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Doublecortin-Like Kinases Promote Neuronal Survival and Induce Growth Cone Reformation via Distinct Mechanisms
Abstract
After axotomy, neuronal survival and growth cone re-formation are required for axon regeneration. We discovered that doublecortin-like kinases (DCLKs), members of the doublecortin (DCX) family expressed in adult retinal ganglion cells (RGCs), play critical roles in both processes, through distinct mechanisms. Overexpression of DCLK2 accelerated growth cone re-formation in vitro and enhanced the initiation and elongation of axon re-growth after optic nerve injury. These effects depended on both the microtubule (MT)-binding domain and the serine-proline-rich (S/P-rich) region of DCXs in-cis in the same molecules. While the MT-binding domain is known to stabilize MT structures, we show that the S/P-rich region prevents F-actin destabilization in injured axon stumps. Additionally, while DCXs synergize with mTOR to stimulate axon regeneration, alone they can promote neuronal survival possibly by regulating the retrograde propagation of injury signals. Multifunctional DCXs thus represent potential targets for promoting both survival and regeneration of injured neurons.
... In addition to their role in neuronal migration, Dcx and Dclk1 have been shown to function in the formation of axons and dendrites [17,18,[20][21][22][23]. These roles in neuronal migration and neurite formation have been attributed to the ability of Dcx and Dclk1 to bind microtubules, regulate their polymerization and affect the function of other microtubule-binding proteins [10,14,15,20,[23][24][25][26][27][28][29]. Dcx and Dclk1/2 have also been shown to interact with actin filaments and this property has been proposed to contribute to the regulation of axon guidance [28,[30][31][32]. ...
... These roles in neuronal migration and neurite formation have been attributed to the ability of Dcx and Dclk1 to bind microtubules, regulate their polymerization and affect the function of other microtubule-binding proteins [10,14,15,20,[23][24][25][26][27][28][29]. Dcx and Dclk1/2 have also been shown to interact with actin filaments and this property has been proposed to contribute to the regulation of axon guidance [28,[30][31][32]. Outside the nervous system, dcx is expressed in muscle cells [33,34] and Dclk1 has attracted considerable attention as a marker for tumour stem cells in a variety of cancers [35,36]. ...
... While our observations suggest a role for NvDclk1 in the regulation of microtubules, we currently cannot rule out that additional functions, e.g. in regulating the actin cytoskeleton [28,30,85], contribute to the phenotype of the NvDclk1 mutants. ...
The complex morphology of neurons requires precise control of their microtubule cytoskeleton. This is achieved by microtubule-associated proteins (MAPs) that regulate the assembly and stability of microtubules, and transport of molecules and vesicles along them. While many of these MAPs function in all cells, some are specifically or predominantly involved in regulating microtubules in neurons. Here we use the sea anemone Nematostella vectensis as a model organism to provide new insights into the early evolution of neural microtubule regulation. As a cnidarian, Nematostella belongs to an outgroup to all bilaterians and thus occupies an informative phylogenetic position for reconstructing the evolution of nervous system development. We identified an ortholog of the microtubule-binding protein doublecortin-like kinase (NvDclk1) as a gene that is predominantly expressed in neurons and cnidocytes (stinging cells), two classes of cells belonging to the neural lineage in cnidarians. A transgenic NvDclk1 reporter line revealed an elaborate network of neurite-like processes emerging from cnidocytes in the tentacles and the body column. A transgene expressing NvDclk1 under the control of the NvDclk1 promoter suggests that NvDclk1 localizes to microtubules and therefore likely functions as a microtubule-binding protein. Further, we generated a mutant for NvDclk1 using CRISPR/Cas9 and show that the mutants fail to generate mature cnidocytes. Our results support the hypothesis that the elaboration of programs for microtubule regulation occurred early in the evolution of nervous systems.
... Knockdown of Porf-2 accelerates axon growth and growth cone formation in retinal explants Because the formation of axon growth cones is a key step in the initiation of axonal regeneration [24][25][26], we next evaluated whether Porf-2 is involved in this process. We employed an ex vivo explant culture system. ...
... For the immunostaining of retina explants, we used a previously published protocol [26,42]. First, retinal explants were fixed in 3% PFA/3% sucrose for 15 min, then blocked with PBS containing 3% bovine serum albumin, 5% normal goat serum, and 0.1% Triton X-100 for 10 min at room temperature. ...
... Σa d , the total number of axons extending distance d in a nerve with a radius of r, was estimated by summing over all the sections of a thickness t (14 µm): Σa d = πr 2 x [average axons/mm]/t. Quantification of retinal explant growth and axon growth cone formation was performed as described previously [26,42]. Anti-Tuj1 and Phalloidin were used to mark the morphology of axons and the growth cones in retinal explants, respectively. ...
Retinal ganglion cells (RGCs), the sole output neurons in the eyes, are vulnerable to diverse insults in many pathological conditions, which can lead to permanent vision dysfunction. However, the molecular and cellular mechanisms that contribute to protecting RGCs and their axons from injuries are not completely known. Here, we identify that Porf-2, a member of the Rho GTPase activating protein gene group, is upregulated in RGCs after optic nerve crush. Knockdown of Porf-2 protects RGCs from apoptosis and promotes long-distance optic nerve regeneration after crush injury in both young and aged mice in vivo. In vitro, we find that inhibition of Porf-2 induces axon growth and growth cone formation in retinal explants. Inhibition of Porf-2 provides long-term and post-injury protection to RGCs and eventually promotes the recovery of visual function after crush injury in mice. These findings reveal a neuroprotective impact of the inhibition of Porf-2 on RGC survival and axon regeneration after optic nerve injury, providing a potential therapeutic strategy for vision restoration in patients with traumatic optic neuropathy.
... We also found the protein doublecortin-like kinase 2 (DCLK2) expressed in NeuN+ cells of all visual targets ( Supplementary Fig. 2e, f). DCLK2 was recently shown to promote axon growth via induction of growth cone reformation 44 , an essential step for the axon to interact with its environment and to respond to guidance cues. ...
... Recent advances in the field of CNS regeneration have led to longdistance regeneration 3,4,44,55,71 . Yet, this achievement comes with an unexpected drawback as most regenerative axons are misguided away from their proper targets, counteracting any attempt of functional recovery 3,4,55 . ...
In the injured adult central nervous system (CNS), activation of pro-growth molecular pathways in neurons leads to long-distance regeneration. However, most regenerative fibers display guidance defects, which prevent reinnervation and functional recovery. Therefore, the molecular characterization of the proper target regions of regenerative axons is essential to uncover the modalities of adult reinnervation. In this study, we use mass spectrometry (MS)-based quantitative proteomics to address the proteomes of major nuclei of the adult visual system. These analyses reveal that guidance-associated molecules are expressed in adult visual targets. Moreover, we show that bilateral optic nerve injury modulates the expression of specific proteins. In contrast, the expression of guidance molecules remains steady. Finally, we show that regenerative axons are able to respond to guidance cues ex vivo, suggesting that these molecules possibly interfere with brain target reinnervation in adult. Using a long-distance regeneration model, we further demonstrate that the silencing of specific guidance signaling leads to rerouting of regenerative axons in vivo. Altogether, our results suggest ways to modulate axon guidance of regenerative neurons to achieve circuit repair in adult.
... The clustered regularly interspaced short palindromic repeats (CRISPR) technology (Cong et al., 2013;Mali et al., 2013) provides a powerful means to conduct such screens in vivo. For analysis of ONC, intravitreal injection of adeno-associated virus 2 (AAV2) vectors efficiently transduces most RGCs with minimal effect on other cell types (Nawabi et al., 2015;Park et al., 2008). In addition, the massive and reproducible RGC loss and the short experimental duration Neuron 110, 2607-2624, August 17, 2022 ª 2022 Elsevier Inc. 2607 ll Figure 1. ...
... Third, these plasmids were used to prepare AAV vectors for transducing RGCs in vivo. We chose serotype 2/2 (AAV2) because it transduces RGCs following intravitreal injection with high efficiency and reasonable selectivity (mostly RGCs and amacrine cells in the ganglion cell layer) (Nawabi et al., 2015;Norsworthy et al., 2017;Park et al., 2008). All AAV viral vectors were made by Boston Children's Hospital Viral Core. ...
Regulatory programs governing neuronal death and axon regeneration in neurodegenerative diseases remain poorly understood. In adult mice, optic nerve crush (ONC) injury by severing retinal ganglion cell (RGC) axons results in massive RGC death and regenerative failure. We performed an in vivo CRISPR-Cas9-based genome-wide screen of 1,893 transcription factors (TFs) to seek repressors of RGC survival and axon regeneration following ONC. In parallel, we profiled the epigenetic and transcriptional landscapes of injured RGCs by ATAC-seq and RNA-seq to identify injury-responsive TFs and their targets. These analyses converged on four TFs as critical survival regulators, of which ATF3/CHOP preferentially regulate pathways activated by cytokines and innate immunity and ATF4/C/EBPγ regulate pathways engaged by intrinsic neuronal stressors. Manipulation of these TFs protects RGCs in a glaucoma model. Our results reveal core transcription programs that transform an initial axonal insult into a degenerative process and suggest novel strategies for treating neurodegenerative diseases.
... How TP53mut cells bypass TP53 control of NUC also remains to be answered. In addition, DCX and LIS1 functions are not confined to NUC because of their involvement in MT transport and regeneration in PNS (Nawabi et al, 2015;Hines et al, 2018). The defects in LP and lack of significant alterations in N-C distance, which we observed in DCX-KD cells, conform with this idea. ...
... Nevertheless, in ADRN NB, MT function lies upstream of actomyosin forces. MT-binding proteins such as DCX can serve as bridges between MTs and actin in neurons (Nawabi et al, 2015); MT destabilisation in ADRN NB cells might disrupt these links, crushing the entire migration machinery. The N-C-inversion mode requires the concerted action of process maturation and dynamic N-C attachment. ...
The migrational propensity of neuroblastoma is affected by cell identity, but the mechanisms behind the divergence remain unknown. Using RNAi and time-lapse imaging, we show that ADRN-type NB cells exhibit RAC1- and kalirin-dependent nucleokinetic (NUC) migration that relies on several integral components of neuronal migration. Inhibition of NUC migration by RAC1 and kalirin-GEF1 inhibitors occurs without hampering cell proliferation and ADRN identity. Using three clinically relevant expression dichotomies, we reveal that most of up-regulated mRNAs in RAC1- and kalirin–GEF1–suppressed ADRN-type NB cells are associated with low-risk characteristics. The computational analysis shows that, in a context of overall gene set poverty, the upregulomes in RAC1- and kalirin–GEF1–suppressed ADRN-type cells are a batch of AU-rich element–containing mRNAs, which suggests a link between NUC migration and mRNA stability. Gene set enrichment analysis–based search for vulnerabilities reveals prospective weak points in RAC1- and kalirin–GEF1–suppressed ADRN-type NB cells, including activities of H3K27- and DNA methyltransferases. Altogether, these data support the introduction of NUC inhibitors into cancer treatment research.
... In humans, two members of DCLK1 and DCLK2 are present and are known to play the similar or in some cases redundant roles in neuronal survival, growth cone regeneration, and axon regeneration. [7][8][9] More recently, DCLK1 has been reported to contribute to plasticity regulation of both cancer cells and stem cells in the mouse pancreas model. 5 There, DCLK1 positive cells appear to proliferate in response to both chemical and physical damage, and promote regeneration under the normal condition. ...
... 2 | RESULTS AND DISCUSSION 2.1 | Identification of DCLK1 and two genes in the sea urchin embryo Both human DCLK1 and DCLK2 are known to undergo alternative transcription and splicing, and may be present as a truncated version that contains only the N-terminus (DCX domain) or the C-terminus (Kinase domain) of each protein. 8,[10][11][12] In the human database, DCLK1 is reported to have five isoforms. Human DCLK1 isoforms 1 and 5 appear to have the full length while isoforms 2 and 3 appear to be the shorter form only with the C terminus (Data S1). ...
