Directed conversion of Alzheimer's disease patient skin fibroblasts into functional neurons.

Directed conversion of mature human cells, as from fibroblasts to neurons, is of potential clinical utility for neurological disease modeling as well as cell therapeutics. Here, we describe the efficient generation of human-induced neuronal (hiN) cells from adult skin fibroblasts of unaffected individuals and Alzheimer's patients, using virally transduced transcription regulators and extrinsic support factors. hiN cells from unaffected individuals display morphological, electrophysiological, and gene expression profiles that typify glutamatergic forebrain neurons and are competent to integrate functionally into the rodent CNS. hiN cells from familial Alzheimer disease (FAD) patients with presenilin-1 or -2 mutations exhibit altered processing and localization of amyloid precursor protein (APP) and increased production of Aß, relative to the source patient fibroblasts or hiN cells from unaffected individuals. Together, our findings demonstrate directed conversion of human fibroblasts to a neuronal phenotype and reveal cell type-selective pathology in hiN cells derived from FAD patients.

Vascularized Brain Assembloids With Enhanced Cellular Complexity Provide Insights Into the Cellular Deficits of Tauopathy.

Advanced technologies have enabled the engineering of self-organized 3-dimensional (3D) cellular structures from human induced pluripotent stem cells (hiPSCs), namely organoids, which recapitulate some key features of tissue development and functions of the human central nervous system (CNS). While hiPSC-derived 3D CNS organoids hold promise in providing a human-specific platform for studying CNS development and diseases, most of them do not incorporate the full range of implicated cell types, including vascular cell components and microglia, limiting their ability to accurately recreate the CNS environment and their utility in the study of certain aspects of the disease. Here we have developed a novel approach, called vascularized brain assembloids, for constructing hiPSC-derived 3D CNS structures with a higher level of cellular complexity. This is achieved by integrating forebrain organoids with common myeloid progenitors and phenotypically stabilized human umbilical vein endothelial cells (VeraVecs), which can be cultured and expanded in serum-free conditions. Compared with organoids, these assembloids exhibited enhanced neuroepithelial proliferation, advanced astrocytic maturation, and increased synapse numbers. Strikingly, the assembloids derived from hiPSCs harboring the tauP301S mutation exhibited increased levels of total tau and phosphorylated tau, along with a higher proportion of rod-like microglia-like cells and enhanced astrocytic activation, when compared to the assembloids derived from isogenic hiPSCs. Additionally, the tauP301S assembloids showed an altered profile of neuroinflammatory cytokines. This innovative assembloid technology serves as a compelling proof-of-concept model, opening new avenues for unraveling the intricate complexities of the human brain and accelerating progress in the development of effective treatments for neurological disorders.

Modeling gain-of-function and loss-of-function components of SPAST-based hereditary spastic paraplegia using transgenic mice.

Hereditary spastic paraplegia (HSP) is a disease in which dieback degeneration of corticospinal tracts, accompanied by axonal swellings, leads to gait deficiencies. SPG4-HSP, the most common form of the disease, results from mutations of human spastin gene (SPAST), which is the gene that encodes spastin, a microtubule-severing protein. The lack of a vertebrate model that recapitulates both the etiology and symptoms of SPG4-HSP has stymied the development of effective therapies for the disease. hSPAST-C448Y mice, which express human mutant spastin at the ROSA26 locus, display corticospinal dieback and gait deficiencies but not axonal swellings. On the other hand, mouse spastin gene (Spast)-knockout (KO) mice display axonal swellings but not corticospinal dieback or gait deficiencies. One possibility is that reduced spastin function, resulting in axonal swellings, is not the cause of the disease but exacerbates the toxic effects of the mutant protein. To explore this idea, Spast-KO and hSPAST-C448Y mice were crossbred, and the offspring were compared with the parental lines via histological and behavioral analyses. The crossbred animals displayed axonal swellings as well as earlier onset, worsened gait deficiencies and corticospinal dieback compared with the hSPAST-C448Y mouse. These results, together with observations on changes in histone deacetylases 6 and tubulin modifications in the axon, indicate that each of these three transgenic mouse lines is valuable for investigating a different component of the disease pathology. Moreover, the crossbred mice are the best vertebrate model to date for testing potential therapies for SPG4-HSP.

