Pathogenesis Of Hereditary Spastic Paraplegia Research Articles

Loss of Fic causes progressive neurodegeneration in a Drosophila model of hereditary spastic paraplegia

Hereditary Spastic Paraplegia (HSP) is a group of rare inherited disorders characterized by progressive weakness and spasticity of the legs. Recent newly discovered biallelic variants in the gene FICD were found in patients with a highly similar phenotype to early onset HSP. FICD encodes filamentation induced by cAMP domain protein. FICD is involved in the AMPylation and deAMPylation protein modifications of the endoplasmic reticulum (ER) chaperone BIP, a major constituent of the ER that regulates the unfolded protein response. Although several biochemical properties of FICD have been characterized, the neurological function of FICD and the pathological mechanism underlying HSP are unknown. We established a Drosophila model to gain mechanistic understanding of the function of FICD in HSP pathogenesis, and specifically the role of BIP in neuromuscular physiology. Our studies on Drosophila Fic null mutants uncovered that loss of Fic resulted in locomotor impairment and reduced levels of BIP in the motor neuron circuitry, as well as increased reactive oxygen species (ROS) in the ventral nerve cord of Fic null mutants. Finally, feeding Drosophila Fic null mutants with chemical chaperones PBA or TUDCA, or treatment of patient fibroblasts with PBA, reduced the ROS accumulation. The neuronal phenotypes of Fic null mutants recapitulate several clinical features of HSP patients and further reveal cellular patho-mechanisms. By modeling FICD in Drosophila, we provide potential targets for intervention for HSP, and advance fundamental biology that is important for understanding related rare and common neuromuscular diseases.

ITPR1 variant-induced autosomal dominant hereditary spastic paraplegia in a Chinese family.

Hereditary spastic paraplegia (HSP) is a rare neurodegenerative disease prominently characterized by slowly progressive lower limb weakness and spasticity. The significant genotypic and phenotypic heterogeneity of this disease makes its accurate diagnosis challenging. In this study, we identified the NM_001168272: c.2714A > G (chr3.hg19: g.4716912A > G, N905S) variant in the ITPR1 gene in a three-generation Chinese family with multiple individuals affected by HSP, which we believed to be associated with HSP pathogenesis. To confirm, we performed whole exome sequencing, copy number variant assays, dynamic mutation analysis of the entire family, and protein structure prediction. The variant identified in this study was in the coupling domain, and this is the first corroborated report assigning ITPR1 variants to HSP. These findings expand the clinical and genetic spectrum of HSP and provide important data for its genetic analysis and diagnosis.

KCNJ3 is a novel candidate gene for autosomal dominant pure hereditary spastic paraplegia identified using whole genome sequencing.

Hereditary spastic paraplegia (HSP) is a group of familial diseases characterized by progressive corticospinal tract degeneration. Clinically, patients present with lower-limb spasticity and weakness. To date, more than 80 genetic HSP types have been identified. Despite advances in molecular genetics, novel HSP gene discoveries are ongoing, with a low genetic diagnostic yield. In this study, we aimed to determine pathogenic variants in a family with HSP, which was not diagnosed through conventional genetic testing. We clinically characterized a large family and conducted whole genome sequencing (WGS) analysis of four affected and three unaffected individuals in the family to identify the genetic cause of HSP. This family had autosomal dominant pure (uncomplicated) late childhood-onset HSP. The patients' symptoms accelerated between the ages of 20 and 30. Brain magnetic resonance images typically showed white matter changes, a thin corpus callosum, and cerebellar atrophy. We identified a heterozygous missense variant, KCNJ3 c.1297T>G (p.Leu433Val), through WGS and family genetic analysis, confirmed by Sanger sequencing. We suggest that the identification of KCNJ3 c.1297T>G (p.Leu433Val) constitutes the discovery of a potential novel gene responsible for HSP in this family. This is the first study to report the possible role of a KCNJ3 variant in HSP pathogenesis. Our findings further expand the phenotypic and genotypic spectrum of HSP.