Background
Doublecortin‐like kinase1 and 2 (DCLKs) are protein Ser/Thr kinases important for neuronal development. More recently, they are also reported to regulate plasticity such as cell proliferation and differentiation of stem cells and cancer cells, but the details of their functions in this biological context are still unclear. With an attempt to reveal the functions of DCLKs in plasticity regulation, we here used the sea urchin embryo that undergoes highly regulative development as an experimental model.
Results
We found that both the transcripts and the proteins of DCLKs are uniformly present during early embryogenesis and with some enrichment in mesenchymal cells after gastrula stage. Knockdown of DCLKs induced general developmental delay and defects at day 2. Further, the damage on the embryo/larva induced ectopic expression of DCLKs in the ectoderm where the damage was most severe. Under a tumor‐prone or ‐suppressive condition, DCLKs expression was upregulated or downregulated, respectively, after damage. In both cases, the embryos showed severe developmental defects.
Conclusions
Taken together, a transient upregulation of DCLKs appears to be involved in a damage response both during normal and abnormal development, and which could result in different phenotypes in a context dependent manner.
... The transcriptional function of STAT3 is crucial for CNS axon regeneration, and MEK regulates the localization and function of STAT3, with PTEN deletion enhancing its role in promoting optic nerve regeneration (117). Moreover, combining PTEN deletion with gene overexpression, such as B-RAF, DCLK2, and Sox11, has yielded promising regenerative outcomes (118)(119)(120). Identifying genes or pathways that protect RGCs or specific subtypes from cell death is equally important, as Sox11 promotes regeneration in non-α-RGCs, which are resistant to PTEN deletion-induced regeneration (2,120). ...
Retinal ganglion cells (RGCs) generally fail to regenerate axons, resulting in irreversible vision loss after optic nerve injury. While many studies have shown that modulating specific genes can enhance RGCs survival and promote optic nerve regeneration, inducing long-distance axon regeneration in vivo through single-gene manipulation remains challenging. Nevertheless, combined multi-gene therapies have proven effective in significantly enhancing axonal regeneration. At present, research on promoting optic nerve regeneration remains slow, with most studies unable to achieve axonal growth beyond the optic chiasm or reestablish connections with the brain. Future research priorities include directing axonal growth along correct pathways, facilitating synapse formation and myelination, and modifying the inhibitory microenvironment. These strategies are crucial not only for optic nerve regeneration but also for broader applications in central nervous system repair. In this review, we discuss multifactors therapeutic strategies for optic nerve regeneration, offering insights into advancing nerve regeneration research.
... Moreover, Tppp3 promotes axon regeneration in zebrafish [20]. Since stabilizing microtubules improves the transport of essential elements to the growth cone [8,31] and TPPP3 appears to play a role in regulating microtubule dynamics and neuronal function [1,20], TPPP3 could potentially have a positive role in mammalian axon regeneration. Here, we tested the hypothesis that Tppp3 overexpression could promote axon regeneration and RGC survival in a mouse optic nerve crush (ONC) model. ...
Mammalian central nervous system (CNS) axons cannot spontaneously regenerate after injury, creating an unmet need to identify molecular regulators to promote axon regeneration and reduce the lasting impact of CNS injuries. While tubulin polymerization promoting protein family member 3 (Tppp3) is known to promote axon outgrowth in amphibians, its role in mammalian axon regeneration remains unknown. Here we investigated Tppp3 in retinal ganglion cells (RGCs) neuroprotection and axonal regeneration using an optic nerve crush (ONC) model in the rodent. Single-cell RNA sequencing identified the expression of Tppp3 in RGCs of mice, macaques, and humans. Tppp3 overexpression enhanced neurite outgrowth in mouse primary RGCs in vitro, promoted axon regeneration, and improved RGC survival after ONC. Bulk RNA sequencing indicated that Tppp3 overexpression upregulates axon regeneration genes such as Bmp4 and neuroinflammatory pathways. Our findings advance regenerative medicine by developing a new therapeutic strategy for RGC neuroprotection and axon regeneration.
... Thus, DCLK1, ARHGAP1, and TPPP3 showed downregulation in Rpe65 −/− samples but were upregulated in rd10 and P23H. DCLK1 is a doublecortin-like kinase that regulates microtubule binding (55), and it has been shown to promote neuronal survival, growth cone formation, and axon regeneration in retinal ganglion cells (RGCs) after axotomy (56). ARHGAP1 belongs to the family of Rho GTPase-activating proteins that play a role in axon guidance (57). ...
Inherited retinal degenerations (IRDs) are a leading cause of blindness among the population of young people in the developed world. Approximately half of IRDs initially manifest as gradual loss of night vision and visual fields, characteristic of retinitis pigmentosa (RP). Due to challenges in genetic testing, and the large heterogeneity of mutations underlying RP, targeted gene therapies are an impractical largescale solution in the foreseeable future. For this reason, identifying key pathophysiological pathways in IRDs that could be targets for mutation-agnostic and disease-modifying therapies (DMTs) is warranted. In this study, we investigated the retinal proteome of three distinct IRD mouse models, in comparison to sex- and age-matched wild-type mice. Specifically, we used the Pde6βRd10 (rd10) and RhoP23H/WT (P23H) mouse models of autosomal recessive and autosomal dominant RP, respectively, as well as the Rpe65−/− mouse model of Leber’s congenital amaurosis type 2 (LCA2). The mice were housed at two distinct institutions and analyzed using LC-MS in three separate facilities/instruments following data-dependent and data-independent acquisition modes. This cross-institutional and multi-methodological approach signifies the reliability and reproducibility of the results. The large-scale profiling of the retinal proteome, coupled with in vivo electroretinography recordings, provided us with a reliable basis for comparing the disease phenotypes and severity. Despite evident inflammation, cellular stress, and downscaled phototransduction observed consistently across all three models, the underlying pathologies of RP and LCA2 displayed many differences, sharing only four general KEGG pathways. The opposite is true for the two RP models in which we identify remarkable convergence in proteomic phenotype even though the mechanism of primary rod death in rd10 and P23H mice is different. Our data highlights the cAMP and cGMP second-messenger signaling pathways as potential targets for therapeutic intervention. The proteomic data is curated and made publicly available, facilitating the discovery of universal therapeutic targets for RP.
... 51 Based on the literature CG17528 and Apc play important roles in regulating actin and microtubule cytoskeleton, while dop encodes a kinase that regulates protein localization and transport. [52][53][54][55][56][57][58][59][60][61][62][63][64][65][66] If Fat2 intracellular domain indeed interacts with these proteins, we imagine that Fat2 gathers the appropriate cytoskeletal effectors to regulate the cytoskeletal framing of axon terminals and thus affect axon behavior. We further investigated whether any of these genes genetically interacted with fat2 by asking if reducing the dose of fat2 by half in the ORN-specific RNAi knockdown background enhanced or suppressed phenotypes observed (Figures 7A and 7B). ...
The process of how neuronal identity confers circuit organization is intricately related to the mechanisms underlying neurodegeneration and neuropathologies. Modeling this process, the olfactory circuit builds a functionally organized topographic map, which requires widely dispersed neurons with the same identity to converge their axons into one a class-specific neuropil, a glomerulus. In this article, we identified Fat2 (also known as Kugelei) as a regulator of class-specific axon organization. In fat2 mutants, axons belonging to the highest fat2-expressing classes present with a more severe phenotype compared to axons belonging to low fat2-expressing classes. In extreme cases, mutations lead to neural degeneration. Lastly, we found that Fat2 intracellular domain interactors, APC1/2 (Adenomatous polyposis coli) and dop (Drop out), likely orchestrate the cytoskeletal remodeling required for axon condensation. Altogether, we provide a potential mechanism for how cell surface proteins’ regulation of cytoskeletal remodeling necessitates identity specific circuit organization.
... 24,25 Overexpression of DCLKs (especially DCLK2) can also promote neuronal survival and growth cone regeneration after injury. 26 In cancer, DCLK1 is a cancer stem cell (CSC) marker. It is expressed in CSCs but not normal stem cells (NSCs), as determined by lineage tracing techniques in the murine intestine. ...
... Our research showed that the mRNA level of PTEN, SOCS3, Klf9, and Mdm4 decreased signi cantly in AAV-STAT3 group. Overexpression of Dclk2, Armcx1, c-myc, and Nrn1 are closely related to the regeneration of RGCs axon (34)(35)(36)(37). The mRNA level of Dclk2, Armcx1, c-myc, and Nrn1 signi cantly increased in the AAV-STAT3 group. ...
Müller differentiated RGCs have potential therapeutic value for glaucoma. However, axonal regeneration of differentiated RGCs has been a difficult problem. Retinal stem cells were differenticated from rat retinal Müller cells. The stem cells were randomly divided into five groups (control group, AAV-STAT3 group, shSTAT3 group, Y27632 group and AAV-STAT3 + Y27632 group). Stem cells in different groups were injected into rat model of glaucoma. The length of axon regeneration in STAT3 combined with Y27632 group was significantly longer than that in other experimental groups. The AAV-STAT3 transfected RGCs treated with Y27632 significantly increased the mRNA levels of Esrrb, Prdm14, Sox2, and Rex1, while decreasing the mRNA levels of Nestin, Eomes, Mixl1, and Gata4. Meanwhile, Socs3, Pten, Klf9, and Mdm4 were significantly lowered, while Dclk2, Armcx1, C-MYC, and Nrn1 were elevated. After injecting differentiated RGCs into the glaucoma model rat eyes, the axon length, RGC layer thickness and the electrophysiology were superior to the glaucoma model group. These findings suggested that STAT3 combined with Y27632 can significantly improve the axonal growth level of Müller differentiated RGCs, and reveal the potential mechanism to induce pluripotency of RGCs.
... The mRNA level of Prox1, a marker of early hippocampal development 12) , was found to be higher in The mRNA level of doublecortin (Dcx), a neuroregeneration marker 24) , was found to be higher in MCAO2 than MCAO1 ( Figure 4B). WT had mRNA levels of 0.59 (±0.24 ...
Objective:
Markers of neuroinflammation during ischemic stroke are well characterized, but additional markers of neural damage are lacking. The study identified associations of behavioral disorders after stroke with histologic neural damage and molecular biological change.
Methods:
8-week-old, 25g male mice of the C57BL/6J strain were subjected to middle cerebral artery occlusion (MCAO) to induce ischemic stroke. The control group was a healthy wild type (WT), and the experimental group were designed as a low severity MCAO1 and a high severity MCAO2 based on post-stroke neurological scoring. All groups underwent behavioral tests, real-time polymerase chain reaction (rt-PCR), triphenyltetrazolium chloride (TTC) staining and hematoxylin and eosin (H&E) staining. One-way analysis of variance (ANOVA) was used to analyze statistical significance between groups.
Results:
In TTC staining, MCAO1 showed 29.02% and MCAO2 showed 38.94% infarct volume (p<0.0001). The pro-inflammatory cytokine interleukin (IL)-1β was most highly expressed in MCAO2 (WT 0.44 vs MCAO1 2.69 vs MCAO2 5.02, p<0.0001). From the distance to target in the Barnes maze test, WT had a distance of 178 cm, MCAO1 had a distance of 276 cm, and MCAO2 had a distance of 1051 (p=0.0015). The latency to target was 13.3 seconds for WT, 27.9 seconds for MCAO1, and 87.9 seconds for MCAO2 (p=0.0007). Prospero homeobox 1 (Prox1) was most highly expressed in MCAO2 (p=0.0004). Doublecortin (Dcx) was most highly expressed in MCAO2 (p<0.0001).