A Visible Light Responsive Smart Covalent Organic Framework with a Bridged Azobenzene Backbone.

Condensation of 3,3'-diamino-2,2'-ethylene-bridged azobenzene with 1,2,4,5-tetrakis-(4-formylphenyl) benzene produces a visible light responsive porous 2D covalent organic framework, COF-bAzo-TFPB, with a large surface area, good crystallinity, and thermal and chemical stability. The results demonstrate that the elaborated designed linker can make azo unit on the COF-bAzo-TFPB skeleton undergo reversible photoisomerization. This work expands the application scope of covalent organic frameworks in photo-controlled release, uptake of guest molecules, dynamic photoswitching, and UV-sensitive functions.

A cellular approach to understanding and treating Gulf War Illness.

Gulf War Illness (GWI), a disorder suffered by approximately 200,000 veterans of the first Gulf War, was caused by exposure to low-level organophosphate pesticides and nerve agents in combination with battlefield stress. To elucidate the mechanistic basis of the brain-related symptoms of GWI, human-induced pluripotent stem cells (hiPSCs) derived from veterans with or without GWI were differentiated into forebrain glutamatergic neurons and then exposed to a Gulf War (GW) relevant toxicant regimen consisting of a sarin analog and cortisol, a human stress hormone. Elevated levels of total and phosphorylated tau, reduced microtubule acetylation, altered mitochondrial dynamics/transport, and decreased neuronal activity were observed in neurons exposed to the toxicant regimen. Some of the data are consistent with the possibility that some veterans may have been predisposed to acquire GWI. Wistar rats exposed to a similar toxicant regimen showed a mild learning and memory deficit, as well as cell loss and tau pathology selectively in the CA3 region of the hippocampus. These cellular responses offer a mechanistic explanation for the memory loss suffered by veterans with GWI and provide a cell-based model for screening drugs and developing personalized therapies for these veterans.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated kif15 mutations accelerate axonal outgrowth during neuronal development and regeneration in zebrafish.

KIF15, the vertebrate kinesin-12, is best known as a mitotic motor protein, but continues to be expressed in neurons. Like KIF11 (the vertebrate kinesin-5), KIF15 interacts with microtubules in the axon to limit their sliding relative to one another. Unlike KIF11, KIF15 also regulates interactions between microtubules and actin filaments at sites of axonal branch formation and in growth cones. Our original work on these motors was done on cultured rat neurons, but we are now using zebrafish to extend these studies to an in vivo model. We previously studied kif15 in zebrafish by injecting splice-blocking morpholinos injected into embryos. Consistent with the cell culture work, these studies demonstrated that axons grow faster and longer when KIF15 levels are reduced. In the present study, we applied CRISPR/Cas9-based knockout technology to create kif15 mutants and labeled neurons with Tg(mnx1:GFP) transgene or transient expression of elavl3:EGFP-alpha tubulin. We then compared by live imaging the homozygotic, heterozygotic mutants to their wildtype siblings to ascertain the effects of depletion of kif15 during Caudal primary motor neuron and Rohon-Beard (R-B) sensory neuron development. The results showed, compared to the kif15 wildtype, the number of branches was reduced while axon outgrowth was accelerated in kif15 homozygotic and heterozygotic mutants. In R-B sensory neurons, after laser irradiation, injured axons with loss of kif15 displayed significantly greater regenerative velocity. Given these results and the fact that kif15 drugs are currently under development, we posit kif15 as a novel target for therapeutically augmenting regeneration of injured axons.

