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Hereditary spastic paraplegias (HSPs) are degenerative motor neuron diseases characterized by progressive spasticity and weakness in the lower limbs. The most common form of HSP is due to SPG4 gene haploinsufficiency. SPG4 encodes the microtubule severing enzyme spastin. Although, there is no cure for SPG4-HSP, strategies to induce a spastin recovery are emerging as promising therapeutic approaches. Spastin protein levels are regulated by poly-ubiquitination and proteasomal-mediated degradation, in a neddylation-dependent manner. However, the molecular players involved in this regulation are unknown. Here, we show that the Cullin-4-RING E3 ubiquitin ligase complex (CRL4) regulates spastin stability. Inhibition of CRL4 increases spastin levels by preventing its poly-ubiquitination and subsequent degradation in spastin-proficient and in patient derived SPG4 haploinsufficient cells. To evaluate the role of CRL4 complex in spastin regulation in vivo , we developed a Drosophila melanogaster model of SPG4 haploinsufficiency which show alterations of synapse morphology and locomotor activity, recapitulating phenotypical defects observed in patients. Downregulation of the CRL4 complex, highly conserved in Drosophila , rescues spastin levels and the phenotypical defects observed in flies. As a proof of concept of possible pharmacological treatments, we demonstrate a recovery of spastin levels and amelioration of the SPG4-HSP-associated defects both in the fly model and in patient-derived cells by chemical inactivation of the CRL4 complex with NSC1892. Taken together, these findings show that CRL4 contributes to spastin stability regulation and that it is possible to induce spastin recovery and rescue of SPG4-HSP defects by blocking the CRL4-mediated spastin degradation. [ABSTRACT FROM AUTHOR]

Background: Hereditary spastic paraplegia (HSP) is a rare genetically heterogeneous neurodegenerative disorder. The most common type of HSP is caused by pathogenic variants in the SPAST gene. Various hypotheses regarding the pathogenic mechanisms of HSP-SPAST have been proposed. However, a single hypothesis may not be sufficient to explain HSP-SPAST. Objective: To determine the causative gene of autosomal dominant HSP-SPAST in a pure pedigree and to study its underlying pathogenic mechanism. Methods: A four-generation Chinese family was investigated. Genetic testing was performed for the causative gene, and a splice site variant was identified. In vivo and in vitro experiments were conducted separately. Western blotting and immunofluorescence were performed after transient transfection of cells with the wild-type (WT) or mutated plasmid. The developmental expression pattern of zebrafish spasts was assessed via whole-mount in situ hybridization. The designed guide RNA (gRNA) and an antisense oligo spast-MO were microinjected into Tg(hb9:GFP) zebrafish embryos, spinal cord motor neurons were observed, and a swimming behavioral analysis was conducted. Results: A novel heterozygous intron variant, c.1004 + 5G > A, was identified in a pure HSP-SPAST pedigree and shown to cosegregate with the disease phenotypes. This intron splice site variant skipped exon 6, causing a frameshift mutation that resulted in a premature termination codon. In vitro, the truncated protein was evenly distributed throughout the cytoplasm, formed filamentous accumulations around the nucleus, and colocalized with microtubules. Truncated proteins diffusing in the cytoplasm appeared denser. No abnormal microtubule structures were observed, and the expression levels of α-tubulin remained unchanged. In vivo, zebrafish larvae with this mutation displayed axon pathfinding defects, impaired outgrowth, and axon loss. Furthermore, spast-MO larvae exhibited unusual behavioral preferences and increased acceleration. Conclusion: The adverse effects of premature stop codon mutations in SPAST result in insufficient levels of functional protein, and the potential toxicity arising from the intracellular accumulation of spastin serves as a contributing factor to HSP-SPAST. [ABSTRACT FROM AUTHOR]

Abstract Background Hereditary spastic paraplegia (HSP) is a rare genetically heterogeneous neurodegenerative disorder. The most common type of HSP is caused by pathogenic variants in the SPAST gene. Various hypotheses regarding the pathogenic mechanisms of HSP-SPAST have been proposed. However, a single hypothesis may not be sufficient to explain HSP-SPAST. Objective To determine the causative gene of autosomal dominant HSP-SPAST in a pure pedigree and to study its underlying pathogenic mechanism. Methods A four-generation Chinese family was investigated. Genetic testing was performed for the causative gene, and a splice site variant was identified. In vivo and in vitro experiments were conducted separately. Western blotting and immunofluorescence were performed after transient transfection of cells with the wild-type (WT) or mutated plasmid. The developmental expression pattern of zebrafish spasts was assessed via whole-mount in situ hybridization. The designed guide RNA (gRNA) and an antisense oligo spast-MO were microinjected into Tg(hb9:GFP) zebrafish embryos, spinal cord motor neurons were observed, and a swimming behavioral analysis was conducted. Results A novel heterozygous intron variant, c.1004 + 5G > A, was identified in a pure HSP-SPAST pedigree and shown to cosegregate with the disease phenotypes. This intron splice site variant skipped exon 6, causing a frameshift mutation that resulted in a premature termination codon. In vitro, the truncated protein was evenly distributed throughout the cytoplasm, formed filamentous accumulations around the nucleus, and colocalized with microtubules. Truncated proteins diffusing in the cytoplasm appeared denser. No abnormal microtubule structures were observed, and the expression levels of α-tubulin remained unchanged. In vivo, zebrafish larvae with this mutation displayed axon pathfinding defects, impaired outgrowth, and axon loss. Furthermore, spast-MO larvae exhibited unusual behavioral preferences and increased acceleration. Conclusion The adverse effects of premature stop codon mutations in SPAST result in insufficient levels of functional protein, and the potential toxicity arising from the intracellular accumulation of spastin serves as a contributing factor to HSP-SPAST.

Surface trafficking of AMPA receptors (AMPARs) is one of the important mechanisms mediating synaptic plasticity which is essential for cognitive functions such as learning and memory. Spastin, as a novel binding partner for the AMPAR, has been reported to regulate AMPAR surface expression and synaptic function. Additionally, Spastin undergoes two posttranslational modifications, phosphorylation and SUMOylation, both of which are crucial for synaptic function. However, gaps exist in our knowledge of how Spastin phosphorylation cross-talks with its SUMOylation in the regulation of AMPAR surface expression and synaptic function. Here, we reported that deSUMOylation of Spastin at Lys427 increased the surface level of AMPAR GluA2 subunit, the amplitude and frequency of miniature excitatory synaptic currents (mEPSC), and facilitated the morphological maturation of dendritic spines in cultured hippocampal neurons. Further studies demonstrated that Spastin phosphorylation at Ser210 further increased the enhancement of GluA2 surface expression and synaptic function by deSUMOylated Spastin, while dephosphorylation had the opposite effect. Simultaneously, deSUMOylation at Lys427 significantly increased the promoting effect of Spastin phosphorylation on synaptic function. In conclusion, our study suggests that cooperative interactions between phosphorylated and deSUMOylated Spastin are novel pathways to enhance synaptic function. [ABSTRACT FROM AUTHOR]

Abstract Objective Haploinsufficiency is widely accepted as the pathogenic mechanism of hereditary spastic paraplegias type 4 (SPG4). However, there are some cases that cannot be explained by reduced function of the spastin protein encoded by SPAST. The aim of this study was to identify the causative variant of SPG4 in a large Chinese family and explore its pathological mechanism. Materials and methods A five-generation family with 49 members including nine affected (4 males and 5 females) and 40 unaffected individuals in Mongolian nationality was recruited. Whole exome sequencing was employed to investigate the genetic etiology. Western blotting and immunofluorescence were used to analyze the effects of the mutant proteins in vitro. Results A novel frameshift variant NM_014946.4: c.483_484delinsC (p.Val162Leufs*2) was identified in SPAST from a pedigree with SPG4. The variant segregated with the disease in the family and thus determined as the disease-causing variant. The c.483_484delinsC variant produced two truncated mutants (mutant M1 and M87 isoforms). They accumulated to a higher level and presented increased stability than their wild-type counterparts and may lost the microtubule severing activity. Conclusion SPAST mutations leading to premature stop codons do not always act through haploinsufficiency. The potential toxicity to the corticospinal tract caused by the intracellular accumulation of truncated spastin should be considered as the pathological mechanism of SPG4.