Conclusion:
The study demonstrated that histological damage to neural cells and changes in brain mRNA expression were associated with behavioral impairment after ischemic stroke. Prox1 and Dcx may be biomarkers of neural damage associated with long-term cognitive decline, and increased expression at the mRNA level was consistent with neural damage and long-term cognitive dysfunction.
... 8,10 Overexpression of doublecortin-like kinase (DCLK) can also promote growth cone formation by maintaining microtubule homeostasis. 11 Deletion of phosphatase and tensin homolog (PTEN) antagonizes the action of PI3K and inactivates mTOR signaling, thereby promoting axonal regeneration in adult retinal ganglion cells (RGCs) and adult sensory neurons. 12,13 Deletion of suppressor of cytokine signaling 3 (SOCS3) alone promotes optic nerve axon growth by inhibiting the Recently, single-cell RNA sequencing, as a high-throughput and efficient technical means, can detect the heterogeneity of samples at different developmental and pathological stages at very high resolution, and explore the different transcriptional states of single cells. ...
Spinal motor neurons, the distinctive neurons of the central nervous system, extend into the peripheral nervous system and have outstanding ability of axon regeneration after injury. Here, we explored the heterogeneity of spinal ventral horn cells after rat sciatic nerve crush via single-nuclei RNA sequencing. Interestingly, regeneration mainly occurred in a Sncg+ and Anxa2+ motor neuron subtype (MN2) surrounded by a newly emerged microglia subtype (Mg6) after injury. Subsequently, microglia depletion slowed down the regeneration of sciatic nerve. OPCs were also involved into the regeneration process. Knockdown of Cacna2d2 in vitro and systemic blocking of Cacna2d2 in vivo improved the axon growth ability, hinting us the importance of Ca2+. Ultimately, we proposed three possible phases of motor neuron axon regeneration: preparation stage, early regeneration stage, and regeneration stage. Taken together, our study provided a resource for deciphering the underlying mechanism of motor neuron axon regeneration in a single cell dimension.
... Experiments were performed as described in Nawabi and colleagues [59]. Probes were cloned in pGEMT vector from cDNA extracted from cerebellum: pGEM-T_RNAprobeRSK1; pGEM-T_RNAprobeRSK2; pGEM-T_RNAprobeRSK3; pGEM-T_RNAprobeRSK4 Sequence used for the probe was described in S1 Table. ...
Unlike immature neurons and the ones from the peripheral nervous system (PNS), mature neurons from the central nervous system (CNS) cannot regenerate after injury. In the past 15 years, tremendous progress has been made to identify molecules and pathways necessary for neuroprotection and/or axon regeneration after CNS injury. In most regenerative models, phosphorylated ribosomal protein S6 (p-RPS6) is up-regulated in neurons, which is often associated with an activation of the mTOR (mammalian target of rapamycin) pathway. However, the exact contribution of posttranslational modifications of this ribosomal protein in CNS regeneration remains elusive. In this study, we demonstrate that RPS6 phosphorylation is essential for PNS and CNS regeneration in mice. We show that this phosphorylation is induced during the preconditioning effect in dorsal root ganglion (DRG) neurons and that it is controlled by the p90S6 kinase RSK2. Our results reveal that RSK2 controls the preconditioning effect and that the RSK2-RPS6 axis is key for this process, as well as for PNS regeneration. Finally, we demonstrate that RSK2 promotes CNS regeneration in the dorsal column, spinal cord synaptic plasticity, and target innervation leading to functional recovery. Our data establish the critical role of RPS6 phosphorylation controlled by RSK2 in CNS regeneration and give new insights into the mechanisms related to axon growth and circuit formation after traumatic lesion.
... Cleavage at these sites is expected to generate N-terminal fragments of~35 kDa consisting of both DCX domains and C-terminal fragments of 49 kDa consisting of the kinase domain and the C-terminal tail. The cleavage was predicted to dysregulate the kinase and microtubule assembly functions of DCLK1 (91)(92)(93). In agreement with the TAILS findings, a Western blot of the cell lysates derived from the control and glutamate-treated neurons revealed the enhanced formation of truncated DCLK1 fragments of~50 to 56 kDa (supplemental Fig. S12B). ...
Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by over-stimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIβ (CaMKIIβ). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIβ, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.
... Similarly, the SNP rs28413051 on 4q31.23 (multivariate z = 6.28, P = 3.47 × 10 −10 ) is an eQTL of DCLK2 that is important for axon growth cone formation and neural migration (40) and is also within a region interacting with the promoter of DCLK2 in neural progenitor cells (36). As a further example, allele C of rs13107325 on 4q24 (multivariate z = 5.77, P = 7.99 × 10 −9 in the node-level connectivity mvGWAS and multivariate z = 8.74, P = 2.37 × 10 −18 in the edge-level connectivity mvGWAS) is a missense coding variant in the gene SLC39A8 that showed a high combined annotation-dependent depletion score of 23.1, which indicates that this SNP is deleterious (its frequency was 7.01%). ...
White matter tracts form the structural basis of large-scale brain networks. We applied brain-wide tractography to diffusion images from 30,810 adults (U.K. Biobank) and found significant heritability for 90 node-level and 851 edge-level network connectivity measures. Multivariate genome-wide association analyses identified 325 genetic loci, of which 80% had not been previously associated with brain metrics. Enrichment analyses implicated neurodevelopmental processes including neurogenesis, neural differentiation, neural migration, neural projection guidance, and axon development, as well as prenatal brain expression especially in stem cells, astrocytes, microglia, and neurons. The multivariate association profiles implicated 31 loci in connectivity between core regions of the left-hemisphere language network. Polygenic scores for psychiatric, neurological, and behavioral traits also showed significant multivariate associations with structural connectivity, each implicating distinct sets of brain regions with trait-relevant functional profiles. This large-scale mapping study revealed common genetic contributions to variation in the structural connectome of the human brain.
... Because these microtubule-stabilizing agents also reduce the fibrotic scar, it is not clear whether enhanced regeneration following their application can be attributed more to effects on neurons or on the fibrotic scar. In RGCs, doublecortin-like kinases (DCLKs) promote neuronal survival and axon regeneration through distinct mechanisms, including microtubule dynamics and retrograde injury signalling 135 . ...
One hundred years ago, Ramón y Cajal, considered by many as the founder of modern neuroscience, stated that neurons of the adult central nervous system (CNS) are incapable of regenerating. Yet, recent years have seen a tremendous expansion of knowledge in the molecular control of axon regeneration after CNS injury. We now understand that regeneration in the adult CNS is limited by (1) a failure to form cellular or molecular substrates for axon attachment and elongation through the lesion site; (2) environmental factors, including inhibitors of axon growth associated with myelin and the extracellular matrix; (3) astrocyte responses, which can both limit and support axon growth; and (4) intraneuronal mechanisms controlling the establishment of an active cellular growth programme. We discuss these topics together with newly emerging hypotheses, including the surprising finding from transcriptomic analyses of the corticospinal system in mice that neurons revert to an embryonic state after spinal cord injury, which can be sustained to promote regeneration with neural stem cell transplantation. These gains in knowledge are steadily advancing efforts to develop effective treatment strategies for spinal cord injury in humans. The inability of the mammalian central nervous system to functionally regenerate after injury is largely attributable to the limited capacity of injured neurons to regrow axons. In the spinal cord, recent work on the mechanisms restricting axon regrowth suggests new therapeutic avenues to promote functional recovery after damage.
... The present work also indicates the DCLK2 as a novel regulator of endothelial cell proliferation. DCLK1 and two are understudied kinases, with the majority of work being upon DCKL1 and its role in cancer (Ferguson et al., 2020), neuronal survival (Nawabi et al., 2015), and maintenance of intestinal crypt cell stemness (Chandrakesan et al., 2017). DCLK2 was identified as having synthetic lethal interaction with mutant Ras in colorectal cancer (Luo et al., 2009), suggesting that it may be an enticing target for cancers drive by the Ras oncogene. ...
Antiangiogenic therapy began as an effort to inhibit VEGF signaling, which was thought to be the sole factor driving tumor angiogenesis. It has become clear that there are more pro-angiogenic growth factors that can substitute for VEGF during tumor vascularization. This has led to the development of multi-kinase inhibitors which simultaneously target multiple growth factor receptors. These inhibitors perform better than monotherapies yet to date no multi-kinase inhibitor targets all receptors known to be involved in pro-angiogenic signaling and resistance inevitably occurs. Given the large number of pro-angiogenic growth factors identified, it may be impossible to simultaneously target all pro-angiogenic growth factor receptors. Here we search for kinase targets, some which may be intracellularly localized, that are critical in endothelial cell proliferation irrespective of the growth factor used. We develop a quantitative endothelial cell proliferation assay and combine it with “kinome regression” or KIR, a recently developed method capable of identifying kinases that influence a quantitative phenotype. We report the kinases implicated by KIR and provide orthogonal evidence of their importance in endothelial cell proliferation. Our approach may point to a new strategy to develop a more complete anti-angiogenic blockade.
... Similarly, the SNP rs28413051 on 4q31.23 (multivariate z = 6.28, P = 3.47 × 10 −10 ) is an eQTL of DCLK2 that is important for axon growth cone formation and neural migration (40) and is also within a region interacting with the promoter of DCLK2 in neural progenitor cells (36). As a further example, allele C of rs13107325 on 4q24 (multivariate z = 5.77, P = 7.99 × 10 −9 in the node-level connectivity mvGWAS and multivariate z = 8.74, P = 2.37 × 10 −18 in the edge-level connectivity mvGWAS) is a missense coding variant in the gene SLC39A8 that showed a high combined annotation-dependent depletion score of 23.1, which indicates that this SNP is deleterious (its frequency was 7.01%). ...
... Recent studies have shown that multiple steps of signal initiation and transduction, including receptor availability [139] and signal propagation [140,141], or silencing inhibitory pathways [142,143], can be targeted to enhance axon regeneration of the mature neuron (Fig. 2C). For example, a combination of osteopontin/insulin-like growth factor 1 (IGF1) induces robust sprouting of CST axons and associated behavioral recovery after injuries [144]. ...
hemisphere. Animal experiments have revealed that regrowth of ipsilateral descending fibers from the unaffected hemisphere to denervated motor neurons plays a significant role in the restoration of motor function. In addition, several clinical treatments have been designed to restore ipsilateral motor control, including brain stimulation, nerve transfer surgery, and brain-computer interface systems. Here, we comprehensively review the neural mechanisms as well as translational applications of ipsilateral motor control upon rehabilitation after CNS injuries.
... Progress has been made in modifying the hostile central nervous system (CNS) microenvironment by neutralizing inhibitory molecules 1-3 and enhancing intrinsic growth capacity by introducing foreign genes through viral vector to facilitate axon regeneration [4][5][6][7][8][9][10][11][12] . Knowledge of the major CNS inhibitors has increased remarkably over the past decades. ...
Adult mammalian injured axons regenerate over short-distance in the peripheral nervous system (PNS) while the axons in the central nervous system (CNS) are unable to regrow after injury. Here, we demonstrated that Lycium barbarum polysaccharides (LBP), purified from Wolfberry, accelerated long-distance axon regeneration after severe peripheral nerve injury (PNI) and optic nerve crush (ONC). LBP not only promoted intrinsic growth capacity of injured neurons and function recovery after severe PNI, but also induced robust retinal ganglion cell (RGC) survival and axon regeneration after ONC. By using LBP gene expression profile signatures to query a Connectivity map database, we identified a Food and Drug Administration (FDA)-approved small-molecule glycopyrrolate, which promoted PNS axon regeneration, RGC survival and sustained CNS axon regeneration, increased neural firing in the superior colliculus, and enhanced visual target re-innervations by regenerating RGC axons leading to a partial restoration of visual function after ONC. Our study provides insights into repurposing of FDA-approved small molecule for nerve repair and function recovery.