The Reelin receptor ApoER2 is a cargo for the adaptor protein complex AP-4: Implications for Hereditary Spastic Paraplegia

Adaptor protein complex 4 (AP-4) is a heterotetrameric complex that promotes export of selected cargo proteins from the trans-Golgi network. Mutations in each of the AP-4 subunits cause a complicated form of Hereditary Spastic Paraplegia (HSP). Herein, we report that ApoER2, a receptor in the Reelin signaling pathway, is a cargo of the AP-4 complex. We identify the motif ISSF/Y within the ApoER2 cytosolic domain as necessary for interaction with the canonical signal-binding pocket of the µ4 (AP4M1) subunit of AP-4. AP4E1- knock-out (KO) HeLa cells and hippocampal neurons from Ap4e1-KO mice display increased co-localization of ApoER2 with Golgi markers. Furthermore, hippocampal neurons from Ap4e1-KO mice and AP4M1-KO human iPSC-derived cortical i3Neurons exhibit reduced ApoER2 protein expression. Analyses of biosynthetic transport of ApoER2 reveal differential post-Golgi trafficking of the receptor, with lower axonal distribution in KO compared to wild-type neurons, indicating a role of AP-4 and the ISSF/Y motif in the axonal localization of ApoER2. Finally, analyses of Reelin signaling in mouse hippocampal and human cortical KO neurons show that AP4 deficiency causes no changes in Reelin-dependent activation of the AKT pathway and only mild changes in Reelin-induced dendritic arborization, but reduces Reelin-induced ERK phosphorylation, CREB activation, and Golgi deployment. This work thus establishes ApoER2 as a novel cargo of the AP-4 complex, suggesting that defects in the trafficking of this receptor and in the Reelin signaling pathway could contribute to the pathogenesis of HSP caused by mutations in AP-4 subunits.

Identification of c.1495C > T mutation in SPAST gene in a family of Han Chinese with hereditary spastic paraplegia

BackgroundHereditary spastic paraplegia 4 (SPG4) caused by spastin (SPAST) gene mutations accounts for 40–45% of hereditary spastic paraplegia (HSP) cases. To search for more genetic evidences for the pathogenesis of HSP, the SPAST genotype and clinical phenotype of a Chinese Han SPG4 family were analysed in this study. MethodsThe clinical data of the proband and his family members were collected. Whole genomic DNA was extracted from peripheral blood, and the gene detection and pathogenicity analysis of mutations were conducted using whole-exome sequencing technology. Suspected pathogenic mutations were identified. Verification within this family was conducted by Sanger sequencing. ResultsEight (4 males and 4 females) of 20 members in 4 generations had SPG4. All patients presented with the high feet arches (pes cavus), the abnormal gait, the active tendon reflexes of the upper limbs, the hyperreflexia of the lower limbs, and the positive ankle clonus and Babinski’s signs bilaterally. In the proband, we found a heterozygous mutation c.1495C > T in SPAST gene, which was associated with the autosomal dominant SPG4. Both the daughters and granddaughters of the proband in this family were verified to carry this mutation. The clinical characteristics of the SPG4 patients in this family are in line with the simple type of HSP. Heterozygous c.1495C > T is a pathogenic mutation in this family. ConclusionIn this study, we identified a c.1495C > T mutation in the SPAST gene in a Han Chinese family, enriching the mutation spectrum of SPG4.

SARM1 deletion delays cerebellar but not spinal cord degeneration in an enhanced mouse model of SPG7 deficiency.