The endosomal sorting complexes required for transport (ESCRT) system is an ancient and ubiquitous membrane scission machinery that catalyzes the budding and scission of membranes. ESCRT-mediated scission events, exemplified by those involved in the budding of HIV-1, are usually directed away from the cytosol ("reverse topology"), but they can also be directed toward the cytosol ("normal topology"). The ESCRT-III subunits CHMP1B and IST1 can coat and constrict positively curved membrane tubes, suggesting that these subunits could catalyze normal topology membrane severing. CHMP1B and IST1 bind and recruit the microtubule-severing AAA+ ATPase spastin, a close relative of VPS4, suggesting that spastin could have a VPS4-like role in normal-topology membrane scission. Here, we reconstituted the process in vitro using membrane nanotubes pulled from giant unilamellar vesicles using an optical trap in order to determine whether CHMP1B and IST1 are capable of membrane severing on their own or in concert with VPS4 or spastin. CHMP1B and IST1 copolymerize on membrane nanotubes, forming stable scaffolds that constrict the tubes, but do not, on their own, lead to scission. However, CHMP1B-IST1 scaffolded tubes were severed when an additional extensional force was applied, consistent with a friction-driven scission mechanism. We found that spastin colocalized with CHMP1B-enriched sites but did not disassemble the CHMP1B-IST1 coat from the membrane. VPS4 resolubilized CHMP1B and IST1 without leading to scission. These observations show that the CHMP1B-IST1 ESCRT-III combination is capable of severing membranes by a friction-driven mechanism that is independent of VPS4 and spastin.

Objective: Haploinsufficiency is widely accepted as the pathogenic mechanism of hereditary spastic paraplegias type 4 (SPG4). However, there are some cases that cannot be explained by reduced function of the spastin protein encoded by SPAST. The aim of this study was to identify the causative variant of SPG4 in a large Chinese family and explore its pathological mechanism. Materials and methods: A five-generation family with 49 members including nine affected (4 males and 5 females) and 40 unaffected individuals in Mongolian nationality was recruited. Whole exome sequencing was employed to investigate the genetic etiology. Western blotting and immunofluorescence were used to analyze the effects of the mutant proteins in vitro. Results: A novel frameshift variant NM_014946.4: c.483_484delinsC (p.Val162Leufs*2) was identified in SPAST from a pedigree with SPG4. The variant segregated with the disease in the family and thus determined as the disease-causing variant. The c.483_484delinsC variant produced two truncated mutants (mutant M1 and M87 isoforms). They accumulated to a higher level and presented increased stability than their wild-type counterparts and may lost the microtubule severing activity. Conclusion: SPAST mutations leading to premature stop codons do not always act through haploinsufficiency. The potential toxicity to the corticospinal tract caused by the intracellular accumulation of truncated spastin should be considered as the pathological mechanism of SPG4. [ABSTRACT FROM AUTHOR]

Developing effective and long-term treatment strategies for rare and complex neurodegenerative diseases is challenging. One of the major roadblocks is the extensive heterogeneity among patients. This hinders understanding the underlying disease-causing mechanisms and building solutions that have implications for a broad spectrum of patients. One potential solution is to develop personalized medicine approaches based on strategies that target the most prevalent cellular events that are perturbed in patients. Especially in patients with a known genetic mutation, it may be possible to understand how these mutations contribute to problems that lead to neurodegeneration. Protein–protein interaction analyses offer great advantages for revealing how proteins interact, which cellular events are primarily involved in these interactions, and how they become affected when key genes are mutated in patients. This line of investigation also suggests novel druggable targets for patients with different mutations. Here, we focus on alsin and spastin, two proteins that are identified as “causative” for amyotrophic lateral sclerosis and hereditary spastic paraplegia, respectively, when mutated. Our review analyzes the protein interactome for alsin and spastin, the canonical pathways that are primarily important for each protein domain, as well as compounds that are either Food and Drug Administration–approved or are in active clinical trials concerning the affected cellular pathways. This line of research begins to pave the way for personalized medicine approaches that are desperately needed for rare neurodegenerative diseases that are complex and heterogeneous.

One means by which cells reutilize neutral lipids stored in lipid droplets is to degrade them by autophagy. This process involves spartin, mutations of which cause the rare inherited disorder Troyer syndrome (or spastic paraplegia-20, SPG20). A recently published paper from the team led by Karin Reinsich (Yale) suggests that the molecular function of spartin and its unique highly conserved "senescence" domain is as a lipid transfer protein. Spartin binds to and transfers all lipid species found in lipid droplets, from phospholipids to triglycerides and sterol esters. This lipid transfer activity correlates with spartin's ability to sustain lipid droplet turnover. The senescence domain poses an intriguing question around the wide range of its cargoes, but intriguingly it has yet to yield up its secrets because attempts at crystallization failed and AlphaFold's prediction is unconvincing. [ABSTRACT FROM AUTHOR]

Axon regeneration is abortive in the central nervous system following injury. Orchestrating microtubule dynamics has emerged as a promising approach to improve axonal regeneration. The microtubule severing enzyme spastin is essential for axonal development and regeneration through remodeling of microtubule arrangement. To date, however, little is known regarding the mechanisms underlying spastin action in neural regeneration after spinal cord injury. Here, we use glutathione transferase pulldown and immunoprecipitation assays to demonstrate that 14-3-3 interacts with spastin, both in vivo and in vitro, via spastin Ser233 phosphorylation. Moreover, we show that 14-3-3 protects spastin from degradation by inhibiting the ubiquitination pathway and upregulates the spastin-dependent severing ability. Furthermore, the 14-3-3 agonist Fusicoccin (FC-A) promotes neurite outgrowth and regeneration in vitro which needs spastin activation. Western blot and immunofluorescence results revealed that 14-3-3 protein is upregulated in the neuronal compartment after spinal cord injury in vivo. In addition, administration of FC-A not only promotes locomotor recovery, but also nerve regeneration following spinal cord injury in both contusion and lateral hemisection models; however, the application of spastin inhibitor spastazoline successfully reverses these phenomena. Taken together, these results indicate that 14-3-3 is a molecular switch that regulates spastin protein levels, and the small molecule 14-3-3 agonist FC-A effectively mediates the recovery of spinal cord injury in mice which requires spastin participation.