... Moreover, eEF1A proteins possess actin-binding domains that enable a role in actinbundling and cytoskeleton organization in vivo [16][17][18][19][20]. Along these lines, the most recent data have revealed that eEF1A2 deficient neurons exhibit shorter neurite length [21]. This is relevant following axonal damage since actin dynamics have a key role in the formation of a competent growth cone, which is vital for proper axon growth and regeneration [22][23][24]. Overall, the literature has implicated a role for eEF1A proteins in cellular processes deemed important for axonal repair. ...
Although protein synthesis is hypothesized to have a pivotal role in axonal repair after central nervous system (CNS) injury, the role of core components of the protein synthesis machinery has not been examined. Notably, some elongation factors possess non-canonical functions that may further impact axonal repair. Here, we examined whether overexpressing eukaryotic elongation factor 1 alpha (eEF1A) proteins enhances the collateral sprouting of corticospinal tract (CST) neurons after unilateral pyramidotomy, along with the underlying molecular mechanisms. We found that overexpressing eEF1A proteins in CST neurons increased the levels of pS6, an indicator for mTOR activity, but not pSTAT3 and pAKT levels, in neuronal somas. Strikingly, overexpressing eEF1A2 alone, but neither eEF1A1 alone nor both factors simultaneously, increased protein synthesis and actin rearrangement in CST neurons. While eEF1A1 overexpression only slightly enhanced CST sprouting after pyramidotomy, eEF1A2 overexpression substantially enhanced this sprouting. Surprisingly, co-overexpression of both eEF1A1 and eEF1A2 led to a sprouting phenotype similar to wild-type controls, suggesting an antagonistic effect of overexpressing both proteins. These data provide the first evidence that overexpressing a core component of the translation machinery, eEF1A2, enhances CST sprouting, likely by a combination of increased protein synthesis, mTOR signaling and actin cytoskeleton rearrangement.
... The retinas were then immuno-stained with anti-βIII-tubulin. Fluorescence images of different regions (at least 5 regions on each retina) of the retina were taken using a Leica DM4 B fluorescent microscope (20× objective) [30]. The number of RGCs was blindly counted. ...
Traumatic optic neuropathy (TON) is the major contributor to optic nerve damage, where the retinal ganglion cells (RGCs) are substantially lost. However, the underlying pathological mechanisms for these conditions remain largely elusive. Present work conducted a study on TON rat model, where the iron-dependent cyclooxygenase-2 (COX-2) overexpression and lipid peroxidation were observed in RGCs, suggesting ferroptosis, an iron-dependent non-apoptotic cell death, is involved in TON-induced death of RGCs. Hence, the newly formulated hyaluronic acid (HA)-based deferoxamine (DFO) nanoparticles (DFO-NPs) were intravitreally administrated in the rat model. It was hypothesized that the effective delivery of DFO, iron chelator, to the RGCs might rescue RGC ferroptosis from TON-induced injury. Also, since DFO is poor in bioavailability and of very short half-life in vivo, its safe and efficient intravitreal delivery is critical. Therefore, DFO-NPs were prepared by chemical grafting DFO onto HA molecules, and then crosslinking them in microemulsion bubbles for nanoparticles formulation. The nanoparticles were highly accumulated around the ganglionic cells and DFO uptake was increased in RGCs, accompanied by the significantly inhibited the overexpression of COX-2 and inactivation of glutathione peroxidase 4 (GPX4). These results indicate that DFO-NPs acted as an effective ferroptosis inhibitor, for the prevention of TON-induced RGC death. The current study provides new insights into the underlying mechanism of TON-induced RGC death, which may help to explore a novel strategy for the treatment of TON.
... Cleavage at these sites is expected to generate N-terminal fragments of ~35 kDa conisisting of both DCX domains and C-terminal fragments of ~49 kDa conisisting of the kinase domain and the C-terminal tail. The cleavage was predicted to dysregulate the kinase and microtubule assembly functions of DCLK1 (Burgess & Reiner, 2001) (Patel et al., 2016) (Nawabi, Belin et al., 2015). In agreement with the TAILS findings, Western blot of the cell lysates derived from the control and glutamate-treated neurons revealed enhanced formation of truncated DCLK1 fragments of ~50-56 kDa ( Figure S12B). ...
Excitotoxicity is a neuronal death process initiated by over-stimulation of ionotropic glutamate receptors. Although dysregulation of proteolysis and protein phosphorylation signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remains unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. Our investigation led to the discovery of proteins that, upon proteolysis by calpains, perturb synaptic organization and function. These included key synaptic regulatory proteins including CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIβ (CaMKIIβ), which we found to undergo calpain-catalyzed proteolytic processing to generate stable truncated fragments with altered activities. We further show that blockade of proteolysis of Src by calpains could protect against neuronal loss in a rat model of neurotoxicity, and that CaMKIIβ and its isoform CaMKIIα undergo differential processing by calpains in mouse brains under physiological conditions and during ischemic stroke. Our findings thus reveal new insights into excitotoxic neuronal death mechanisms and suggest potential therapeutic targets for neurological disorders.
One Sentence Summary
Proteolytic events important for excitotoxic neuronal death as potential novel therapeutic targets.
Graphical Abstract
In Brief
Ameen, et al. used an N-terminomic method to identify neuronal proteins and the exact cleavage sites proteolyzed by calpains during excitotoxicity. Upon proteolysis, these proteins generate stable truncated fragments, which potentially induce synaptic dysfunction and loss, eventually leading to neuronal death. As such, some of these proteins such as protein kinases Src and CaMKII are potential targets for neuroprotection.
Highlights
Over 300 neuronal proteins are cleaved by calpains to form stable truncated fragments during excitotoxicity.
The calpain cleavage sites of these proteins unveil for the first time the preferred cleavage sequences of calpains in neurons.
These pathological proteolytic events potentially induce synaptic dysfunction and loss, which leads to excitotoxic neuronal death.
Some of the neuronal proteins proteolyzed by calpains are potential targets of neuroprotection.
... The proteins with the biggest fold change in differentiated cells were neuromodulin (GAP43), a protein involved in neuronal and axonal growth and regeneration which is upregulated by BDNF and whose expression has been found to be decreased in PD patient brains (Saal et al., 2017;Chung et al., 2020), doublecortinlike kinase 1 (DCLK1), a protein kinase part of the doublecortin family that participates in neuronal migration and neurogenesis (Shu et al., 2006;Nawabi et al., 2015;Patel et al., 2016), and eukaryotic elongation factor 1 alpha 2 (EEF1A2), a protein implicated in protein translation elongation and autophagy that inhibits apoptotic cell death and may be important for DAn survival (Khwanraj et al., 2016;Prommahom and Dharmasaroja, 2021). These three proteins, along with vesicle amine transport 1 like (VAT1L), were also the only proteins among those with the largest fold change in differentiated cells whose genes also exhibited a significant fold change in the NGS study (data not shown). ...
Parkinson’s disease (PD) is an age-associated neurodegenerative disorder for which there is currently no cure. Cell replacement therapy is a potential treatment for PD; however, this therapy has more clinically beneficial outcomes in younger patients with less advanced PD. In this study, hVM1 clone 32 cells, a line of human neural stem cells, were characterized and subsequently transplanted in middle-aged Parkinsonian mice in order to examine cell replacement therapy as a treatment for PD. In vitro analyses revealed that these cells express standard dopamine-centered markers as well as others associated with mitochondrial and peroxisome function, as well as glucose and lipid metabolism. Four months after the transplantation of the hVM1 clone 32 cells, striatal expression of tyrosine hydroxylase was minimally reduced in all Parkinsonian mice but that of dopamine transporter was decreased to a greater extent in buffer compared to cell-treated mice. Behavioral tests showed marked differences between experimental groups, and cell transplant improved hyperactivity and gait alterations, while in the striatum, astroglial populations were increased in all groups due to age and a higher amount of microglia were found in Parkinsonian mice. In the motor cortex, nonphosphorylated neurofilament heavy was increased in all Parkinsonian mice. Overall, these findings demonstrate that hVM1 clone 32 cell transplant prevented motor and non-motor impairments and that PD is a complex disorder with many influencing factors, thus reinforcing the idea of novel targets for PD treatment that tend to be focused on dopamine and nigrostriatal damage.
... Recent work demonstrates that destabilized and disorganized microtubules cause active growth cones to become dystrophic, an effect that can be overcome by ll administering epothilone B, an FDA-approved drug, or by specifically deleting a small GTPase ras homolog gene family member A (RhoA) in neurons (Ruschel et al., 2015;Stern et al., 2021). In the visual system, other mechanisms have been shown to be effective in initiating a growth cone; for example, following ONC, overexpression of doublecortin-like kinases promotes growth cone initiation, resulting in more axons regenerating past the lesion site in the optic nerve (Nawabi et al., 2015). Ultimately, growth cone formation is an important aspect of axon regeneration involving a cascade of events, some of which overlap with mechanisms activated by axonal responses to injury (Bradke et al., 2012). ...
Neurons of the mammalian central nervous system fail to regenerate. Substantial progress has been made toward identifying the cellular and molecular mechanisms that underlie regenerative failure and how altering those pathways can promote cell survival and/or axon regeneration. Here, we summarize those findings while comparing the regenerative process in the central versus the peripheral nervous system. We also highlight studies that advance our understanding of the mechanisms underlying neural degeneration in response to injury, as many of these mechanisms represent primary targets for restoring functional neural circuits.
... In their recent genome-wide association study of 11,492 U.S. soldiers, Stein et al. reported that a locus upstream of doublecortin-like kinase 2 (DCLK2) was significantly associated, at a genome-wide level, with resilience following deployment [12]. Like DCX, DCLK2 is involved in dendritic remodeling and growth cone dynamics and, in rodents, has been shown to partially compensate for the loss of DCX [13,14]. Still, in the absence of any experimental characterization of DCX's relationship to behavior, its potential relevance to non-neurogenesis-related processing remains unclear. ...
Doublecortin (DCX) has long been implicated in, and employed as a marker for, neurogenesis, yet little is known about its function in non-neurogenic brain regions, including the amygdala. This study sought first to explore, in rodents, whether fear learning and extinction modulate amygdala DCX expression and, second, to assess the utility of peripheral DCX correlates as predictive biomarkers of trauma response in rodents and humans. Pavlovian conditioning was found to alter DCX protein levels in mice 24 h later, resulting in higher DCX expression associated with enhanced learning in paradigms examining both the acquisition and extinction of fear (p < 0.001). This, in turn, is associated with differences in freezing on subsequent fear expression tests, and the same relationship between DCX and fear extinction was replicated in rats (p < 0.001), with higher amygdala DCX levels associated with more rapid extinction of fear. RNAseq of amygdala and blood from mice identified 388 amygdala genes that correlated with DCX (q < 0.001) and which gene ontology analyses revealed were significantly over-represented for neurodevelopmental processes. In blood, DCX-correlated genes included the Wnt signaling molecule Cdk14 which was found to predict freezing during both fear acquisition (p < 0.05) and brief extinction protocols (p < 0.001). High Cdk14 measured in blood immediately after testing was also associated with less freezing during fear expression testing (p < 0.01). Finally, in humans, Cdk14 expression in blood taken shortly after trauma was found to predict resilience in males for up to a year post-trauma (p < 0.0001). These data implicate amygdala DCX in fear learning and suggest that Cdk14 may serve as a predictive biomarker of trauma response.