Hereditary spastic paraplegia is a neurological condition characterized by predominant axonal degeneration in long spinal tracts, leading to weakness and spasticity in the lower limbs. The nicotinamide adenine dinucleotide (NAD+)-consuming enzyme SARM1 has emerged as a key executioner of axonal degeneration upon nerve transection and in some neuropathies. An increase in the nicotinamide mononucleotide/NAD+ ratio activates SARM1, causing catastrophic NAD+ depletion and axonal degeneration. However, the role of SARM1 in the pathogenesis of hereditary spastic paraplegia has not been investigated. Here, we report an enhanced mouse model for hereditary spastic paraplegia caused by mutations in SPG7. The eSpg7 knockout mouse carries a deletion in both Spg7 and Afg3l1, a redundant homologue expressed in mice but not in humans. The eSpg7 knockout mice recapitulate the phenotypic features of human patients, showing progressive symptoms of spastic-ataxia and degeneration of axons in the spinal cord as well as the cerebellum. We show that the lack of SPG7 rewires the mitochondrial proteome in both tissues, leading to an early onset decrease in mito-ribosomal subunits and a remodelling of mitochondrial solute carriers and transporters. To interrogate mechanisms leading to axonal degeneration in this mouse model, we explored the involvement of SARM1. Deletion of SARM1 delays the appearance of ataxic signs, rescues mitochondrial swelling and axonal degeneration of cerebellar granule cells and dampens neuroinflammation in the cerebellum. The loss of SARM1 also prevents endoplasmic reticulum abnormalities in long spinal cord axons, but does not halt the degeneration of these axons. Our data thus reveal a neuron-specific interplay between SARM1 and mitochondrial dysfunction caused by lack of SPG7 in hereditary spastic paraplegia.

The interconnection of endoplasmic reticulum and microtubule and its implication in Hereditary Spastic Paraplegia

The endoplasmic reticulum (ER) and microtubule (MT) network form extensive contact with each other and their interconnection plays a pivotal role in ER maintenance and distribution as well as MT stability. The ER participates in a variety of biological processes including protein folding and processing, lipid biosynthesis, and Ca2+ storage. MTs specifically regulate cellular architecture, provide routes for transport of molecules or organelles, and mediate signaling events. The ER morphology and dynamics are regulated by a class of ER shaping proteins, which also provide the physical contact structure for linking of ER and MT. In addition to these ER-localized and MT-binding proteins, specific motor proteins and adaptor-linking proteins also mediate bidirectional communication between the two structures. In this review, we summarize the current understanding of the structure and function of ER-MT interconnection. We further highlight the morphologic factors which coordinate the ER-MT network and maintain the normal physiological function of neurons, with their defect causing neurodegenerative diseases such as Hereditary Spastic Paraplegia (HSP). These findings promote our understanding of the pathogenesis of HSP and provide important therapeutic targets for treatment of these diseases.

FBXL17/spastin axis as a novel therapeutic target of hereditary spastic paraplegia

BackgroundSpastin significantly influences microtubule regulation in neurons and is implicated in the pathogenesis of hereditary spastic paraplegia (HSP). However, post-translational regulation of the spastin protein remains nebulous. The association between E3 ubiquitin ligase and spastin provides a potential therapeutic strategy.ResultsAs evidenced by protein chip analysis, FBXL17 inversely correlated with SPAST-M1 at the protein level in vitro and, also in vivo during embryonic developmental stage. SPAST-M1 protein interacted with FBXL17 specifically via the BTB domain at the N-terminus of SPAST-M1. The SCFFBXL17 E3 ubiquitin ligase complex degraded SPAST-M1 protein in the nuclear fraction in a proteasome-dependent manner. SPAST phosphorylation occurred only in the cytoplasmic fraction by CK2 and was involved in poly-ubiquitination. Inhibition of SCFFBXL17 E3 ubiquitin ligase by small chemical and FBXL17 shRNA decreased proteasome-dependent degradation of SPAST-M1 and induced axonal extension. The SPAST Y52C mutant, harboring abnormality in BTB domain could not interact with FBXL17, thereby escaping protein regulation by the SCFFBXL17 E3 ubiquitin ligase complex, resulting in loss of functionality with aberrant quantity. Although this mutant showed shortening of axonal outgrowth, low rate proliferation, and poor differentiation capacity in a 3D model, this phenotype was rescued by inhibiting SCFFBXL17 E3 ubiquitin ligase.ConclusionsWe discovered that a novel pathway, FBXL17-SPAST was involved in pathogenicity of HSP by the loss of function and the quantitative regulation. This result suggested that targeting FBXL17 could provide new insight into HSP therapeutics.