Promoting axonal regeneration is an effective strategy for recovery from traumatic spinal cord injury (SCI). Spastin, a microtubule-severing protein, modulates axonal outgrowth and branch formation by regulating microtubule dynamics. However, the exact role of spastin during recovery from SCI remains unknown. Therefore, we utilized a hemisection injury model of the mouse spinal cord and explored the effect of spastin using a spastin inhibitor, spastazoline. Results showed that spastazoline significantly suppressed the microtubule-severing activity of spastin in COS-7 cells and inhibited the promoting effect of spastin on neurite outgrowth in primarily cultured hippocampal neurons. The protein expression level of spastin was significantly upregulated in the injured spinal cord. Injured mice showed impaired motor functions, which included increased toe-off angle and foot fault steps and decreased stride length and Basso mouse scale score. Notably, these motor function impairments were aggravated by the application of spastazoline. Inhibition of spastin exacerbated neurogenesis impairment, as demonstrated by neuronal nuclei antigen staining, the inflammatory response, as shown by Iba-1 and GFAP staining, and axonal regeneration impairment, as shown by 5-hydroxytryptamine staining. Furthermore, mass spectrometry analysis revealed that the inhibition of spastin resulted in numerous dysregulated differentially expressed proteins that were closely associated with vesicle organization and transport. Taken together, our data suggest that spastin is critical for recovery from SCI and may be a potential target for the treatment of SCI.

Dictyostelium amoebae perform a semi-closed mitosis, in which the nuclear envelope is fenestrated at the insertion sites of the mitotic centrosomes and around the central spindle during karyokinesis. During late telophase the centrosome relocates to the cytoplasmic side of the nucleus, the central spindle disassembles and the nuclear fenestrae become closed. Our data indicate that Dictyostelium spastin (DdSpastin) is a microtubule-binding and severing type I membrane protein that plays a role in this process. Its mitotic localization is in agreement with a requirement for the removal of microtubules that would hinder closure of the fenestrae. Furthermore, DdSpastin interacts with the HeH/ LEM-family protein Src1 in BioID analyses as well as the inner nuclear membrane protein Sun1, and shows subcellular co-localizations with Src1, Sun1, the ESCRT component CHMP7 and the IST1-like protein filactin, suggesting that the principal pathway of mitotic nuclear envelope remodeling is conserved between animals and Dictyostelium amoebae.

Abstract Chromosome 3-linked frontotemporal dementia (FTD3) is caused by a gain-of-function mutation in CHMP2B, resulting in the production of a truncated toxic protein, CHMP2BIntron5. Loss-of-function mutations in spastin are the most common genetic cause of hereditary spastic paraplegias (HSP). How these proteins might interact with each other to drive pathology remains to be explored. Here we found that spastin binds with greater affinity to CHMP2BIntron5 than to CHMP2BWT and colocalizes with CHMP2BIntron5 in p62-positive aggregates. In cultured cells expressing CHMP2BIntron5, spastin level in the cytoplasmic soluble fraction is decreased while insoluble spastin level is increased. These pathological features of spastin are validated in brain neurons of a mouse model of FTD3. Moreover, genetic knockdown of spastin enhances CHMP2BIntron5 toxicity in a Drosophila model of FTD3, indicating the functional significance of their association. Thus, our study reveals that the enhanced association between mutant CHMP2B and spastin represents a novel potential pathological link between FTD3 and HSP.

Tau is an abundant microtubule-associated protein in neurons. Tau aggregation into insoluble fibrils is a hallmark of Alzheimer's disease and other types of dementia1, yet the physiological state of tau molecules within cells remains unclear. Using single-molecule imaging, we directly observe that the microtubule lattice regulates reversible tau self-association, leading to localized, dynamic condensation of tau molecules on the microtubule surface. Tau condensates form selectively permissible barriers, spatially regulating the activity of microtubule-severing enzymes and the movement of molecular motors through their boundaries. We propose that reversible self-association of tau molecules, gated by the microtubule lattice, is an important mechanism of the biological functions of tau, and that oligomerization of tau is a common property shared between the physiological and disease-associated forms of the molecule.

Microtubule-severing enzymes generate internal breaks in microtubules. They are conserved in eukaryotes from ciliates to mammals, and their function is important in diverse cellular processes ranging from cilia biogenesis to cell division, phototropism, and neurogenesis. Their mutation leads to neurodegenerative and neurodevelopmental disorders in humans. All three known microtubule-severing enzymes, katanin, spastin, and fidgetin, are members of the meiotic subfamily of AAA ATPases that also includes VPS4, which disassembles ESCRTIII polymers. Despite their conservation and importance to cell physiology, the cellular and molecular mechanisms of action of microtubule-severing enzymes are not well understood. Here we review a subset of cellular processes that require microtubule-severing enzymes as well as recent advances in understanding their structure, biophysical mechanism, and regulation.

Investigating novel mechanisms of neurite outgrowth via cytoskeleton is critical for developing therapeutic strategies against neural disorders. Rab3A is a vesicle-related protein distributed throughout the nervous system, but the detailed mechanism related to cytoskeleton remains largely unknown. Our previous reports show that spastin serves microtubule to regulate neurite outgrowth. Here, we asked whether Rab3A could function via modulating spastin during neuronal development. The results revealed that Rab3A colocalized with spastin in cultured hippocampal neurons. Immunoprecipitation assays showed that Rab3A physically interacted with spastin in rat brain lysates. Rab3A overexpression significantly induced spastin degradation; this effect was reversed by leupeptin- or MG-132- administration, suggesting the lysosomal and ubiquitin-mediated degradation system. Immunofluorescence staining further confirmed that Rab3A and spastin immune-colocalized with the lysosome marker lysotracker. In COS7 cells, Rab3A overexpression significantly downregulated spastin expression and abolished the spastin-mediated microtubule severing. Furthermore, overexpression inhibited while genetic knockdown of Rab3A promoted neurite outgrowth. However, this inhibitory effect on neurite outgrowth in hippocampal neurons could be reversed via co-transfection of spastin, indicating that Rab3A functions via its interaction protein spastin. In general, our data identify an interaction between Rab3A and spastin, and this interaction affects the protein stability of spastin and eliminates its microtubule severing function, thereby modulating neurite outgrowth. • Rab3A colocalizes and interacts with spastin in hippocampal neurons. • Rab3A induces spastin degradation and alleviates the microtubule severing activity of spastin. • Overexpression inhibits while genetic knockdown of Rab3A promotes neurite outgrowth. • Rab3A/Spastin regulates neurite outgrowth in hippocampal neurons. [ABSTRACT FROM AUTHOR]

Microtubule-severing protein Spastin has been shown to co-localize with actin in migratory glioblastoma cells and is linked to glioblastomas' migration and invasion capacity. However, the effectiveness of Spastin in glioblastoma migration and the molecular mechanism underpinning the orientation of Spastin towards actin filaments remain unknown. Here, we demonstrated that Spastin plays an active role in glioblastoma migration by showing a reduced migratory potential of T98G glioblastoma cells using real-time cell analysis (RTCA) in Spastin-depleted cells. Pull-down assays revealed that a cis–trans isomerase Pin1 interacts with Spastin through binding to the phosphorylated Pin1 recognition motifs in the microtubule-binding domain (MBD), and immunocytochemistry analysis showed that interaction with Pin1 directs Spastin to actin filaments in extended cell regions. Consequently, by utilizing RTCA, we proved that the migration and invasion capacity of T98G glioblastoma cells significantly increased with the overexpression of Spastin, of which the Pin1 recognition motifs in MBD are constitutively phosphorylated, while the overexpression of phospho-mutant form did not have a significant effect on migration and invasion of T98G glioblastoma cells. These findings demonstrate that Pin1 is a novel interaction partner of Spastin, and their interaction drives Spastin to actin filaments, allowing Spastin to contribute to the glioblastomas' migration and invasion abilities. [ABSTRACT FROM AUTHOR]