... Our results showed that OGD significantly reduced the number of DCX+ cells while exposure to OGD/R conditions reversed those effects, probably due to the endogenous mechanisms of restoration mentioned previously. Recently, new actions for the DCX family have been associated to cell survival and regeneration [83]; therefore, it is possible that GH neuroprotective effects include DCX involvement, since we found that GH treatment during OGD injury significantly increased DCX-IR, to reach similar levels as in the normoxic controls. Likewise, GH addition during OGD/R maintained the effect observed during OGD harm. ...
As a classical growth promoter and metabolic regulator, growth hormone (GH) is involved in development of the central nervous system (CNS). This hormone might also act as a neurotrophin, since GH is able to induce neuroprotection, neurite growth, and synaptogenesis during the repair process that occurs in response to neural injury. After an ischemic insult, the neural tissue activates endogenous neuroprotective mechanisms regulated by local neurotrophins that promote tissue recovery. In this work, we investigated the neuroprotective effects of GH in cultured hippocampal neurons exposed to hypoxia-ischemia injury and further reoxygenation. Hippocampal cell cultures obtained from chick embryos were incubated under oxygen-glucose deprivation (OGD
... A previous study reported that doublecortin-like kinases, known as doublecortin and CaM kinase-like, are essential growth cone reformation-associated proteins. In addition, doublecortin members regulate F-actin dynamics in injured axonal stumps [42]. Another limitation of this study was that we only evaluated the in vitro effect of CES on the neurotherapeutic potential. ...
Cervus elaphus sibericus (CES), commonly known as deer antler, has been used as a medicinal herb because of its various pharmacological activities, including its anti-infective, anti-arthritic, anti-allergic, and anti-oxidative properties. However, the precise mechanisms by which CES functions as a potent anti-oxidative agent remain unknown; particularly, the effects of CES on cortical neurons and its neurobiological mechanism have not been examined. We used primary cortical neurons from the embryonic rat cerebral cortex and hydrogen peroxide to induce oxidative stress and damage in neurons. After post-treatment of CES at three concentrations (10, 50, and 200 µg/mL), the influence of CES on the neurobiological mechanism was assessed by immunocytochemistry, flow cytometry, and real-time PCR. CES effectively prevented neuronal death caused by hydrogen peroxide-induced damage by regulating oxidative signaling. In addition, CES significantly induced the expression of brain-derived neurotrophic factor and neurotrophin nerve growth factor, as well as regeneration-associated genes. We also observed newly processing elongated axons after CES treatment under oxidative conditions. In addition, filopodia tips generally do not form a retraction bulb, called swollen endings. Thus, CES shows therapeutic potential for treating neurological diseases by stimulating neuron repair and regeneration.
... For last, we have a negative correlation of DCKL2 with lnc-uc.147. The DCLK2 is a welldescribed gene in neuronal activity, and in only one recent study, its higher expression was associated with reduced patient survival n chronic lymphocytic leukaemia [105][106][107]. So, it is not clear the correlation expression of this gene and the lnc-uc.147. ...
The human genome contains 481 ultraconserved regions (UCRs), which are genomic stretches of over 200 base pairs conserved among human, rat, and mouse. The majority of these regions are transcriptionally active (T-UCRs), and several have been found to be differentially expressed in tumours. Some T-UCRs have been functionally characterized, but of those few have been associated to breast cancer (BC). Using TCGA data, we found 302 T-UCRs related to clinical features in BC: 43% were associated with molecular subtypes, 36% with oestrogen-receptor positivity, 17% with HER2 expression, 12% with stage, and 10% with overall survival. The expression levels of 12 T-UCRs were further analysed in a cohort of 82 Brazilian BC patients using RT-qPCR. We found that uc.147 is high expressed in luminal A and B patients. For luminal A, a subtype usually associated with better prognosis, high uc.147 expression was associated with a poor prognosis and suggested as an independent prognostic factor. The lncRNA from uc.147 (lnc-uc.147) is located in the nucleus. Northern blotting results show that uc.147 is a 2,8 kb monoexonic trancript, and its sequence was confirmed by RACE. The silencing of uc.147 increases apoptosis, arrests cell cycle, and reduces cell viability and colony formation in BC cell lines. Additionally, we identifed 19 proteins that interact with lnc-uc.147 through mass spectrometry and demonstrated a high correlation of lnc-uc.147 with the neighbour gene expression and miR-18 and miR-190b. This is the first study to analyse the expression of all T-UCRs in BC and to functionally assess the lnc-uc.147.
... Although TAC-1 and ZYG-8 are centrosomal proteins, they can both localize in axons and are each required for axon regeneration [74]. Xenopus TACC3 was shown to promote axon outgrowth in embryonic cultured neural crest cells [114], and DCLK is required for axon regrowth in mice [115]. Compared to wildtype, tac-1 and zyg-8 mutants demonstrated fewer growing microtubules in uninjured axons, and do not exhibit reduced microtubule dynamics in response to injury. ...
Axon regeneration after injury is a conserved biological process that involves a large number of molecular pathways, including rapid calcium influx at injury sites, retrograde injury signaling, epigenetic transition, transcriptional reprogramming, polarized transport, and cytoskeleton reorganization. Despite the numerous efforts devoted to understanding the underlying cellular and molecular mechanisms of axon regeneration, the search continues for effective target molecules for improving axon regeneration. Although there have been significant historical efforts towards characterizing pro-regenerative factors involved in axon regeneration, the pursuit of intrinsic inhibitors is relatively recent. EFA6 (exchange factor for ARF6) has been demonstrated to inhibit axon regeneration in different organisms. EFA6 inhibition could be a promising therapeutic strategy to promote axon regeneration and functional recovery after axon injury. This review summarizes the inhibitory role on axon regeneration through regulating microtubule dynamics and through affecting ARF6 (ADP-ribosylation factor 6) GTPase-mediated integrin transport.
... An indepth discussion of these is beyond the scope of this review and has been discussed elsewhere. [75][76][77] Briefly, deletion of PTEN, 78 SOCS3, 79 c-myc, 80 DCLK, 81 and members of the KLF family 82 have been reported to robustly increase the amount of regeneration of RGCs following optic nerve crush, and combinations of these manipulations have been reported to further augment this growth. 21,80 Recent work has led to the identification of different combinations of growth factors, such as Insulin-like growth factor-1 (IGF-1), osteopontin (OPN), and CNTF, which act on similar molecular pathways and yield similar degrees of regeneration, [23][24][25]50 a key step toward eventual clinical translation. ...
There have been tremendous advances in identifying cellular and molecular mechanisms constraining axon growth and strategies have been developed to overcome regenerative failure. However, reproducible and meaningful functional recovery remains elusive. An emerging reason is that neurons possess subtype-specific activation requirements. Much of this evidence comes from studying retinal ganglion cells following optic nerve injury. This review summarizes key neuropathologic events following spinal cord injury, and draws on findings from the optic nerve to suggest how a similar framework may be used to dissect and manipulate the heterogeneous and subtype-specific responses of neurons useful to target for spinal cord injury.
... This work also indicates the DCAMKL2 as a novel regulator of endothelial cell proliferation. DCAMKL1 and 2 are understudied kinases, with the majority of work being upon DCAMKL1 and its role in cancer (Ferguson et al. 2020), neuronal survival (Nawabi et al. 2015), and maintenance of intestinal crypt cell stemness (Chandrakesan et al. 2017). It will be of interest to further explore the role of DCAMKL2 in endothelial cell biology. ...
Antiangiogenic therapy began as an effort to inhibit VEGF signaling, which was thought to be the sole factor driving tumor angiogenesis. It has become clear that there are more pro-angiogenic growth factors that can substitute for VEGF during tumor vascularization. This has led to the development of multi-kinase inhibitors which simultaneously target multiple growth factor receptors. These inhibitors perform better than monotherapies yet to date no multi-kinase inhibitor targets all receptors known to be involved in pro-angiogenic signaling and resistance inevitably occurs. Given the large number of pro-angiogenic growth factors identified, it may be impossible to simultaneously target all pro-angiogenic growth factor receptors. Here we search for kinase targets, some which may be intracellularly localized, that are critical in endothelial cell proliferation irrespective of the growth factor used. We develop a quantitative endothelial cell proliferation assay and combine it with "kinome regression" or KIR, a recently developed method capable of identifying kinases that influence a quantitative phenotype. We report the kinases implicated by KIR and provide orthogonal evidence of their importance in endothelial cell proliferation. Our approach points to a new strategy to develop a more complete anti-angiogenic blockade.
... This indicates the extent of injury in the retina due to oxidative damage and the expected further complications of aging and its associated macular degeneration in retinal tissue remodeling during glaucoma [47][48][49][50]. An increase further follows the neuronal injury in the expression of doublecortin-like kinases (DCLK1), which promotes neuronal survival and activates the cone forma-tion in the retinal cells [51]. The retinal cells do recover from the retinal injury with the treatment of cells with BG and are mainly due to the low cytotoxicity exhibited in the treated cells [52]. ...
Background:
The use of phytochemicals for the treatment of various bodily ailments has been in practice since ancient days. Even though in practice, scientific studies on the protective effect of β-glucogallin (BG) against glaucoma is limited.
Objectives:
In the present study, the in vitro glaucoma model (hydrostatic pressure) using PC12 neuronal cells exposed to BG were used to elucidate its protective effects.
Method:
The cultured cells were analyzed for the mitochondrial responses, oxidant-antioxidant status, and expression of caveolin-1, ANGPTL7, the glaucoma markers, and cytokines.
Results:
We demonstrated a significant increase in the expression of glial fibrillary acidic protein, ANGPTL7, with altered mitochondrial enzymes in glaucoma cells compared to the control. Moreover, cells predisposed to hydrostatic pressure demonstrated an increase in oxidative stress with augmented (p < 0.01) inflammatory cytokines such as IL-2, CXCR4, IL-6, IL-8, MCP-1, and TNF-α. On the other hand, cells pretreated with BG attenuated the reactive oxygen species levels with improved antioxidant enzymes. Simultaneously, the levels of inflammatory cytokines and ANGPTL7 proteins were found attenuated with restored mitochondrial responses in BG pretreated cells.
Conclusion:
Thus, the results of the present study demonstrate that the use of BG on retinal cells against relieving the intraocular pressure may be a promising therapeutic for controlling the disease progression.
TANK-binding kinase 1 (TBK1) is a serine/threonine kinase with well-established roles as a central player in innate immune signaling. Dysregulation of TBK1 activity has been implicated in a variety of pathophysiologic conditions, including cancer. Generally, TBK1 acts as an oncogene and increased TBK1 activity, indicated by increased phosphorylation at the Ser172 residue, can be observed in multiple human cancers. TBK1 can be activated either by autophosphorylation of Ser172 or transphosphorylation at this site by upstream kinases. Serving as a hub for integrating numerous extracellular and intracellular signals, TBK1 can be activated through multiple signaling pathways. However, the direct upstream kinase responsible for TBK1 activation remains elusive, which limits our comprehensive understanding of its activation mechanism and potential therapeutic application targeting TBK1-related signaling especially in cancer. In this review, we summarize the findings on mechanisms of TBK1 activation in cancer cells and recent discoveries that shed light on the direct upstream kinases promoting TBK1 activation.
Cytoplasmic accumulation and aggregation of TDP-43 is a hallmark of ∼97% of ALS cases. Formation of TDP-43 insoluble aggregates is suggested to either directly or indirectly cause motor neuron loss and progressive neuromuscular degeneration, although how this occurs is not precisely understood. Cytoplasmic TDP-43 is observed in stress granules (SG). SGs are ribonucleoprotein (RNP) complexes formed during stress conditions, consisting of mRNAs and RNA-binding proteins (RNPs). Chronic TDP-43/SG formation may play a role in neuromuscular degeneration in ALS. The composition of in vivo TDP-43-asscociated SGs in ALS not known. This knowledge may provide insights into the molecular pathways impaired by TDP-43-associated SGs and suggest disease modifying mechanisms. The aim of this study was to isolate and analyse the proteome of the insoluble TDP-43-associated SG fraction from brain tissue of end-stage TDP-43ΔNLS mice. Proteomic analysis identified 134 enriched and 17 depleted proteins in the TDP-43ΔNLS mice, when compared to the control mice. Bioinformatics analyses of the impacted proteins from the SG preparation suggested that brain tissue from end-stage NEFH-TDP-43ΔNLS mice have sustained SG formation, CLUH granule recruitment and impaired mitochondrial metabolism. This is the first time that CLUH granule recruitment has been demonstrated in ALS and the known role of CLUH suggests that cell starvation is a potential mechanism of motor neuron loss that could be targeted in ALS.