MYO1H is a novel candidate gene for autosomal dominant pure hereditary spastic paraplegia.

In this study, we aimed to determine the genetic basis of a Turkish family related to hereditary spastic paraplegia (HSP) by exome sequencing. HSP is a progressive neurodegenerative disorder and displays genetic and clinical heterogeneity. The major symptoms are muscle weakness and spasticity, especially in the lower extremities. We studied seven affected and seven unaffected family members, as well as a clinically undetermined member, to identify the disease-causing gene. Exome sequencing was performed for four affected and two unaffected individuals. The variants were firstly filtered for HSP-associated genes, and we found a common variant in the ZFYVE27 gene, which has been previously implied for association with HSP. Due to the incompletely penetrant segregation pattern of the ZFYVE27 variant, revealed by Sanger sequencing, with the disease in this family, filtering was re-performed according to the mode of inheritance and allelic frequencies. The resulting 14 rare variants were further evaluated in terms of their cellular functions, and three candidate variants in ATAD3C, VPS16, and MYO1H genes were selected as possible causative variants, which were analyzed for their familial segregation. ATAD3C and VPS16 variants were eliminated due to incomplete penetrance. Eventually, the MYO1H variant NM_001101421.3:c.2972_2974del (p.Glu992del, rs372231088) was found as the possible disease-causing deletion for HSP in this family. This is the first study reporting the possible role of a MYO1H variant in HSP pathogenesis. Further studies on the cellular roles of Myo1h protein are needed to validate the causality of MYO1H gene at the onset of HSP.

Insights into Clinical, Genetic, and Pathological Aspects of Hereditary Spastic Paraplegias: A Comprehensive Overview.

Hereditary spastic paraplegias (HSP) are a heterogeneous group of motor neurodegenerative disorders that have the core clinical presentation of pyramidal syndrome which starts typically in the lower limbs. They can present as pure or complex forms with all classical modes of monogenic inheritance reported. To date, there are more than 100 loci/88 spastic paraplegia genes (SPG) involved in the pathogenesis of HSP. New patterns of inheritance are being increasingly identified in this era of huge advances in genetic and functional studies. A wide range of clinical symptoms and signs are now reported to complicate HSP with increasing overall complexity of the clinical presentations considered as HSP. This is especially true with the emergence of multiple HSP phenotypes that are situated in the borderline zone with other neurogenetic disorders. The genetic diagnostic approaches and the utilized techniques leave a diagnostic gap of 25% in the best studies. In this review, we summarize the known types of HSP with special focus on those in which spasticity is the principal clinical phenotype (“SPGn” designation). We discuss their modes of inheritance, clinical phenotypes, underlying genetics, and molecular pathways, providing some observations about therapeutic opportunities gained from animal models and functional studies. This review may pave the way for more analytic approaches that take into consideration the overall picture of HSP. It will shed light on subtle associations that can explain the occurrence of the disease and allow a better understanding of its observed variations. This should help in the identification of future biomarkers, predictors of disease onset and progression, and treatments for both better functional outcomes and quality of life.

A novel insertion mutation in atlastin 1 is associated with spastic quadriplegia, increased membrane tethering, and aberrant conformational switching