Dictyostelium amoebae perform a semi-closed mitosis, in which the nuclear envelope is fenestrated at the insertion sites of the mitotic centrosomes and around the central spindle during karyokinesis. During late telophase the centrosome relocates to the cytoplasmic side of the nucleus, the central spindle disassembles and the nuclear fenestrae become closed. Our data indicate that Dictyostelium spastin (DdSpastin) is a microtubule-binding and severing type I membrane protein that plays a role in this process. Its mitotic localization is in agreement with a requirement for the removal of microtubules that would hinder closure of the fenestrae. Furthermore, DdSpastin interacts with the HeH/ LEM-family protein Src1 in BioID analyses as well as the inner nuclear membrane protein Sun1, and shows subcellular co-localizations with Src1, Sun1, the ESCRT component CHMP7 and the IST1-like protein filactin, suggesting that the principal pathway of mitotic nuclear envelope remodeling is conserved between animals and Dictyostelium amoebae. [ABSTRACT FROM AUTHOR]

Spastin, a microtubule-severing enzyme, is known to be important for neurite outgrowth. However, the role of spastin post-translational modification, particularly its phosphorylation regulation in neuronal outgrowth, remains unclear. This study aimed to investigate the effects of eliminating spastin phosphorylation on the neurite outgrowth of rat hippocampal neurons. To accomplish this, we constructed a spastin mutant with eleven potential phosphorylation sites mutated to alanine. The phosphorylation levels of the wildtype spastin (WT) and the mutant (11A) were then detected using Phos-tag SDS-PAGE. The spastin constructs were transfected into COS7 cells for the observation of microtubule severing, and into rat hippocampal neurons for the detection of neuronal outgrowth. The results showed that compared to the spastin WT, the phosphorylation levels were significantly reduced in the spastin 11A mutant. The spastin mutant 11A impaired its ability to promote neurite length, branching, and complexity in hippocampal neurons, but did not affect its ability to sever microtubules in COS7 cells. In conclusion, the data suggest that mutations at multiple phosphorylation sites of spastin do not impair its microtubule cleavage ability in COS7 cells, but reduce its ability to promote neurite outgrowth in rat hippocampal neurons.

Chromosome 3-linked frontotemporal dementia (FTD3) is caused by a gain-of-function mutation in CHMP2B, resulting in the production of a truncated toxic protein, CHMP2BIntron5. Loss-of-function mutations in spastin are the most common genetic cause of hereditary spastic paraplegias (HSP). How these proteins might interact with each other to drive pathology remains to be explored. Here we found that spastin binds with greater affinity to CHMP2BIntron5 than to CHMP2BWT and colocalizes with CHMP2BIntron5 in p62-positive aggregates. In cultured cells expressing CHMP2BIntron5, spastin level in the cytoplasmic soluble fraction is decreased while insoluble spastin level is increased. These pathological features of spastin are validated in brain neurons of a mouse model of FTD3. Moreover, genetic knockdown of spastin enhances CHMP2BIntron5 toxicity in a Drosophila model of FTD3, indicating the functional significance of their association. Thus, our study reveals that the enhanced association between mutant CHMP2B and spastin represents a novel potential pathological link between FTD3 and HSP. [ABSTRACT FROM AUTHOR]

Pathogenic variants in SPAST, the gene coding for spastin, are the single most common cause of hereditary spastic paraplegia, a progressive motor neuron disease. Spastin regulates key cellular functions, including microtubule-severing and endoplasmic reticulum-morphogenesis. However, it remains unclear how alterations in these cellular functions due to SPAST pathogenic variants result in motor neuron dysfunction. Since spastin influences both microtubule network and endoplasmic reticulum structure, we hypothesized that spastin is necessary for the regulation of Ca2+ homeostasis via store-operated calcium entry. Here, we show that the lack of spastin enlarges the endoplasmic reticulum and reduces store-operated calcium entry. In addition, elevated levels of different spastin variants induced clustering of STIM1 within the endoplasmic reticulum, altered the transport of STIM1 to the plasma membrane and reduced store-operated calcium entry, which could be rescued by exogenous expression of STIM1. Importantly, store-operated calcium entry was strongly reduced in induced pluripotent stem cell-derived neurons from hereditary spastic paraplegia patients with pathogenic variants in SPAST resulting in spastin haploinsufficiency. These neurons developed axonal swellings in response to lack of spastin. We were able to rescue both store-operated calcium entry and axonal swellings in SPAST patient neurons by restoring spastin levels, using CRISPR/Cas9 to correct the pathogenic variants in SPAST. These findings demonstrate that proper amounts of spastin are a key regulatory component for store-operated calcium entry mediated Ca2+ homeostasis and suggest store-operated calcium entry as a disease relevant mechanism of spastin-linked motor neuron disease. [ABSTRACT FROM AUTHOR]

IntroductionHereditary spastic paraplegias (HSPs) are genetic neurodegenerative diseases. The most common form of pure HSP that is inherited in an autosomal dominant manner is spastic paraplegia type 4 (SPG4), which is caused by mutations in the SPAST gene. Different theories have been proposed as the mechanism underlying SPAST-HSP for different types of genetic mutations, including gain- and loss-of-function mechanisms. To better understand the mutation mechanisms, we performed genetic analysis and investigated a truncating SPAST variant that segregated with disease in one family.Objectives and methodsWe described a pure HSP pedigree with family members across four generations. We performed genetic analysis and investigated a novel frameshift pathogenic variant (c.862_863dupAC, p. H289Lfs*27) in this family. We performed reverse transcription-polymerase chain reaction (RT-PCR), Sanger sequencing, and quantitative RT-PCR using total RNA from an Epstein-Barr virus-induced lymphoblastoid cell line produced from the proband. We also performed Western blotting on cell lysates to investigate if the protein expression of spastin is affected by this variant.ResultsThis variant (c.862_863dupAC, p. H289Lfs*27) co-segregated with pure HSP in this family and is not registered in any public database. Measurement of SPAST transcripts in lymphoblasts from the proband demonstrated a reduction of SPAST transcript levels through likely nonsense-mediated mRNA decay. Immunoblot analyses demonstrated a reduction of spastin protein expression levels in lymphoblasts.ConclusionWe report an SPG4 family with a novel heterozygous frameshift variant p.H289Lfs*27 in SPAST. Our study implies haploinsufficiency as the pathogenic mechanism for this variant and expands the known mutation spectrum of SPAST.

The maintenance of appropriate endoplasmic reticulum (ER) homeostasis is critical to effective spinal cord injury (SCI) repair. In previous reports, protein disulfide isomerase A6 (PDIA6) demonstrated to serve as a reversible functional modulator of ER stress responses, while spastin can coordinate ER organization through the modulation of the dynamic microtubule network surrounding this organelle. While both PDIA6 and spastin are thus important regulators of the ER, whether they interact with one another for SCI repair still needs to be determined. Here a proteomics analysis identified PDIA6 as being related to SCI repair, and protein interaction mass spectrometry further confirmed the ability of PDIA6 and spastin to interact with one another. Pull-down and co-immunoprecipitation assays were further performed to validate and characterize the interactions between these two proteins. The RNAi-based knockdown of PDIA6 in COS-7 cells inhibited the activity of spastin-dependent microtubule severing. PDIA6 was also found to promote injured neuron repair, while spastin knockdown reversed this reparative activity. Together, these results thus confirm that PDIA6 and spastin function together as critical mediators of nerve repair, highlighting their potential value as validated targets for efforts to promote SCI repair. [ABSTRACT FROM AUTHOR]