Highlights
We present a detailed a protocol for the extraction of cross-linked TDP containing stress granules from brain tissue.
We present proteomics data from the insoluble fraction from brain tissue of an ALS mouse model.
We identify the mitochondrial mRNA transport protein CLUH and CLUH targets trapped in insoluble SG fraction of brain.
Reanalysing proteomics data from axonal soluble fraction supports a link between proteins trapped in the brain and depleted from the axons.
Propose a model where metabolic mitochondrial enzymes trapped in the insoluble fraction from the brain via a CLUH dependent mechanism results in motor neuron death by starvation in ALS.
Chronic cerebral ischemia (CCI) is a clinical syndrome characterised by brain dysfunction due to decreased chronic cerebral perfusion. CCI initiates several inflammatory pathways, including pyroptosis. RNA-binding proteins (RBPs) play important roles in CCI. This study aimed to explore whether the interaction between RBP-Cpeb4 and Dclk2 affected Ehf phosphorylation to regulate neuronal pyroptosis. HT22 cells and mice were used to construct oxygen glucose deprivation (OGD)/CCI models. We found that Cpeb4 and Dclk2 were upregulated in OGD-treated HT22 cells and CCI-induced hippocampal CA1 tissues. Cpeb4 upregulated Dclk2 expression by increasing Dclk2 mRNA stability. Knockdown of Cpeb4 or Dclk2 inhibited neuronal pyroptosis in OGD-treated HT22 cells and CCI-induced hippocampal CA1 tissues. By binding to the promoter regions of Caspase1 and Caspase3, the transcription factor Ehf reduced their promoter activities and inhibited the transcription. Dclk2 phosphorylated Ehf and changed its nucleoplasmic distribution, resulting in the exit of p-Ehf from the nucleus and decreased Ehf levels. It promoted the expression of Caspase1 and Caspase3 and stimulated neuronal pyroptosis of HT22 cells induced by OGD. Cpeb4/Dclk2/Ehf pathway plays an important role in the regulation of cerebral ischemia-induced neuronal pyroptosis.
Cancer cells have been shown to exploit neurons to modulate their survival and growth, including through establishment of neural circuits within the central nervous system (CNS) 1-3. Here, we report a distinct pattern of cancer-nerve interactions between the peripheral nervous system (PNS) and gastric cancer (GC). In multiple GC mouse models, nociceptive nerves demonstrated the greatest degree of nerve expansion in an NGF-dependent manner. Neural tracing identified CGRP+ peptidergic neurons as the primary gastric sensory neurons. Three-dimensional co-culture models showed that sensory neurons directly connect with gastric cancer spheroids through synapse-like structures. Chemogenetic activation of sensory neurons induced the release of calcium into the cytoplasm of cancer cells, promoting tumor growth and metastasis. Pharmacological ablation of sensory neurons or treatment with CGRP inhibitors suppressed tumor growth and extended survival. Depolarization of gastric tumor membranes through in vivo optogenetic activation led to enhanced calcium flux in nodose ganglia and CGRP release, defining a cancer cell-peptidergic neuronal circuit. Together, these findings establish the functional connectivity between cancer and sensory neurons, identifying this pathway as a potential therapeutic target.
The complex morphology of neurons requires precise control of their microtubule cytoskeleton. This is achieved by microtubule-associated proteins (MAPs) that regulate the assembly and stability of microtubules, and transport of molecules and vesicles along them. While many of these MAPs function in all cells, some are specifically or predominantly involved in regulating microtubules in neurons. Here we use the sea anemone Nematostella vectensis as a model organism to provide new insights into the early evolution of neural microtubule regulation. As a cnidarian, Nematostella belongs to an outgroup to all bilaterians and thus occupies an informative phylogenetic position for reconstructing the evolution of nervous system development. We identified an ortholog of the microtubule-binding protein doublecortin-like kinase ( NvDclk1 ) as a gene that is predominantly expressed in neurons and cnidocytes (stinging cells), two classes of cells belonging to the neural lineage in cnidarians. A transgenic NvDclk1 reporter line revealed an elaborate network of neurite-like processes emerging from cnidocytes in the tentacles and the body column. A transgene expressing NvDclk1 under the control of the NvDclk1 promoter suggests that NvDclk1 is indeed a microtubule-binding protein. Further, we generated a mutant for NvDclk1 using CRISPR/Cas9 and show that the mutants fail to generate mature cnidocytes. Our results support the hypothesis that the elaboration of programs for microtubule regulation occurred early in the evolution of nervous systems.
Nerve injuries may result in neurological dysfunction or even permanent disability, which poses various challenges to physicians. In the peripheral nervous system (PNS), only small nerve injuries can be regenerated spontaneously in vivo, while larger nerve injuries must be treated surgically with biomaterials or nerve grafts harvested. Attributed to the influence of inhibiting factors such as inflammation and microenvironment changes, a solution to completely repair central nervous system (CNS) injuries has not been discovered. Hence, most bioengineering strategies for PNS have been focused on the guidance of regenerative nerves, whereas the efforts for CNS have been focused on creating a suitable regenerating microenvironment in vivo. Recent advances in neurology, tissue engineering, biomaterials, gene transfection, and multifactor combinations offer optimistic prospects for the development of nerve regeneration. In this chapter, we firstly examine the current understanding of the neurophysiology and factors that are critical for nerve regeneration, and discuss their implications for promoting axon regeneration. Then, the current approaches, challenges, and future perspectives of biomaterials being explored to aid PNS and CNS regeneration are highlighted.
Aim: To explore the potential role of hsa_circ_0005480 as a marker for gastric cancer (GC). Methods: GSE83521, GSE93541 and GSE131414 were combined to screen the most potential circRNAs in GC. The expression of hsa_circ_0005480 was verified in clinical plasma samples and its diagnostic performance was evaluated by receiver operating characteristic (ROC) curve. Results: Hsa_circ_0005480 was upregulated in newly diagnosed GC patients, and performed well in distinguishing GC patients from healthy donors. Combination of hsa_circ_0005480 with traditional laboratory metrics showed increased sensitivity and accuracy than standalone application. In addition, hsa_circ_0005480 expression was low in GC patients treated with chemoradiotherapy or surgery, and decreased dynamically about 1 week postoperatively. Conclusion: Hsa_circ_0005480 may prove to be a marker for auxiliary diagnosis and prognostic evaluation of GC.
A unique and defining characteristic of the olfactory sensory circuit is its functionally organized topographic map, which requires widely dispersed olfactory sensory neurons with the same identity to converge their axons into one neuropil, a class-specific glomerulus. Understanding the process of how neuronal identity confers circuit organization is a major endeavor in the field of neurobiology due to its intricate connection to neurodegeneration and neuronal dysfunction. In the olfactory system, many aspects of circuit organization, from axon guidance to synaptic matching, are regulated by a variety of cell surface proteins (for example Robo/Slit and Toll receptors). In this paper, we have identified a novel atypical cadherin protein, Fat2 (also known as Kugelei), as a regulator of class-specific axon organization. Fat2 is expressed in olfactory receptor neurons (ORNs) and local interneurons (LNs) within the olfactory circuits, but little to no expression is found in projection neurons (PNs). Fat2 expression levels vary in a neuronal class-specific manner and peak during pupal development. In fat2 null mutants, ORN axon terminals belonging to different ORN classes present with varying phenotypic severity with the highest fat2 expressing classes being most severely affected. In the most extreme cases, fat2 mutations lead to ORN degeneration. We then show evidence that suggests Fat2 intracellular domain is necessary for Fat2 function in ORN axon organization. Within the developmental context, Fat2 is required starting at early stages of olfactory circuit development specifically for precise axon retraction to further condense class-specific glomeruli. We have also shown that PN and LN expression of Fat2 likely does not contribute to ORN organization . Lastly, we narrow down potential Fat2 intracellular domain interactors, APC family proteins (Adenomatous polyposis coli) and dop (Drop out), that likely orchestrate the cytoskeletal remodeling required for axon retraction during protoglomerular development. Altogether, we provide a foundational understanding of how Fat2 functions in olfactory circuit organization and implicate the critical role of axon retraction during glomerular maturation.
Background
Doublecortin-like kinase 2 (DCLK2) is a microtubule-associated protein kinase that participates in neural development and maturation; however, whether it is involved in tumour progression remains unclear.
MethodsDCLK2 overexpression and knockdown clones were established by lentivirus transfection. Western blot, PCR assays and bioinformatics analyses were conducted to observe the expression of DCLK2. CCK8, colony formation, scratch migration and Transwell assays were used to detect cell proliferation, migration and invasion, respectively. Tumour metastasis was evaluated in vivo using a tail vein metastasis model. Bioinformatics analyses were performed to analyse the expression correlation between DCLK2 and TCF4, or EMT markers in breast cancer.
ResultsOur data indicate that DCLK2 is highly expressed in breast cancer cells and is associated with poor prognosis. Silencing DCLK2 does not affect the proliferation rate of tumour cells, but significantly suppresses migration and invasion as well as lung metastasis processes. Overexpression of DCLK2 can enhance the migratory and invasive abilities of normal breast epithelial cells. Moreover, TCF4/β-catenin inhibitor LF3 downregulates the expression of DCLK2 and inhibits the migration and invasion of breast cancer cells. Furthermore, we found that the downregulation of DCLK2 blocks the epithelial–mesenchymal transition (EMT) process.Conclusion
Our study indicates that DCLK2 plays an important role in EMT, cell invasion and metastasis, suggesting that DCLK2 is a potential target for the treatment of metastatic breast cancer.
Axon regeneration holds great promise for neural repair of CNS axonopathies, including glaucoma. Pten deletion in retinal ganglion cells (RGCs) promotes potent optic nerve regeneration, but only a small population of Pten-null RGCs are actually regenerating RGCs (regRGCs); most surviving RGCs (surRGCs) remain non-regenerative. Here, we developed a strategy to specifically label and purify regRGCs and surRGCs, respectively, from the same Pten-deletion mice after optic nerve crush, in which they differ only in their regeneration capability. Smart-Seq2 single-cell transcriptome analysis revealed novel regeneration-associated genes that significantly promote axon regeneration. The most potent of these, Anxa2, acts synergistically with its ligand tPA in Pten-deletion-induced axon regeneration. Anxa2, its downstream effector ILK, and Mpp1 dramatically protect RGC somata and axons and preserve visual function in a clinically relevant model of glaucoma, demonstrating the exciting potential of this innovative strategy to identify novel effective neural repair candidates.