Hereditary spastic paraplegia (HSP) comprises a heterogeneous group of neuropathies affecting upper motor neurons and causing progressive gait disorder. Mutations in the gene SPG3A/atlastin-1 (ATL1), encoding a dynamin superfamily member, which utilizes the energy from GTP hydrolysis for membrane tethering and fusion to promote the formation of a highly branched, smooth endoplasmic reticulum (ER), account for approximately 10% of all HSP cases. The continued discovery and characterization of novel disease mutations are crucial for our understanding of HSP pathogenesis and potential treatments. Here, we report a novel disease-causing, in-frame insertion in the ATL1 gene, leading to inclusion of an additional asparagine residue at position 417 (N417ins). This mutation correlates with complex, early-onset spastic quadriplegia affecting all four extremities, generalized dystonia, and a thinning of the corpus callosum. We show using limited proteolysis and FRET-based studies that this novel insertion affects a region in the protein central to intramolecular interactions and GTPase-driven conformational change, and that this insertion mutation is associated with an aberrant prehydrolysis state. While GTPase activity remains unaffected by the insertion, membrane tethering is increased, indicative of a gain-of-function disease mechanism uncommon for ATL1-associated pathologies. In conclusion, our results identify a novel insertion mutation with altered membrane tethering activity that is associated with spastic quadriplegia, potentially uncovering a broad spectrum of molecular mechanisms that may affect neuronal function.

ER Morphology in the Pathogenesis of Hereditary Spastic Paraplegia

The endoplasmic reticulum (ER) is the most abundant and widespread organelle in cells. Its peculiar membrane architecture, formed by an intricate network of tubules and cisternae, is critical to its multifaceted function. Regulation of ER morphology is coordinated by a few ER-specific membrane proteins and is thought to be particularly important in neurons, where organized ER membranes are found even in the most distant neurite terminals. Mutation of ER-shaping proteins has been implicated in the neurodegenerative disease hereditary spastic paraplegia (HSP). In this review we discuss the involvement of these proteins in the pathogenesis of HSP, focusing on the experimental evidence linking their molecular function to disease onset. Although the precise biochemical activity of some ER-related HSP proteins has been elucidated, the pathological mechanism underlying ER-linked HSP is still undetermined and needs to be further investigated.

Lipid Droplets in the Pathogenesis of Hereditary Spastic Paraplegia.

Hereditary spastic paraplegias (HSPs) are genetically heterogeneous conditions caused by the progressive dying back of the longest axons in the central nervous system, the corticospinal axons. A wealth of data in the last decade has unraveled disturbances of lipid droplet (LD) biogenesis, maturation, turnover and contact sites in cellular and animal models with perturbed expression and function of HSP proteins. As ubiquitous organelles that segregate neutral lipid into a phospholipid monolayer, LDs are at the cross-road of several processes including lipid metabolism and trafficking, energy homeostasis, and stress signaling cascades. However, their role in brain cells, especially in neurons remains enigmatic. Here, we review experimental findings linking LD abnormalities to defective function of proteins encoded by HSP genes, and discuss arising questions in the context of the pathogenesis of HSP.

Microtubule-dependent and independent roles of spastin in lipid droplet dispersion and biogenesis.

Lipid droplets (LDs) are metabolic organelles that store neutral lipids and dynamically respond to changes in energy availability by accumulating or mobilizing triacylglycerols (TAGs). How the plastic behavior of LDs is regulated is poorly understood. Hereditary spastic paraplegia is a central motor axonopathy predominantly caused by mutations in SPAST, encoding the microtubule-severing protein spastin. The spastin-M1 isoform localizes to nascent LDs in mammalian cells; however, the mechanistic significance of this targeting is not fully explained. Here, we show that tightly controlled levels of spastin-M1 are required to inhibit LD biogenesis and TAG accumulation. Spastin-M1 maintains the morphogenesis of the ER when TAG synthesis is prevented, independent from microtubule binding. Moreover, spastin plays a microtubule-dependent role in mediating the dispersion of LDs from the ER upon glucose starvation. Our results reveal a dual role of spastin to shape ER tubules and to regulate LD movement along microtubules, opening new perspectives for the pathogenesis of hereditary spastic paraplegia.

Spastin MIT Domain Disease-Associated Mutations Disrupt Lysosomal Function.