Microtubules tightly regulate various cellular activities. Our understanding of microtubules is largely based on experiments using microtubule‐targeting agents, which, however, are insufficient to dissect the dynamic mechanisms of specific microtubule populations, due to their slow effects on the entire pool of microtubules. To overcome this technological limitation, we have used chemo and optogenetics to disassemble specific microtubule subtypes, including tyrosinated microtubules, primary cilia, mitotic spindles, and intercellular bridges, by rapidly recruiting engineered microtubule‐cleaving enzymes onto target microtubules in a reversible manner. Using this approach, we show that acute microtubule disassembly swiftly halts vesicular trafficking and lysosomal dynamics. It also immediately triggers Golgi and ER reorganization and slows the fusion/fission of mitochondria without affecting mitochondrial membrane potential. In addition, cell rigidity is increased after microtubule disruption owing to increased contractile stress fibers. Microtubule disruption furthermore prevents cell division, but does not cause cell death during interphase. Overall, the reported tools facilitate detailed analysis of how microtubules precisely regulate cellular architecture and functions. SYNOPSIS: Characterization of microtubules in cells via microtubule‐targeting agents is not ideal for assessing dynamics of specific microtubule populations. Here, a novel approach using an engineered microtubule‐severing enzyme allows spatiotemporal manipulation of microtubule disassembly triggered by either chemicals or illumination. A triple glutamine mutation in Spastin (dNSpastin3Q) reduces its association with microtubules.Engineered dNSpastin3Q acts as a microtubule‐severing enzyme in living cells.Recruitment of dNSpastin3Q to microtubules can be controlled by chemical‐ or light‐induced dimerization.Induced dimerization of dNSpastin3Q leads to acute disassembly of target microtubule subtypes. [ABSTRACT FROM AUTHOR]

Cells generate a highly diverse microtubule network to carry out different activities. This network is comprised of distinct tubulin isotypes, tubulins with different post-translational modifications, and many microtubule-based structures. Defects in this complex system cause numerous human disorders. However, how different microtubule subtypes in this network regulate cellular architectures and activities remains largely unexplored. Emerging tools such as photosensitive pharmaceuticals, chemogenetics, and optogenetics enable the spatiotemporal manipulation of structures, dynamics, post-translational modifications, and cross-linking with actin filaments in target microtubule subtypes. This review summarizes the design rationale and applications of these new approaches and aims to provide a roadmap for researchers navigating the intricacies of microtubule dynamics and their post-translational modifications in cellular contexts, thereby opening new avenues for therapeutic interventions. [ABSTRACT FROM AUTHOR]

Synaptic plasticity is essential for cognitive functions such as learning and memory. One of the mechanisms involved in synaptic plasticity is the dynamic delivery of AMPA receptors (AMPARs) in and out of synapses. Mutations of SPAST , which encodes SPASTIN, a microtubule-severing protein, are considered the most common cause of hereditary spastic paraparesis (HSP). In some cases, patients with HSP also manifest cognitive impairment. In addition, mice with Spastin depletion exhibit working and associative memory deficits and reduced AMPAR levels. However, the exact effect and molecular mechanism of Spastin on AMPARs trafficking has remained unclear. Here, we report that Spastin interacts with AMPAR, and phosphorylation of Spastin enhances its interaction with AMPAR subunit GluA2. Further study shows that phosphorylation of Spastin can increase AMPAR GluA2 surface expression and the amplitude and frequency of miniature excitatory synaptic currents (mEPSC) in cultured hippocampal neurons. Moreover, phosphorylation of Spastin at Ser210 is crucial for GluA2 surface expression. Phosphorylation of Spastin K353A, which obliterates microtubule-severing activity, also promotes AMPAR GluA2 subunit trafficking to the surface and increases the amplitude and frequency of mEPSCs in cultured neurons. Taken together, our data demonstrate that Spastin phosphorylation promotes the surface delivery of the AMPAR GluA2 subunit independent of microtubule dynamics. [ABSTRACT FROM AUTHOR]

Background: Haploinsufficiency is widely accepted as the pathogenic mechanism of spastic paraplegia type 4 (SPG4). However, there are some cases that cannot be explained by reduced function of the spastin protein encoded by SPAST. Objectives: To identify the causative gene of autosomal dominant hereditary spastic paraplegia in three large Chinese families and explore the pathological mechanism of a spastin variant. Methods: Three large Chinese hereditary spastic paraplegia families with a total of 247 individuals (67 patients) were investigated, of whom 59 members were recruited to the study. Genetic testing was performed to identify the causative gene. Western blotting and immunofluorescence were used to analyze the effects of the mutant proteins in vitro. Results: In the three hereditary spastic paraplegia families, of whom three index cases were misdiagnosed as other types of neurological diseases, a novel c.985dupA (p.Met329Asnfs*3) variant in SPAST was identified and was shown to cosegregate with the phenotype in the three families. The c.985dupA mutation produced two truncated mutants (mutant M1 and M87 isoforms) that accumulated to a higher level than their wild‐type counterparts. Furthermore, the mutant M1 isoform heavily decorated the microtubules and rendered them resistant to depolymerization. In contrast, the mutant M87 isoform was diffusely localized in both the nucleus and the cytoplasm, could not decorate microtubules, and was not able to promote microtubule disassembly. Conclusions: SPAST mutations leading to premature stop codons do not always act through haploinsufficiency. The truncated spastin may damage the corticospinal tracts through an isoform‐specific toxic effect. [ABSTRACT FROM AUTHOR]

Spastic paraplegia type 4 (SPG4) is the most common type of hereditary spastic paraplegia (HSP) caused by the mutations in the SPAST gene, which encodes a microtubule-severing protein named spastin. Spastin regulates the number and mobility of microtubules and is essential for axonal outgrowth and neuronal morphogenesis. Herein, we report a patient with SPG4 harboring a novel donor splice site mutation in the SPAST gene (c.1616+1dupG). Although SPG4 usually manifests itself as a pure form of HSP, this patient exhibited a slow progressive cognitive decline and also developed narcolepsy type 2 (narcolepsy without cataplexy) prior to the onset of SPG4. Recently, cognitive decline has attracted attention as a main non-motor symptom of SPG4. However, this is the first reported case of a patient developing both SPG4 and narcolepsy, although it remains unclear whether the manifestation of the two diseases is a coincidence or an association. In this report, we describe the clinical symptoms and genetic background of the patient.

Cytoskeletal microtubule rearrangement and movement are crucial in the repair of spinal cord injury. Spastin plays an important role in the regulation of microtubule severing. Both spastin and collapsin response mediator proteins can regulate neurite growth and branching; however, whether spastin interacts with collapsin response mediator protein 3 (CRMP3) during this process remains unclear, as is the mechanism by which CRMP3 participates in the repair of spinal cord injury. In this study, we used a proteomics approach to identify key proteins associated with spinal cord injury repair. We then employed liquid chromatography-mass spectrometry to identify proteins that were able to interact with glutathione S-transferase-spastin. Then, co-immunoprecipitation and staining approaches were used to evaluate potential interactions between spastin and CRMP3. Finally, we co-transfected primary hippocampal neurons with CRMP3 and spastin to evaluate their role in neurite outgrowth. Mass spectrometry identified the role of CRMP3 in the spinal cord injury repair process. Liquid chromatography-mass spectrometry pulldown assays identified three CRMP3 peptides that were able to interact with spastin. CRMP3 and spastin were co-expressed in the spinal cord and were able to interact with one another in vitro and in vivo. Lastly, CRMP3 overexpression was able to enhance the ability of spastin to promote neurite growth and branching. Therefore, our results confirm that spastin and CRMP3 play roles in spinal cord injury repair by regulating neurite growth and branching. These proteins may therefore be novel targets for spinal cord injury repair. The Institutional Animal Care and Use Committee of Jinan University, China approved this study (approval No. IACUS-20181008-03) on October 8, 2018.