Hereditary spastic paraplegia: gain-of-function mechanisms revealed by new transgenic mouse.

Mutations of the SPAST gene, which encodes the microtubule-severing protein spastin, are the most common cause of hereditary spastic paraplegia (HSP). Haploinsufficiency is the prevalent opinion as to the mechanism of the disease, but gain-of-function toxicity of the mutant proteins is another possibility. Here, we report a new transgenic mouse (termed SPASTC448Y mouse) that is not haploinsufficient but expresses human spastin bearing the HSP pathogenic C448Y mutation. Expression of the mutant spastin was documented from fetus to adult, but gait defects reminiscent of HSP (not observed in spastin knockout mice) were adult onset, as is typical of human patients. Results of histological and tracer studies on the mouse are consistent with progressive dying back of corticospinal axons, which is characteristic of the disease. The C448Y-mutated spastin alters microtubule stability in a manner that is opposite to the expectations of haploinsufficiency. Neurons cultured from the mouse display deficits in organelle transport typical of axonal degenerative diseases, and these deficits were worsened by depletion of endogenous mouse spastin. These results on the SPASTC448Y mouse are consistent with a gain-of-function mechanism underlying HSP, with spastin haploinsufficiency exacerbating the toxicity of the mutant spastin proteins. These findings reveal the need for a different therapeutic approach than indicated by haploinsufficiency alone.

Pharmacologically increasing microtubule acetylation corrects stress-exacerbated effects of organophosphates on neurons.

Many veterans of the 1990-1991 Gulf War contracted Gulf War Illness (GWI), a multisymptom disease that primarily affects the nervous system. Here, we treated cultures of human or rat neurons with diisopropyl fluorophosphate (DFP), an analog of sarin, one of the organophosphate (OP) toxicants to which the military veterans were exposed. All observed cellular defects produced by DFP were exacerbated by pretreatment with corticosterone or cortisol, which, in rat and human neurons, respectively, serves in our experiments to mimic the physical stress endured by soldiers during the war. To best mimic the disease, DFP was used below the level needed to inhibit acetylcholinesterase. We observed a diminution in the ratio of acetylated to total tubulin that was correctable by treatment with tubacin, a drug that inhibits HDAC6, the tubulin deacetylase. The reduction in microtubule acetylation was coupled with deficits in microtubule dynamics, which were correctable by HDAC6 inhibition. Deficits in mitochondrial transport and dopamine release were also improved by tubacin. Thus, various negative effects of the toxicant/stress exposures were at least partially correctable by restoring microtubule acetylation to a more normal status. Such an approach may have therapeutic benefit for individuals suffering from GWI or other neurological disorders linked to OP exposure.

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.

Integrative genomics identifies APOE ε4 effectors in Alzheimer's disease.

Late-onset Alzheimer's disease (LOAD) risk is strongly influenced by genetic factors such as the presence of the apolipoprotein E ε4 allele (referred to here as APOE4), as well as non-genetic determinants including ageing. To pursue mechanisms by which these affect human brain physiology and modify LOAD risk, we initially analysed whole-transcriptome cerebral cortex gene expression data in unaffected APOE4 carriers and LOAD patients. APOE4 carrier status was associated with a consistent transcriptomic shift that broadly resembled the LOAD profile. Differential co-expression correlation network analysis of the APOE4 and LOAD transcriptomic changes identified a set of candidate core regulatory mediators. Several of these--including APBA2, FYN, RNF219 and SV2A--encode known or novel modulators of LOAD associated amyloid beta A4 precursor protein (APP) endocytosis and metabolism. Furthermore, a genetic variant within RNF219 was found to affect amyloid deposition in human brain and LOAD age-of-onset. These data implicate an APOE4 associated molecular pathway that promotes LOAD.

Depletion of kinesin-12, a myosin-IIB-interacting protein, promotes migration of cortical astrocytes.