The hereditary spastic paraplegias (HSPs) are genetic motor neuron diseases characterized by progressive degeneration of corticospinal tract axons. Mutations in SPAST, encoding the microtubule-severing ATPase spastin, are the most common causes of HSP. The broad SPAST mutational spectrum indicates a haploinsufficiency pathogenic mechanism in most cases. Most missense mutations cluster in the ATPase domain, where they disrupt the protein's ability to sever microtubules. However, several putative missense mutations in the protein's microtubule interacting and trafficking (MIT) domain have also been described, but the pathogenicity of these mutations has not been verified with functional studies. Spastin promotes endosomal tubule fission, and defects in this lead to lysosomal enzyme mistrafficking and downstream lysosomal abnormalities. We investigated the function of three disease-associated spastin MIT mutants and found that none was able to promote normal endosomal tubule fission, lysosomal enzyme receptor trafficking, or lysosomal morphology. One of the mutations affected recruitment of spastin to endosomes, a property that requires the canonical function of the MIT domain in binding endosomal sorting complex required for transport (ESCRT)-III proteins. However, the other mutants did not affect spastin's endosomal recruitment, raising the possibility of pathologically important non-canonical roles for the MIT domain. In conclusion, we demonstrate that spastin MIT mutants cause functional abnormalities related to the pathogenesis of HSP. These mutations do not directly affect spastin's microtubule-severing capacity, and so we identify a new molecular pathological mechanism by which spastin mutations may cause disease.

Management of Hereditary Spastic Paraplegia: A Systematic Review of the Literature.

The term hereditary spastic paraplegia (HSP) embraces a clinically and genetically heterogeneous group of neurodegenerative diseases characterized by progressive spasticity and weakness of the lower limbs. There currently exist no specific therapies for HSP, and treatment is exclusively symptomatic, aimed at reducing muscle spasticity, and improving strength and gait. The authors set out to perform a comprehensive systematic review of the available scientific literature on the treatment of HSP, applying Cochrane Collaboration methods. The Google Scholar, PubMed and Scopus electronic databases were searched to find relevant randomized control trials (RCTs) and open-label interventional studies, prospective, and retrospective observational studies of supplements, medications, and physical therapy, as well as case reports and case series. Two authors independently analyzed 27 articles selected on the basis of a series of inclusion criteria. Applying a best-evidence synthesis approach, they evaluated these articles for methodological quality. A standardized scoring system was used to obtain interrater assessments. Disagreements were resolved by discussion. The 27 articles focused on pharmacological treatment (n = 17 articles), physical therapy (n = 5), surgical treatment (n = 5). The drugs used in the 17 articles on pharmacological therapy were: gabapentin, progabide, dalfampridine, botulinum toxin, L-Dopa, cholesterol-lowering drugs, betaine, and folinic acid. Gabapentin, progabide, dalfampridine, and botulinum toxin were used as antispastic agents; the study evaluating gabapentin efficacy was well-designed, but failed to demonstrate any significant improvement. L-Dopa, cholesterol-lowering drugs, betaine, and folinic acid were only used in specific HSP subtypes. Two of the three studies evaluating cholesterol-lowering drugs (in SPG5 patients) were well-designed and showed a significant reduction of specific serum biomarkers (oxysterols), but clinical outcomes were not evaluated. The articles focusing on physical treatment and surgical therapy were found to be of low/medium quality and, accordingly, failed to clarify the role of these approaches in HSP. Despite recent advances in understanding of the pathogenesis of HSP and the possibility, in several centers, of obtaining more precise and rapid molecular diagnoses, there is still no adequate evidence base for recommending the various published therapies. Well-designed RCTs are needed to evaluate the efficacy of both symptomatic and pathogenetic treatments.

Pathogenic TFG Mutations Underlying Hereditary Spastic Paraplegia Impair Secretory Protein Trafficking and Axon Fasciculation

SUMMARYLength-dependent axonopathy of the corticospinal tract causes lower limb spasticity and is characteristic of several neurological disorders, including hereditary spastic paraplegia (HSP) and amyotrophic lateral sclerosis. Mutations in Trk-fused gene (TFG) have been implicated in both diseases, but the pathomechanisms by which these alterations cause neuropathy remain unclear. Here, we biochemically and genetically define the impact of a mutation within the TFG coiled-coil domain, which underlies earlyonset forms of HSP. We find that the TFG (p.R106C) mutation alters compaction of TFG ring complexes, which play a critical role in the export of cargoes from the endoplasmic reticulum (ER). Using CRISPR-mediated genome editing, we engineered human stem cells that express the mutant form of TFG at endogenous levels and identified specific defects in secretion from the ER and axon fasciculation following neuronal differentiation. Together, our data highlight a key role for TFG-mediated protein transport in the pathogenesis of HSP.