Synaptic plasticity is essential for cognitive functions such as learning and memory. One of the mechanisms involved in synaptic plasticity is the dynamic delivery of AMPA receptors (AMPARs) in and out of synapses. Mutations of SPAST, which encodes SPASTIN, a microtubule-severing protein, are considered the most common cause of hereditary spastic paraparesis (HSP). In some cases, patients with HSP also manifest cognitive impairment. In addition, mice with Spastin depletion exhibit working and associative memory deficits and reduced AMPAR levels. However, the exact effect and molecular mechanism of Spastin on AMPARs trafficking has remained unclear. Here, we report that Spastin interacts with AMPAR, and phosphorylation of Spastin enhances its interaction with AMPAR subunit GluA2. Further study shows that phosphorylation of Spastin can increase AMPAR GluA2 surface expression and the amplitude and frequency of miniature excitatory synaptic currents (mEPSC) in cultured hippocampal neurons. Moreover, phosphorylation of Spastin at Ser210 is crucial for GluA2 surface expression. Phosphorylation of Spastin K353A, which obliterates microtubule-severing activity, also promotes AMPAR GluA2 subunit trafficking to the surface and increases the amplitude and frequency of mEPSCs in cultured neurons. Taken together, our data demonstrate that Spastin phosphorylation promotes the surface delivery of the AMPAR GluA2 subunit independent of microtubule dynamics.

Microtubule-severing protein (MTSP) is critical for the survival of both mitotic and postmitotic cells. However, the study of MTSP during meiosis of mammalian oocytes has not been reported. We found that spastin, a member of the MTSP family, was highly expressed in oocytes and aggregated in spindle microtubules. After knocking down spastin by specific siRNA, the spindle microtubule density of meiotic oocytes decreased significantly. When the oocytes were cultured in vitro, the oocytes lacking spastin showed an obvious maturation disorder. Considering the microtubule-severing activity of spastin, we speculate that spastin on spindles may increase the number of microtubule broken ends by severing the microtubules, therefore playing a nucleating role, promoting spindle assembly and ensuring normal meiosis. In addition, we found the colocalization and interaction of collapsin response mediator protein 5 (CRMP5) and spastin in oocytes. CRMP5 can provide structural support and promote microtubule aggregation, creating transportation routes, and can interact with spastin in the microtubule activity of nerve cells (30). Knocking down CRMP5 may lead to spindle abnormalities and developmental disorders in oocytes. Overexpression of spastin may reverse the abnormal phenotype caused by the deletion of CRMP5. In summary, our data support a model in which the interaction between spastin and CRMP5 promotes the assembly of spindle microtubules in oocytes by controlling microtubule dynamics, therefore ensuring normal meiosis. [ABSTRACT FROM AUTHOR]

Microtubule-severing protein Spastin has been shown to co-localize with actin in migratory glioblastoma cells and is linked to glioblastomas’ migration and invasion capacity. However, the effectiveness of Spastin in glioblastoma migration and the molecular mechanism underpinning the orientation of Spastin towards actin filaments remain unknown. Here, we demonstrated that Spastin plays an active role in glioblastoma migration by showing a reduced migratory potential of T98G glioblastoma cells using real-time cell analysis (RTCA) in Spastin-depleted cells. Pull-down assays revealed that a cis–trans isomerase Pin1 interacts with Spastin through binding to the phosphorylated Pin1 recognition motifs in the microtubule-binding domain (MBD), and immunocytochemistry analysis showed that interaction with Pin1 directs Spastin to actin filaments in extended cell regions. Consequently, by utilizing RTCA, we proved that the migration and invasion capacity of T98G glioblastoma cells significantly increased with the overexpression of Spastin, of which the Pin1 recognition motifs in MBD are constitutively phosphorylated, while the overexpression of phospho-mutant form did not have a significant effect on migration and invasion of T98G glioblastoma cells. These findings demonstrate that Pin1 is a novel interaction partner of Spastin, and their interaction drives Spastin to actin filaments, allowing Spastin to contribute to the glioblastomas’ migration and invasion abilities.

Sjögren’s syndrome nuclear autoantigen-1 (SSNA1/NA14) is a microtubule-associated protein with important functions in cilia, dividing cells, and developing neurons. However, the direct effects of SSNA1 on microtubules are not known. We employed in vitro reconstitution with purified proteins and TIRF microscopy to investigate the activity of human SSNA1 on dynamic microtubule ends and lattices. Our results show that SSNA1 modulates all parameters of microtubule dynamic instability—slowing down the rates of growth, shrinkage, and catastrophe, and promoting rescue. We find that SSNA1 forms stretches along growing microtubule ends and binds cooperatively to the microtubule lattice. Furthermore, SSNA1 is enriched on microtubule damage sites, occurring both naturally, as well as induced by the microtubule severing enzyme spastin. Finally, SSNA1 binding protects microtubules against spastin’s severing activity. Taken together, our results demonstrate that SSNA1 is both a potent microtubule-stabilizing protein and a novel sensor of microtubule damage; activities that likely underlie SSNA1’s functions on microtubule structures in cells.

Neurons rely on the long-range trafficking of synaptic components to form and maintain the complex neural networks that encode the human experience. With a single neuron capable of forming thousands of distinct en passant synapses along its axon, spatially precise delivery of the necessary synaptic components is paramount. How these synapses are patterned, as well as how the efficient delivery of synaptic components is regulated, remains largely unknown. Here, we reveal a novel role for the microtubule (MT)-severing enzyme spastin in locally enhancing MT polymerization to influence presynaptic cargo pausing and retention along the axon. In human neurons derived from induced pluripotent stem cells (iPSCs), we identify sites stably enriched for presynaptic components along the axon prior to the robust assembly of mature presynapses apposed by postsynaptic contacts. These sites are capable of cycling synaptic vesicles, are enriched with spastin, and are hotspots for new MT growth and synaptic vesicle precursor (SVP) pausing/retention. The disruption of neuronal spastin level or activity, by CRISPRi-mediated depletion, transient overexpression, or pharmacologic inhibition of enzymatic activity, interrupts the localized enrichment of dynamic MT plus ends and diminishes SVP accumulation. Using an innovative human heterologous synapse model, where microfluidically isolated human axons recognize and form presynaptic connections with neuroligin-expressing non-neuronal cells, we reveal that neurons deficient for spastin do not achieve the same level of presynaptic component accumulation as control neurons. We propose a model where spastin acts locally as an amplifier of MT polymerization to pattern specific regions of the axon for synaptogenesis and guide synaptic cargo delivery. [Display omitted] • Developing human axons are punctuated by synaptic accumulations or protosynapses • Axonal presynaptic accumulation occurs irrespective of axodendritic contact • Microtubule growth and presynaptic cargo delivery are enriched at protosynapses • Spastin locally enhances axonal microtubule growth and synaptic accumulation Aiken and Holzbaur reveal a novel role for the microtubule-severing enzyme spastin during neuronal development, demonstrating that the local amplification of microtubule growth by spastin directs presynaptic accumulation along the developing human axon. [ABSTRACT FROM AUTHOR]