Kinesin-12 (also named Kif15) participates in important events during neuronal development, such as cell division of neuronal precursors, migration of young neurons and establishment of axons and dendritic arbors, by regulating microtubule organization. Little is known about the molecular mechanisms behind the functions of kinesin-12, and even less is known about its roles in other cell types of the nervous system. Here, we show that kinesin-12 depletion from cultured rat cortical astrocytes decreases cell proliferation but increases migration. Co-immunoprecipitation, GST pulldown and small interfering RNA (siRNA) experiments indicated that kinesin-12 directly interacts with myosin-IIB through their tail domains. Immunofluorescence analyses indicated that kinesin-12 and myosin-IIB colocalize in the lamellar region of astrocytes, and fluorescence resonance energy transfer analyses revealed an interaction between the two. The phosphorylation at Thr1142 of kinesin-12 was vital for their interaction. Loss of their interaction through expression of a phosphorylation mutant of kinesin-12 promoted astrocyte migration. We suggest that kinesin-12 and myosin-IIB can form a hetero-oligomer that generates force to integrate microtubules and actin filaments in certain regions of cells, and in the case of astrocytes, that this interaction can modulate their migration.

A simple approach to assess N load capacity of rice paddy fields in the southern Taihu Lake watershed.

High nitrogen (N) leaching from irrigated agricultural soils is the result of N input exceeding soil N load capacity (NLC). A simple approach was developed in this research to assess the NLC of paddy soils in the southern Taihu Lake watershed. Paddy soils were classified into four types (Submergenic, Illuvium, Gleyed, and Percogenic) and 28 soil samples representing all four types were collected from across the region. The NLC values of the paddy soils were assessed using a split-line model and the spatial variability of the NLC among various rice paddy soils in the region was also evaluated with Kriging analysis. Results showed the NLC of paddy soils were both soil type and background N content related. The critical N sorption values (NLC plus soil N background) of the Gleyed, Illuvium, Submergenic, and Percogenic paddy soil samples varied from 283.1 to 315.6 mg kg(-1), 203.0 to 270.2 mg kg(-1), 240.6 to 254.4 mg kg(-1), and 177.4 to 186.2 mg kg(-1), respectively. However, on average the NLC of paddy soils in the region was 80.3 mg kg(-1), and the corresponding environmental N load threshold was around 110 kg N ha(-1). Geo-statistic results showed that the NLCs were unevenly distributed throughout the rice paddy dominated areas of the southern Taihu Lake watershed. The NLC assessment approach and spatial distribution information provided helpful guidance to set an environmental N threshold for best N management and hence reduce degradation of water for the whole rice ecosystem.

[Study on the Structure and Photoluminescent Properties of Mono-Zn(â ¡) and Di-Cu(â ) Complexes Constructed from Nitrogen Heterocyclic Ring Ligand].

The mononuclear Zn(Ⅱ) complex [Zn(2,6-PDA)(phen)H2O]·H2O (1) and binuclear Cu(Ⅰ) complex{[Cu(µ-Ⅰ)(phen)H2O]·H2O}2 (2) (2,6-H2PDA=2,6- pyridinedicarboxylic acid，phen=1,10- phenanthroline monohydrate) have been prepared with hydro-thermal synthesis method. These complexes have been characterized with single-crystal X-ray, elemental analysis, and IR spectroscopy. The fluorescence spectra of 1 and 2 are studied in solid-state and dimethyl sulfoxide (DMSO) solution. The maximum absorption peak of 1 and 2 are at 253 nm and 242 nm respectively, which are red shift to that of the phen ligand with inceased intensity. It may be assigned to the intraligand π→π* transition of the phen ligand that is modified by the Zn(Ⅱ) or Cu(Ⅰ) ions. On the basis of the coordination, the absorption of organic ligands in the ultraviolet region is increased, which is better for the energy absorption of the ligand. 1 and 2 all showed blue light emission. The emission peak of 1 and 2 have experienced a red shift (ca. 55 and 23 nm) in the solid state (λem = 407, 434, 467 nm for 1, 442, 469, 501 nm for 2) compared to in DMSO solution (λem = 361, 379, 392 nm for 1, 422, 443, 461 nm for 2). The red shift phenomenon can be attributed to the π-stacking of the aromatic rings and other intermolecular Interactions in these molecules in the solid state. Especially, the strong Cu(Ⅰ)…Cu(Ⅰ) interaction of 2 can decrease the HOMO­LUMO energy gap with the red-shifted emission wavelength.