Intestinal ganglionic cells in the adult enteric nervous system (ENS) are continually exposed to stimuli from the surrounding microenvironment, and need at times to respond to disturbed homeostasis following acute intestinal injury. The kinase DCLK1 and intestinal Dclk1-positive cells have been reported to contribute to intestinal regeneration. While Dclk1-positive cells are present in adult enteric ganglia, their cellular identity and response to acute injury have not been investigated in detail. Here, we reveal the presence of distinct Dclk1-tdTom+/CD49b+ glial-like and Dclk1-tdTom+/CD49b- neuronal cell types in adult myenteric ganglia. These ganglionic cells demonstrate distinct patterns of tracing over time, yet show a similar expansion in response to elevated serotonergic signaling. Interestingly, Dclk1-tdTom+ glial-like and neuronal cell types appear resistant to acute irradiation injury-mediated cell death. Moreover, Dclk1-tdTom+/CD49b+ glial-like cells show prominent changes in gene expression profiles induced by injury, in contrast to Dclk1-tdTom+/CD49b- neuronal cell types. Finally, subsets of Dclk1-tdTom+/CD49b+ glial-like cells demonstrate prominent overlap with Nestin and p75NTR, and strong responses to elevated serotonergic signaling or acute injury. These findings, together with their role in early development and their neural-crest like gene expression signature, suggest the presence of reserve progenitor cells in the adult Dclk1 glial cell lineage.
The anticancer activity of bortezomib (BTZ) has been increasingly studied in a number of indications and promising results for the use of this treatment have been shown in neuroblastoma. As BTZ treatment is usually administered in cycles, the development of resistance and side effects in patients undergoing therapy with BTZ remains a major challenge for the clinical usage of this compound. Common resistance development also means that certain cells are able to survive BTZ treatment and bypass molecular mechanisms that render BTZ anticancer activity. We studied the methylome of neuroblastoma cells that survived BTZ treatment. Our results indicate that BTZ induces pronounced genome wide methylation changes in cells which recovered from the treatment. Functional analyses of identified methylation changes demonstrated they were involved in key cancer pathology pathways. These changes may allow the cells to bypass the primary anticancer activity of BTZ and develop a treatment resistant and proliferative phenotype. To study whether cells surviving BTZ treatment acquire a proliferative phenotype, we repeatedly treated cells which recovered from the first round of BTZ treatment. The repetitive treatment led to induction of the extraordinary proliferative potential of the cells, that increased with subsequent treatments. As we did not observe similar effects in cells that survived treatment with lenalidomide, and non-treated cells cultured under the same experimental conditions, this phenomenon seems to be BTZ specific. Overall, our results indicate that methylation changes may play major role in the development of BTZ resistance.
Long-distance regeneration of the central nervous system (CNS) has been achieved from the eye to the brain through activation of neuronal molecular pathways or pharmacological approaches. Unexpectedly, most of the regenerative fibers display guidance defects, which prevents reinnervation and further functional recovery. Therefore, characterizing the mature neuronal environment is essential to understand the adult axonal guidance in order to complete the circuit reconstruction. To this end, we used mass spectrometry to characterize the proteomes of major nuclei of the adult visual system: suprachiasmatic nucleus (SCN), ventral and dorsal lateral geniculate nucleus (vLGN, dLGN) and superior colliculus (SC)), as well as the optic chiasm. These analyses revealed the presence of guidance molecules and guidance-associated factors in the adult visual targets. Moreover, by performing bilateral optic nerve crush, we showed that the expression of some proteins was significantly modulated by the injury in the visual targets, even in the ones most distal to the lesion site. On another hand, we found that the expression of guidance molecules was not modified upon injury. This implies that these molecules may possibly interfere with the reinnervation of the brain targets. Together, our results provides an extensive characterization of the molecular environment in intact and injured conditions. These findings open new ways to correct regenerating axon guidance notably by manipulating the expression of the corresponding guidance receptors in the nervous system.
Glaucoma leads to irreversible vision loss and current therapeutic strategies are often insufficient to prevent the progression of the disease and consequent blindness. Elevated intraocular pressure is an important risk factor, but not required for the progression of glaucomatous neurodegeneration. The demise of retinal ganglion cells represents the final common pathway of glaucomatous vision loss. Still, lifelong control of intraocular pressure is the only current treatment to prevent severe vision loss, although it frequently fails despite best practices. This scenario calls for the development of neuroprotective and pro-regenerative therapies targeting the retinal ganglion cells as well as the optic nerve. Several experimental studies have shown the potential of gene modulation as a tool for neuroprotection and regeneration. In this context, gene therapy represents an attractive approach as persistent treatment for glaucoma. Viral vectors engineered to promote overexpression of a broad range of cellular factors have been shown to protect retinal ganglion cells and/or promote axonal regeneration in experimental models. Here, we review the mechanisms involved in glaucomatous neurodegeneration and regeneration in the central nervous system. Then, we point out current limitations of gene therapy platforms and review a myriad of studies that use viral vectors to manipulate genes in retinal ganglion cells, as a strategy to promote neuroprotection and regeneration. Finally, we address the potential of combining neuroprotective and regenerative gene therapies as an approach to glaucomatous neurodegeneration.
After central nervous system (CNS) injury, inhibitory factors in the lesion scar and poor axon growth potential prevent axon regeneration. Microtubule stabilization reduces scarring and promotes axon growth. However, the cellular mechanisms of this dual effect remain unclear. Here, delayed systemic administration of a blood-brain barrier permeable microtubule stabilizing drug, epothilone B (epoB), decreased scarring after rodent spinal cord injury (SCI) by abrogating polarization and directed migration of scar-forming fibroblasts. Conversely, epothilone B reactivated neuronal polarization by inducing concerted microtubule polymerization into the axon tip, which propelled axon growth through an inhibitory environment. Together, these drug elicited effects promoted axon regeneration and improved motor function after SCI. With recent clinical approval, epothilones hold promise for clinical use after CNS injury.
Copyright © 2015, American Association for the Advancement of Science.
Growth cones facilitate the repair of nervous system damage by providing the driving force for axon regeneration. Using single-neuron laser axotomy and in vivo time-lapse imaging, we show that syndecan, a heparan sulfate (HS) proteoglycan, is required for growth cone function during axon regeneration in C. elegans. In the absence of syndecan, regenerating growth cones form but are unstable and collapse, decreasing the effective growth rate and impeding regrowth to target cells. We provide evidence that syndecan has two distinct functions during axon regeneration: (1) a canonical function in axon guidance that requires expression outside the nervous system and depends on HS chains and (2) an intrinsic function in growth cone stabilization that is mediated by the syndecan core protein, independently of HS. Thus, syndecan is a regulator of a critical choke point in nervous system repair.
Motile growth cones lead growing axons through developing tissues to synaptic targets. These behaviors depend on the organization and dynamics of actin filaments that fill the growth cone leading margin [peripheral (P‐) domain]. Actin filament organization in growth cones is regulated by actin‐binding proteins that control all aspects of filament assembly, turnover, interactions with other filaments and cytoplasmic components, and participation in producing mechanical forces. Actin filament polymerization drives protrusion of sensory filopodia and lamellipodia, and actin filament connections to the plasma membrane link the filament network to adhesive contacts of filopodia and lamellipodia with other surfaces. These contacts stabilize protrusions and transduce mechanical forces generated by actomyosin activity into traction that pulls an elongating axon along the path toward its target. Adhesive ligands and extrinsic guidance cues bind growth cone receptors and trigger signaling activities involving Rho GTP ases, kinases, phosphatases, cyclic nucleotides, and [Ca ⁺⁺ ] fluxes. These signals regulate actin‐binding proteins to locally modulate actin polymerization, interactions, and force transduction to steer the growth cone leading margin toward the sources of attractive cues and away from repellent guidance cues. image
Actin filament organization in motile growth cone leading margin (peripheral (P‐) domain) is regulated by actin‐binding proteins that control all aspects of filament assembly, turnover, interactions with other filaments or cytoplasmic components, and produce mechanical forces. This Review outlines key aspects of growth cone actin dynamics, as well as the roles of adhesion‐mediating proteins, like integrins, which stabilize actin‐based protrusions and transduce mechanical forces generated by actomyosin activity into traction that pulls an elongating axon along the path toward its target.
In contrast to neurons in the central nervous system, mature neurons in the mammalian peripheral nervous system (PNS) can regenerate axons after injury, in part, by enhancing intrinsic growth competence. However, the signalling pathways that enhance the growth potential and induce spontaneous axon regeneration remain poorly understood. Here we reveal that phosphatidylinositol 3-kinase (PI3K) signalling is activated in response to peripheral axotomy and that PI3K pathway is required for sensory axon regeneration. Moreover, we show that glycogen synthase kinase 3 (GSK3), rather than mammalian target of rapamycin, mediates PI3K-dependent augmentation of the growth potential in the PNS. Furthermore, we show that PI3K-GSK3 signal is conveyed by the induction of a transcription factor Smad1 and that acute depletion of Smad1 in adult mice prevents axon regeneration in vivo. Together, these results suggest PI3K-GSK3-Smad1 signalling as a central module for promoting sensory axon regeneration in the mammalian nervous system.
Developing approaches to promote the regeneration of descending supraspinal axons represents an ideal strategy for rebuilding neuronal circuits to improve functional recovery after spinal cord injury (SCI). Our previous studies demonstrated that genetic deletion of phosphatase and tensin homolog (PTEN) in mouse corticospinal neurons reactivates their regenerative capacity, resulting in significant regeneration of corticospinal tract (CST) axons after SCI. However, it is unknown whether nongenetic methods of suppressing PTEN have similar effects and how regenerating axons interact with the extrinsic environment. Herein, we show that suppressing PTEN expression with short-hairpin RNA (shRNA) promotes the regeneration of injured CST axons, and these axons form anatomical synapses in appropriate areas of the cord caudal to the lesion. Importantly, this model of increased CST regrowth enables the analysis of extrinsic regulators of CST regeneration in vivo. We find that regenerating axons avoid dense clusters of fibroblasts and macrophages in the lesion, suggesting that these cell types might be key inhibitors of axon regeneration. Furthermore, most regenerating axons cross the lesion in association with astrocytes, indicating that these cells might be important for providing a permissive bridge for axon regeneration. Lineage analysis reveals that these bridge-forming astrocytes are not derived from ependymal stem cells within the spinal cord, suggesting that they are more likely derived from a subset of mature astrocytes. Overall, this study reveals insights into the critical extrinsic and intrinsic regulators of axon regeneration and establishes shRNA as a viable means to manipulate these regulators and translate findings into other mammalian models.
Axon regeneration after injury requires the extensive reconstruction, reorganization, and stabilization of the microtubule cytoskeleton in the growth cones. Here, we identify KIF3C as a key regulator of axonal growth and regeneration by controlling microtubule dynamics and organization in the growth cone. KIF3C is developmentally regulated. Rat embryonic sensory axons and growth cones contain undetect-able levels of KIF3C protein that is locally translated immediately after injury. In adult neurons, KIF3C is axonally transported from the cell body and is enriched at the growth cone where it preferentially binds to tyrosinated microtubules. Functionally, the interaction of KIF3C with EB3 is necessary for its localization at the microtubule plus-ends in the growth cone. Depletion of KIF3C in adult neurons leads to an increase in stable, overgrown and looped microtubules because of a strong decrease in the microtubule frequency of catastrophes, suggesting that KIF3C functions as a microtubule-destabilizing factor. Adult axons lacking KIF3C, by RNA interference or KIF3C gene knock-out, display an impaired axonal outgrowth in vitro and a delayed regeneration after injury both in vitro and in vivo. Murine KIF3C knock-out embryonic axons grow normally but do not regenerate after injury because they are unable to locally translate KIF3C. These data show that KIF3C is an injury-specific kinesin that contributes to axon growth and regeneration by regulating and organizing the microtubule cytoskeleton in the growth cone.
The cell intrinsic factors that determine whether a neuron regenerates or undergoes apoptosis in response to axonal injury are not well defined. Here we show that the mixed-lineage dual leucine zipper kinase (DLK) is an essential upstream mediator of both of these divergent outcomes in the same cell type. Optic nerve crush injury leads to rapid elevation of DLK protein, first in the axons of retinal ganglion cells (RGCs) and then in their cell bodies. DLK is required for the majority of gene expression changes in RGCs initiated by injury, including induction of both proapoptotic and regeneration-associated genes. Deletion of DLK in retina results in robust and sustained protection of RGCs from degeneration after optic nerve injury. Despite this improved survival, the number of axons that regrow beyond the injury site is substantially reduced, even when the tumor suppressor phosphatase and tensin homolog (PTEN) is deleted to enhance intrinsic growth potential. These findings demonstrate that these seemingly contradictory responses to injury are mechanistically coupled through a DLK-based damage detection mechanism.