Polyethylene glycol can connect the distal and proximal ends of an injured nerve at the cellular level through axonal fusion to avoid Wallerian degeneration of the injured distal nerve and promote peripheral nerve regeneration. However, this method can only prevent Wallerian degeneration in 10% of axons because the cytoskeleton is not repaired in a timely fashion. Reconstruction of the cytoskeletal trunk and microtubule network has been suggested to be the key for improving the efficiency of axonal fusion. As a microtubule-severing protein, spastin has been used to enhance cytoskeletal reconstruction. Therefore, we hypothesized that spastin combined with polyethylene glycol can more effectively promote peripheral nerve regeneration. A total of 120 male Sprague-Dawley rats were randomly divided into sham, suture, polyethylene glycol, and polyethylene glycol + spastin groups. In suture group rats, only traditional nerve anastomosis of the end-to-end suture was performed after transection of the sciatic nerve. In polyethylene glycol and polyethylene glycol + spastin groups, 50 μL of polyethylene glycol or 25 μL of polyethylene glycol + 25 μL of spastin, respectively, were injected immediately under the epineurium of the distal suture. Sensory fiber regeneration distance, which was used to assess early nerve regeneration at 1 week after surgery, was shortest in the suture group, followed by polyethylene glycol group and greatest in the polyethylene glycol + spastin group. Behavioral assessment of motor function recovery in rats showed that limb function was restored in polyethylene glycol and polyethylene glycol + spastin groups at 8 weeks after surgery. At 1, 2, 4 and 8 weeks after surgery, sciatic functional index values and percentages of gastrocnemius muscle wet weight were highest in the sham group, followed by polyethylene glycol + spastin and polyethylene glycol groups, and lowest in the suture group. Masson staining was utilized to assess the morphology of muscle tissue. Morphological changes in skeletal muscle were detectable in suture, polyethylene glycol, and polyethylene glycol + spastin groups at 1, 2, 4, and 8 weeks after surgery. Among them, muscular atrophy of the suture group was most serious, followed by polyethylene glycol and polyethylene glycol + spastin groups. Ultrastructure of distal sciatic nerve tissue, as detected by transmission electron microscopy, showed a pattern of initial destruction, subsequent disintegration, and gradual repair in suture, polyethylene glycol, and polyethylene glycol + spastin groups at 1, 2, 4, and 8 weeks after surgery. As time proceeded, axonal ultrastructure gradually recovered. Indeed, the polyethylene glycol + spastin group was similar to the sham group at 8 weeks after surgery. Our findings indicate that the combination of polyethylene glycol and spastin can promote peripheral nerve regeneration. Moreover, the effect of this combination was better than that of polyethylene glycol alone, and both were superior to the traditional neurorrhaphy. This study was approved by the Animal Ethics Committee of the Second Military Medical University, China (approval No. CZ20170216) on March 16, 2017.

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.

Dendritic spines are specialized structures involved in neuronal processes on which excitatory synaptic contact occurs. The microtubule cytoskeleton is vital for maintaining spine morphology and mature synapses. Spastin is related to microtubule-severing proteases and is involved in synaptic bouton formation. However, it is not yet known if spastin can be modified by Small Ubiquitin-like Modifier (SUMO) or how this modification regulates dendritic spines. Spastin was shown to be SUMOylated at K427, and its deSUMOylation promoted microtubule stability. In addition, SUMOylation of spastin was shown to affect signalling pathways associated with long term synaptic depression. SUMOylated spastin promoted the development of dendrites and dendritic spines. Moreover, SUMOylated spastin regulated endocytosis and affected the transport of the AMPA receptor, GluA1. Our findings suggest that SUMOylation of spastin promotes GluA1 internalization and regulates dendritic spine morphology through targeting of microtubule dynamics.

Spastic paraplegia type 4 (SPG4) is the most common type of hereditary spastic paraplegia (HSP) caused by the mutations in the SPAST gene, which encodes a microtubule-severing protein named spastin. Spastin regulates the number and mobility of microtubules and is essential for axonal outgrowth and neuronal morphogenesis. Herein, we report a patient with SPG4 harboring a novel donor splice site mutation in the SPAST gene (c.1616+1dupG). Although SPG4 usually manifests itself as a pure form of HSP, this patient exhibited a slow progressive cognitive decline and also developed narcolepsy type 2 (narcolepsy without cataplexy) prior to the onset of SPG4. Recently, cognitive decline has attracted attention as a main non-motor symptom of SPG4. However, this is the first reported case of a patient developing both SPG4 and narcolepsy, although it remains unclear whether the manifestation of the two diseases is a coincidence or an association. In this report, we describe the clinical symptoms and genetic background of the patient. [ABSTRACT FROM AUTHOR]

Hereditary spastic paraplegia (HSP) is a group of genetically heterogenous neurodegenerative disorders characterized by progressive spasticity and weakness of lower limbs. We report a novel splicing variant (c.1617-2A>C) of the SPAST gene in a heterozygous carrier from an Italian family with autosomal dominant HSP. The case study describes a pure form of spastic paraparesis with the cardinal clinical features of SPG4. The novel variant affects a canonical splice site and is likely to disrupt RNA splicing. We conclude that the c.1617-2A>C substitution is a null variant, which could be classified as pathogenic; its penetrance should be further investigated., Competing Interests: None to declare., (© 2024 The Author(s). Published by Elsevier B.V.)

Purpose: To provide an overview of outcome and complications of selective dorsal rhizotomy (SDR) and intrathecal baclofen pump implantation (ITB) for spasticity treatment in children with hereditary spastic paraplegia (HSP)., Methods: Retrospective study including children with HSP and SDR or ITB. Gross motor function measure (GMFM-66) scores and level of spasticity were assessed., Results: Ten patients were included (most had mutations in ATL1 (n = 4) or SPAST (n = 3) genes). Four walked without and two with walking aids, four were non-walking children. Six patients underwent SDR, three patients ITB, and one both. Mean age at surgery was 8.9 ± 4.5 years with a mean follow-up of 3.4 ± 2.2 years. Five of the SDR patients were walking. Postoperatively spasticity in the legs was reduced in all patients. The change in GMFM-66 score was + 8.0 (0-19.7 min-max). The three ITB patients treated (SPAST (n = 2) and PNPLA6 (n = 1) gene mutation) were children with a progressive disease course. No complications of surgery occurred., Conclusions: SDR is a feasible treatment option in carefully selected children with HSP, especially in walking patients. The majority of patients benefit with respect to gross motor function, complication risk is low. ITB was used in children with severe and progressive disease., (© 2023. The Author(s).)

Spastin, a microtubule-severing enzyme, is important for neurite outgrowth. However, the mechanisms underlying the post-transcriptional regulation of spastin during microtubule-related processes are largely unknown. We demonstrated that the spastin expression level is controlled by a long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/microRNA-30 (miR-30) axis during neurite outgrowth. The miR-30 expression level decreased in hippocampal neurons with increasing days in culture, and miR-30 overexpression suppressed while miR-30 inhibition promoted neurite outgrowth in hippocampal neurons. Spastin was validated as a target gene of miR-30 using the luciferase reporter assay. The protein expression, microtubule severing activity, and neurite promoting effect of spastin were suppressed by the overexpression of miR-30 mimics and increased by miR-30 inhibitors. MALAT1 expression increased during neurite outgrowth and MALAT1 silencing impaired neurite outgrowth. miR-30 was a sponge target of MALAT1 and MALAT1/miR-30 altered neurite outgrowth in hippocampal neurons. MALAT1 overexpression reversed the inhibitory effect of miR-30 on the activity of a luciferase reporter construct containing spastin, as well as spastin mRNA and protein expression, indicating that spastin was a downstream effector of MALAT1/miR-30. The MALAT1/miR-30 cascade also modulated spastin-induced microtubule severing, and the MALAT1/miR-30/spastin axis regulated neurite outgrowth in hippocampal neurons. This study suggests a new mechanism governing neurite outgrowth in hippocampal neurons involving MALAT1/miR-30-regulated spastin expression.