[A purple-emitting luminescence complex [Cd(bpdc)(phen)2(H2O)] . 6H2O: synthesis, structure and spectral properties].

A supramolecular Cd(II) complex[Cd(bpdc) (phen)2 (H2O)] . 6H20 (1) was synthesized with 2, 4'-biphenyldicarboxylic acid (H2bpdc) and 1, 10-phenanthroline (phen) under hydrothermal conditions and characterized by single-crystal X-ray diffraction elemental analysis, and IR spectrum. Single-crystal X-ray analysis reveals complex 1 crystalizes in the triclinic P 1 space group, the metal center Cd(II) ion is six-coordinated and exhibits a distorted octahedron geometry arrangement. 3D supramolecular structure could be formed taking into account two kinds of hydrogen bonds and π--π interactions. At the same time, we discussed the luminescent properties of complex 1 in the solid-state as well as in the solvents at different temperatures. When excited at 350 nm, in the solid state at 298 K, 1 has purple luminescence with emission band at 390 nm; in the solid state at 77 K, 1 displays two emission bands at 380 and 520 nm. Because the vibration structure is more defined at low temperature, at 298 K, 1 also has purple luminescence in DMSO and CH3OH solutions with emission bands at 380 and 375 nm, which are blue-shifted compared with solid-state maximum emission band. These all can be attribute to the π*-->π transition based on the coordinate ligands. The fluorescence decay curves of complex 1 indicate that the processes of decay consist of two components. At 298 K, the lifetime of 1 is longer in DMSO solution (τ1 =1. 73 µs and τ2 =14. 07 µs) than that in CH3OH solution (τ1 =1. 21 µs and τ2 = 12. 44 µs). Moreover, the-lifetime of 1 is longer at 77 K (τ1 =1. 96 µs and τ2= 16. 11 µs) than that at 298 K in the solid state (τ1= 1. 20 µs and τ2 =11. 34 µs). The results might be caused by the increase in radiative rate and decrease in non-radiative rate at low temperature.

Reevaluating tau reduction as a therapeutic approach for tauopathies: Insights and perspectives.

Tau, one of the most abundant microtubule-associated protein in neurons plays a role in regulating microtubule dynamics in axons, as well as shaping the overall morphology of the axon. Recent studies challenge the traditional view of tau as a microtubule stabilizer and shed new light on the complexity of its role in regulating various properties of the microtubule. While reducing tau levels shows therapeutic promise for early tauopathies, efficacy wanes in later stages due to resilient toxic tau aggregates and neurofibrillary tangles. Notably, tauopathies involve factors beyond toxic tau alone, necessitating a broader therapeutic approach. Overexpression of human tau in mouse models, although useful for answering some questions, may not accurately reflect disease mechanisms in patients with tauopathies. Furthermore, the interplay between tau and MAP6, another microtubule-associated protein, adds complexity to tau's regulation of microtubule dynamics. Tau promotes the formation and elongation of labile microtubule domains, vital for cellular processes, while MAP6 stabilizes microtubules. A delicate balance between these proteins is important for neuronal function. Therefore, tau reduction therapies require a comprehensive understanding of disease progression, considering functional tau loss, toxic aggregates, and microtubule dynamics. Stage-dependent application and potential unintended consequences must be carefully evaluated. Restoring microtubule dynamics in late-stage tauopathies may necessitate alternative strategies. This knowledge is valuable for developing effective and safe treatments for tauopathies.

