constant width of 15 nm, and are highly enriched in the ATPase activator LULL1. Further, we define an important role for a highly conserved aromatic motif at the C terminus of Torsins. Mutations in this motif perturb LULL1 binding, reduce ATPase activity, and profoundly limit the induction of sinusoidal structures.