Neurodegenerative mechanisms due to mutations in spastin currently center on neuronal defects, primarily in microtubule and endomembrane regulation. Spastin loss in Drosophila larvae compromises neuronal microtubule distribution, alters synaptic bouton morphology, and weakens synaptic transmission at glutamatergic neuromuscular junction (NMJ) synapses. Pak3, a p21-activated kinase that promotes actin polymerization and filopodial projections, is required for these spastin mutant defects; animals lacking both genes have normal NMJs. Here we show that Pak3 is expressed in central and peripheral glial populations, and reduction of Pak3 specifically in subperineurial glial cells is sufficient to suppress the phenotypes associated with spastin loss. Subperineurial glia in the periphery ensheathe motor neuron axons and have been shown to extend actin-based projections that regulate synaptic terminals during normal NMJ development. We find that these subperineurial glial projections are Pak3-dependent and nearly twice as frequent in spastin mutants, while in Pak3, spastin double mutants, neither glial projections nor synaptic defects are observed. Spastin deficiency thus increases Pak3-dependent subperineurial glia activity, which is in turn required for neuronal defects. Our results demonstrate a central role for Pak3-mediated, altered glial behavior in the neuronal defects due to spastin loss, and suggest that a similar reactive glia-mediated mechanism may underlie human AD-HSP pathogenesis.

Mutations in SPG7 and SPAST are common causes of hereditary spastic paraplegia (HSP). While some SPG7 mutations cause paraplegin deficiency, other SPG7 mutations cause increased paraplegin expression. Mitochondrial function has been studied in models that are paraplegin-deficient (human, mouse, and Drosophila models with large exonic deletions, null mutations, or knockout models) but not in models of mutations that express paraplegin. Here, we evaluated mitochondrial function in olfactory neurosphere-derived cells, derived from patients with a variety of SPG7 mutations that express paraplegin and compared them to cells derived from healthy controls and HSP patients with SPAST mutations, as a disease control. We quantified paraplegin expression and an extensive range of mitochondrial morphology measures (fragmentation, interconnectivity, and mass), mitochondrial function measures (membrane potential, oxidative phosphorylation, and oxidative stress), and cell proliferation. Compared to control cells, SPG7 patient cells had increased paraplegin expression, fragmented mitochondria with low interconnectivity, reduced mitochondrial mass, decreased mitochondrial membrane potential, reduced oxidative phosphorylation, reduced ATP content, increased mitochondrial oxidative stress, and reduced cellular proliferation. Mitochondrial dysfunction was specific to SPG7 patient cells and not present in SPAST patient cells, which displayed mitochondrial functions similar to control cells. The mitochondrial dysfunction observed here in SPG7 patient cells that express paraplegin was similar to the dysfunction reported in cell models without paraplegin expression. The p.A510V mutation was common to all patients and was the likely species associated with increased expression, albeit seemingly non-functional. The lack of a mitochondrial phenotype in SPAST patient cells indicates genotype-specific mechanisms of disease in these HSP patients.

The endoplasmic reticulum (ER) extends as a network of interconnected tubules and sheet-like structures in eukaryotic cells. ER tubules dynamically change their morphology and position within the cells in response to physiological stimuli and these network rearrangements depend on the microtubule (MT) cytoskeleton. Store-operated calcium entry (SOCE) relies on the repositioning of ER tubules to form specific ER-plasma membrane junctions. Indeed, the tips of polymerizing MTs are supposed to provide the anchor for ER tubules to move toward the plasma membrane, however the precise role of the cytoskeleton during SOCE has not been conclusively clarified. Here we exploit an in vivo approach involving the manipulation of MT dynamics in Drosophila melanogaster by neuronal expression of a dominant-negative variant of the MT-severing protein spastin to induce MT hyper-stabilization. We show that MT stabilization alters ER morphology, favoring an enrichment in ER sheets at the expense of tubules. Stabilizing MTs has a negative impact on the process of SOCE and results in a reduced ER Ca2+ content, affecting the flight ability of the flies. Restoring proper MT organization by administering the MT-destabilizing drug vinblastine, chronically or acutely, rescues ER morphology, SOCE and flight ability, indicating that MT dynamics impairment is responsible for all the phenotypes observed.

Spastin, a microtubule-severing enzyme, is important for neurite outgrowth. However, the mechanisms underlying the post-transcriptional regulation of spastin during microtubule-related processes are largely unknown. We demonstrated that the spastin expression level is controlled by a long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/microRNA-30 (miR-30) axis during neurite outgrowth. The miR-30 expression level decreased in hippocampal neurons with increasing days in culture, and miR-30 overexpression suppressed while miR-30 inhibition promoted neurite outgrowth in hippocampal neurons. Spastin was validated as a target gene of miR-30 using the luciferase reporter assay. The protein expression, microtubule severing activity, and neurite promoting effect of spastin were suppressed by the overexpression of miR-30 mimics and increased by miR-30 inhibitors. MALAT1 expression increased during neurite outgrowth and MALAT1 silencing impaired neurite outgrowth. miR-30 was a sponge target of MALAT1 and MALAT1/miR-30 altered neurite outgrowth in hippocampal neurons. MALAT1 overexpression reversed the inhibitory effect of miR-30 on the activity of a luciferase reporter construct containing spastin, as well as spastin mRNA and protein expression, indicating that spastin was a downstream effector of MALAT1/miR-30. The MALAT1/miR-30 cascade also modulated spastin-induced microtubule severing, and the MALAT1/miR-30/spastin axis regulated neurite outgrowth in hippocampal neurons. This study suggests a new mechanism governing neurite outgrowth in hippocampal neurons involving MALAT1/miR-30-regulated spastin expression. [ABSTRACT FROM AUTHOR]

Neurodegenerative mechanisms due to mutations in spastin currently center on neuronal defects, primarily in microtubule and endomembrane regulation. Spastin loss in Drosophila larvae compromises neuronal microtubule distribution, alters synaptic bouton morphology, and weakens synaptic transmission at glutamatergic neuromuscular junction (NMJ) synapses. Pak3, a p21-activated kinase that promotes actin polymerization and filopodial projections, is required for these spastin mutant defects; animals lacking both genes have normal NMJs. Here we show that Pak3 is expressed in central and peripheral glial populations, and reduction of Pak3 specifically in subperineurial glial cells is sufficient to suppress the phenotypes associated with spastin loss. Subperineurial glia in the periphery ensheathe motor neuron axons and have been shown to extend actin-based projections that regulate synaptic terminals during normal NMJ development. We find that these subperineurial glial projections are Pak3-dependent and nearly twice as frequent in spastin mutants, while in Pak3, spastin double mutants, neither glial projections nor synaptic defects are observed. Spastin deficiency thus increases Pak3-dependent subperineurial glia activity, which is in turn required for neuronal defects. Our results demonstrate a central role for Pak3-mediated, altered glial behavior in the neuronal defects due to spastin loss, and suggest that a similar reactive glia-mediated mechanism may underlie human AD-HSP pathogenesis. [ABSTRACT FROM AUTHOR]