ACO2 homozygous missense mutation associated with complicated hereditary spastic paraplegia.

ObjectiveTo identify the clinical characteristics and genetic etiology of a family affected with hereditary spastic paraplegia (HSP).MethodsClinical, genetic, and functional analyses involving genome-wide linkage coupled to whole-exome sequencing in a consanguineous family with complicated HSP.ResultsA homozygous missense mutation was identified in the ACO2 gene (c.1240T>G p.Phe414Val) that segregated with HSP complicated by intellectual disability and microcephaly. Lymphoblastoid cell lines of homozygous carrier patients revealed significantly decreased activity of the mitochondrial aconitase enzyme and defective mitochondrial respiration. ACO2 encodes mitochondrial aconitase, an essential enzyme in the Krebs cycle. Recessive mutations in this gene have been previously associated with cerebellar ataxia.ConclusionsOur findings nominate ACO2 as a disease-causing gene for autosomal recessive complicated HSP and provide further support for the central role of mitochondrial defects in the pathogenesis of HSP.

Pathogenic TFG Mutations Underlying Hereditary Spastic Paraplegia Impair Secretory Protein Trafficking and Axon Fasciculation

Length-dependent axonopathy of the corticospinal tract causes lower limb spasticity and weakness and is characteristic of several neurological disorders, including hereditary spastic paraplegia (HSP) and amyotrophic lateral sclerosis (ALS). Mutations in tropomyosin-receptor kinase fused gene (TFG) have been implicated in both disease states, but the pathomechanisms by which these alterations cause neuropathy remain unclear. Here, we biochemically and genetically define the impact of a mutation within the coiled coil domain of TFG, which underlies early onset forms of HSP. We find that the TFG (p.R106C) mutation alters compaction of TFG ring complexes, which play a critical role in the export of cargoes from the endoplasmic reticulum (ER). Moreover, using CRISPR-mediated genome editing, we engineered human stem cells that express the mutant form of TFG at endogenous levels and identified specific defects in secretion from the ER and axon fasciculation following neuronal differentiation. Together, our data highlight a key role for TFG-mediated protein transport in the pathogenesis of HSP.

Mutant spastin proteins promote deficits in axonal transport through an isoform-specific mechanism involving casein kinase 2 activation.

Mutations of various genes cause hereditary spastic paraplegia (HSP), a neurological disease involving dying-back degeneration of upper motor neurons. From these, mutations in the SPAST gene encoding the microtubule-severing protein spastin account for most HSP cases. Cumulative genetic and experimental evidence suggests that alterations in various intracellular trafficking events, including fast axonal transport (FAT), may contribute to HSP pathogenesis. However, the mechanisms linking SPAST mutations to such deficits remain largely unknown. Experiments presented here using isolated squid axoplasm reveal inhibition of FAT as a common toxic effect elicited by spastin proteins with different HSP mutations, independent of microtubule-binding or severing activity. Mutant spastin proteins produce this toxic effect only when presented as the tissue-specific M1 isoform, not when presented as the ubiquitously-expressed shorter M87 isoform. Biochemical and pharmacological experiments further indicate that the toxic effects of mutant M1 spastins on FAT involve casein kinase 2 (CK2) activation. In mammalian cells, expression of mutant M1 spastins, but not their mutant M87 counterparts, promotes abnormalities in the distribution of intracellular organelles that are correctable by pharmacological CK2 inhibition. Collectively, these results demonstrate isoform-specific toxic effects of mutant M1 spastin on FAT, and identify CK2 as a critical mediator of these effects.