Challenges and Efficacy of Astrocyte-to-Neuron Reprogramming in Spinal Cord Injury: In Vitro Insights and In Vivo Outcomes.

Traumatic spinal cord injury (SCI) leads to the disruption of neural pathways, causing loss of neural cells, with subsequent reactive gliosis and tissue scarring that limit endogenous repair. One potential therapeutic strategy to address this is to target reactive scar-forming astrocytes with direct cellular reprogramming to convert them into neurons, by overexpression of neurogenic transcription factors. Here we used lentiviral constructs to overexpress Ascl1 or a combination of microRNAs (miRs) miR124, miR9/9*and NeuroD1 transfected into cultured and in vivo astrocytes. In vitro experiments revealed cortically-derived astrocytes display a higher efficiency (70%) of reprogramming to neurons than spinal cord-derived astrocytes. In a rat cervical SCI model, the same strategy induced only limited reprogramming of astrocytes. Delivery of reprogramming factors did not significantly affect patterns of breathing under baseline and hypoxic conditions, but significant differences in average diaphragm amplitude were seen in the reprogrammed groups during eupneic breathing, hypoxic, and hypercapnic challenges. These results show that while cellular reprogramming can be readily achieved in carefully controlled in vitro conditions, achieving a similar degree of successful reprogramming in vivo is challenging and may require additional steps.

Is Gulf War Illness a prolonged early phase tauopathy?

The work of the Gulf War Illness (GWI) Consortium and that of basic and clinical researchers across the USA have resulted in a better understanding in recent years of the pathological basis of GWI, as well as of the mechanisms underlying the disorder. Among the most concerning symptoms suffered by veterans with GWI are cognitive decrements including those related to memory functioning. These decrements are not severe enough to meet dementia criteria, but there is significant concern that the mild cognitive impairment of these veterans will progress to dementia as they become older. Recent studies on GWI using human brain organoids as well as a rat model suggest that one potential cause of the cognitive problems may be elevated levels of tau in the brain, and this is supported by high levels of tau autoantibodies in the blood of veterans with GWI. There is urgency in finding treatments and preventive strategies for these veterans before they progress to dementia, with added value in doing so because their current status may represent an early phase of tauopathy common to many neurodegenerative diseases.

Vascularized Brain Assembloids with Enhanced Cellular Complexity Provide Insights into The Cellular Deficits of Tauopathy.

Advanced technologies have enabled the engineering of self-organized 3-dimensional (3D) cellular structures from human induced pluripotent stem cells (hiPSCs), namely organoids, which recapitulate some key features of tissue development and functions of the human central nervous system (CNS). While hiPSC-derived 3D CNS organoids hold promise in providing a human-specific platform for studying CNS development and diseases, most of them do not incorporate the full range of implicated cell types, including vascular cell components and microglia, limiting their ability to accurately recreate the CNS environment and their utility in the study of certain aspects of the disease. Here we've developed a novel approach, called vascularized brain assembloids, for constructing hiPSC-derived 3D CNS structures with a higher level of cellular complexity. This is achieved by integrating forebrain organoids with common myeloid progenitors and phenotypically stabilized human umbilical vein endothelial cells (VeraVecs™), which can be cultured and expanded in serum-free conditions. Compared with organoids, these assembloids exhibited enhanced neuroepithelial proliferation, advanced astrocytic maturation, and increased synapse numbers. Strikingly, the assembloids derived from hiPSCs harboring the tau P301S mutation exhibited increased levels of total tau and phosphorylated tau, along with a higher proportion of rod-like microglia-like cells and enhanced astrocytic activation, when compared to the assembloids derived from isogenic hiPSCs. Additionally, they showed an altered profile of neuroinflammatory cytokines. This innovative assembloid technology serves as a compelling proof-of-concept model, opening new avenues for unraveling the intricate complexities of the human brain and accelerating progress in the development of effective treatments for neurological disorders. Significance Statement: Modeling neurodegeneration in human in vitro systems has proved challenging and requires innovative tissue engineering techniques to create systems that can accurately capture the physiological features of the CNS to enable the study of disease processes. The authors develop a novel assembloid model which integrates neuroectodermal cells with endothelial cells and microglia, two critical cell types that are commonly missing from traditional organoid models. They then apply this model to investigate early manifestations of pathology in the context of tauopathy and uncover early astrocyte and microglia reactivity as a result of the tau P301S mutation.

Fidgetin impacts axonal growth and branching in a local mTOR signal dependent manner.

Neurons require a constant increase in protein synthesis during axonal growth and regeneration. AKT-mTOR is a central pathway for mammalian cell survival and regeneration. Fidgetin (Fign) is an ATP-dependent microtubule (MT)-severing enzyme whose functions are associated with neurite outgrowth, axon regeneration and cell migration. Although most previous studies have indicated that depletion of Fign is involved in those biological activities by increasing labile MT mass, it remains unknown whether mTOR activation contributes to this process. Here, we showed that depletion of Fign enhanced p-mTOR/p-S6K activation, and the mTOR inhibitor Rapamycin inhibited axon outgrowth and p-rpS6 activation. We then investigated the effects of neuronal-specific Fign deletion in a rat spinal cord hemisection model by injecting syn-GFP Fign shRNA virus. BBB values revealed an improvement in functional recovery. The p-mTOR was activated along with neuronal Fign depletion. The syn-mCherry virus showed more sprouting neurites entering the injury region, which was confirmed by immunostaining GAP43 protein. Further, we showed that Fign siRNA treatment promoted axon outgrowth and branching, whose underlying mechanism was firstly attributed to local activation of the mTOR pathway, and increased MT dynamicity. Finally, considering L-leucine, promotes axonal growth and neuronal survival, we applied L-leucine with Fign depletion after spinal cord injury or in chondroitin sulfate proteoglycan inhibitory molecules. The phenomenon of synergistically augmented axon regeneration was observed. In summary, our results indicated a novel local mTOR pathway for fidgetin to impact axon growth and provided a combined strategy in SCI.

Fidgetin interacting with microtubule end binding protein EB3 affects axonal regrowth in spinal cord injury.

Fidgetin, a microtubule-severing enzyme, regulates neurite outgrowth, axonal regeneration, and cell migration by trimming off the labile domain of microtubule polymers. Because maintenance of the microtubule labile domain is essential for axon initiation, elongation, and navigation, it is of interest to determine whether augmenting the microtubule labile domain via depletion of fidgetin serves as a therapeutic approach to promote axonal regrowth in spinal cord injury. In this study, we constructed rat models of spinal cord injury and sciatic nerve injury. Compared with spinal cord injury, we found that expression level of tyrosinated microtubules in the labile portion of microtubules continuously increased, whereas fidgetin decreased after peripheral nerve injury. Depletion of fidgetin enhanced axon regeneration after spinal cord injury, whereas expression level of end binding protein 3 (EB3) markedly increased. Next, we performed RNA interference to knockdown EB3 or fidgetin. We found that deletion of EB3 did not change fidgetin expression. Conversely, deletion of fidgetin markedly increased expression of tyrosinated microtubules and EB3. Deletion of fidgetin increased the amount of EB3 at the end of neurites and thereby increased the level of tyrosinated microtubules. Finally, we deleted EB3 and overexpressed fidgetin. We found that fidgetin trimmed tyrosinated tubulins by interacting with EB3. When fidgetin was deleted, the labile portion of microtubules was elongated, and as a result the length of axons and number of axon branches were increased. These findings suggest that fidgetin can be used as a novel therapeutic target to promote axonal regeneration after spinal cord injury. Furthermore, they reveal an innovative mechanism by which fidgetin preferentially severs labile microtubules.