Glaucoma, a major cause of blindness worldwide, is a neurodegenerative optic neuropathy in which vision loss is caused by loss of retinal ganglion cells (RGCs). To better define the pathways mediating RGC death and identify targets for the development of neuroprotective drugs, we developed a high-throughput RNA interference screen with primary RGCs and used it to screen the full mouse kinome. The screen identified dual leucine zipper kinase (DLK) as a key neuroprotective target in RGCs. In cultured RGCs, DLK signaling is both necessary and sufficient for cell death. DLK undergoes robust posttranscriptional up-regulation in response to axonal injury in vitro and in vivo. Using a conditional knockout approach, we confirmed that DLK is required for RGC JNK activation and cell death in a rodent model of optic neuropathy. In addition, tozasertib, a small molecule protein kinase inhibitor with activity against DLK, protects RGCs from cell death in rodent glaucoma and traumatic optic neuropathy models. Together, our results establish a previously undescribed drug/drug target combination in glaucoma, identify an early marker of RGC injury, and provide a starting point for the development of more specific neuroprotective DLK inhibitors for the treatment of glaucoma, nonglaucomatous forms of optic neuropathy, and perhaps other CNS neurodegenerations.
Dendritic morphogenesis and formation of synapses at appropriate dendritic locations are essential for the establishment of proper neuronal connectivity. Recent imaging studies provide evidence for stabilization of dynamic distal branches of dendrites by the addition of new synapses. However, molecules involved in both dendritic growth and suppression of synapse maturation remain to be identified. Here we report two distinct functions of doublecortin-like kinases, chimeric proteins containing both a microtubule-binding domain and a kinase domain in postmitotic neurons. First, doublecortin-like kinases localize to the distal dendrites and promote their growth by enhancing microtubule bundling. Second, doublecortin-like kinases suppress maturation of synapses through multiple pathways, including reduction of PSD-95 by the kinase domain and suppression of spine structural maturation by the microtubule-binding domain. Thus, doublecortin-like kinases are critical regulators of dendritic development by means of their specific targeting to the distal dendrites, and their local control of dendritic growth and synapse maturation.
Doublecortin (Dcx) is the causative gene for X-linked lissencephaly, which encodes a microtubule-binding protein. Axon tracts are abnormal in both affected individuals and in animal models. To determine the reason for the axon tract defect, we performed a semiquantitative proteomic analysis of the corpus callosum in mice mutant for Dcx. In axons from mice mutant for Dcx, widespread differences are found in actin-associated proteins as compared with wild-type axons. Decreases in actin-binding proteins α-actinin-1 and α-actinin-4 and actin-related protein 2/3 complex subunit 3 (Arp3), are correlated with dysregulation in the distribution of filamentous actin (F-actin) in the mutant neurons with increased F-actin around the cell body and decreased F-actin in the neurites and growth cones. The actin distribution defect can be rescued by full-length Dcx and further enhanced by Dcx S297A, the unphosphorylatable mutant, but not with the truncation mutant of Dcx missing the C-terminal S/P-rich domain. Thus, the C-terminal region of Dcx dynamically regulates formation of F-actin features in developing neurons, likely through interaction with spinophilin, but not through α-actinin-4 or Arp3. We show with that the phenotype of Dcx/Doublecortin-like kinase 1 deficiency is consistent with actin defect, as these axons are selectively deficient in axon guidance, but not elongation.
Doublecortin (Dcx) defines a growing family of microtubule (MT)-associated proteins (MAPs) involved in neuronal migration and process outgrowth. We show that Dcx is essential for the function of Kif1a, a kinesin-3 motor protein that traffics synaptic vesicles. Neurons lacking Dcx and/or its structurally conserved paralogue, doublecortin-like kinase 1 (Dclk1), show impaired Kif1a-mediated transport of Vamp2, a cargo of Kif1a, with decreased run length. Human disease-associated mutations in Dcx's linker sequence (e.g., W146C, K174E) alter Kif1a/Vamp2 transport by disrupting Dcx/Kif1a interactions without affecting Dcx MT binding. Dcx specifically enhances binding of the ADP-bound Kif1a motor domain to MTs. Cryo-electron microscopy and subnanometer-resolution image reconstruction reveal the kinesin-dependent conformational variability of MT-bound Dcx and suggest a model for MAP-motor crosstalk on MTs. Alteration of kinesin run length by MAPs represents a previously undiscovered mode of control of kinesin transport and provides a mechanism for regulation of MT-based transport by local signals.
Doublecortin (DCX) is a microtubule-associated protein that is specifically expressed in neuronal cells. Genetic mutation of DCX causes lissencephaly disease. Although the abnormal cortical lamination in lissencephaly is thought to be attributable to neuronal cell migration defects, the regulatory mechanisms governing interactions between DCX and cytoskeleton in the migration of neuronal progenitor cells remain obscure. In this study we found that the G(s) and protein kinase A (PKA) signal elicited by pituitary adenylate cyclase-activating polypeptide promotes neuronal progenitor cells migration. Stimulation of G(s)-PKA signaling prevented microtubule bundling and induced the dissociation of DCX from microtubules in cells. PKA phosphorylated DCX at Ser-47, and the phospho-mimicking mutant DCX-S47E promoted cell migration. Activation of PKA and DCX-S47E induced lamellipodium formation. Pituitary adenylate cyclase-activating polypeptide and DCX-S47E stimulated the activation of Rac1, and DCX-S47E interacted with Asef2, a guanine nucleotide exchange factor for Rac1. Our data reveal a dual reciprocal role for DCX phosphorylation in the regulation of microtubule and actin dynamics that is indispensable for proper brain lamination.
Axon growth is driven by the movement of a growth cone, a specialized sensory motile structure located at the tip of a growing neurite. Although stalled retraction bulbs have long been recognized as hallmarks of regeneration failure, mechanisms that control the formation and migration of nerve endings are only beginning to be unraveled. Recent studies point to microtubules as key determinants for such processes, and emerging evidence suggests that regulators of actin and microtubule dynamics in the growth cone might serve as attractive targets for controlling both the speed and trajectory of regenerating axons. This review discusses the potential of and recent progress in direct modulation of the growth cone machinery as a novel strategy to promote axon regeneration in the nervous system after injury.
A formidable challenge in neural repair in the adult central nervous system (CNS) is the long distances that regenerating axons often need to travel in order to reconnect with their targets. Thus, a sustained capacity for axon regeneration is critical for achieving functional restoration. Although deletion of either phosphatase and tensin homologue (PTEN), a negative regulator of mammalian target of rapamycin (mTOR), or suppressor of cytokine signalling 3 (SOCS3), a negative regulator of Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, in adult retinal ganglion cells (RGCs) individually promoted significant optic nerve regeneration, such regrowth tapered off around 2 weeks after the crush injury. Here we show that, remarkably, simultaneous deletion of both PTEN and SOCS3 enables robust and sustained axon regeneration. We further show that PTEN and SOCS3 regulate two independent pathways that act synergistically to promote enhanced axon regeneration. Gene expression analyses suggest that double deletion not only results in the induction of many growth-related genes, but also allows RGCs to maintain the expression of a repertoire of genes at the physiological level after injury. Our results reveal concurrent activation of mTOR and STAT3 pathways as key for sustaining long-distance axon regeneration in adult CNS, a crucial step towards functional recovery.
Axon growth potential is highest in young neurons but diminishes with age, thus becoming a significant obstacle to axonal regeneration after injury in maturity. The mechanism for the decline is incompletely understood, and no effective clinical treatment is available to rekindle innate growth capability. Here, we show that Smad1-dependent bone morphogenetic protein (BMP) signaling is developmentally regulated and governs axonal growth in dorsal root ganglion (DRG) neurons. Down-regulation of the pathway contributes to the age-related decline of the axon growth potential. Reactivating Smad1 selectively in adult DRG neurons results in sensory axon regeneration in a mouse model of spinal cord injury (SCI). Smad1 signaling can be effectively manipulated by an adeno-associated virus (AAV) vector encoding BMP4 delivered by a clinically applicable and minimally invasive technique, an approach devoid of unwanted abnormalities in mechanosensation or pain perception. Importantly, transected axons are able to regenerate even when the AAV treatment is delivered after SCI, thus mimicking a clinically relevant scenario. Together, our results identify a therapeutic target to promote axonal regeneration after SCI.
Mature retinal ganglion cells (RGCs) cannot normally regenerate axons into the injured optic nerve but can do so after lens injury. Astrocyte-derived ciliary neurotrophic factor and leukemia inhibitory factor have been identified as essential key factors mediating this effect. However, the outcome of this regeneration is still limited by inhibitors associated with the CNS myelin and the glial scar. The current study demonstrates that Taxol markedly enhanced neurite extension of mature RGCs and PC12 cells by stabilization of microtubules and desensitized axons toward myelin and chondroitin sulfate proteoglycan (CSPG) inhibition in vitro without reducing RhoA activation. In vivo, the local application of Taxol at the injury site of the optic nerve of rats enabled axons to regenerate beyond the lesion site but did not affect the intrinsic regenerative state of RGCs. Furthermore, Taxol treatment markedly increased lens injury-mediated axon regeneration in vivo, delayed glial scar formation, suppressed CSPG expression, and transiently reduced the infiltration of macrophages at the injury site. Thus, microtubule-stabilizing compounds such as Taxol might be promising candidates as adjuvant drugs in the treatment of CNS injuries particularly when combined with interventions stimulating the intrinsic regenerative state of neurons.
Hypertrophic scarring and poor intrinsic axon growth capacity constitute major obstacles for spinal cord repair. These processes
are tightly regulated by microtubule dynamics. Here, moderate microtubule stabilization decreased scar formation after spinal
cord injury in rodents through various cellular mechanisms, including dampening of transforming growth factor–β signaling.
It prevented accumulation of chondroitin sulfate proteoglycans and rendered the lesion site permissive for axon regeneration
of growth-competent sensory neurons. Microtubule stabilization also promoted growth of central nervous system axons of the
Raphe-spinal tract and led to functional improvement. Thus, microtubule stabilization reduces fibrotic scarring and enhances
the capacity of axons to grow.
Axon outgrowth and guidance to the proper target requires the coordination of filamentous (F)-actin and microtubules (MTs), the dynamic cytoskeletal polymers that promote shape change and locomotion. Over the past two decades, our knowledge of the many guidance cues, receptors, and downstream signaling cascades involved in neuronal outgrowth and guidance has increased dramatically. Less is known, however, about how those cascades of information converge and direct appropriate remodeling and interaction of cytoskeletal polymers, the ultimate effectors of movement and guidance. During development, much of the communication that occurs between environmental guidance cues and the cytoskeleton takes place at the growing tip of the axon, the neuronal growth cone. Several articles on this topic focus on the "input" to the growth cone, the myriad of receptor types, and their corresponding cognate ligands. Others investigate the signaling cascades initiated by receptors and propagated by second messenger pathways (i.e., kinases, phosphatases, GTPases). Ultimately, this plethora of information converges on proteins that associate directly with the actin and microtubule cytoskeletons. The role of these cytoskeletal-associated proteins, as well as the cytoskeleton itself in axon outgrowth and guidance, is the subject of this article.
Despite the essential role of the corticospinal tract (CST) in controlling voluntary movements, successful regeneration of large numbers of injured CST axons beyond a spinal cord lesion has never been achieved. We found that PTEN/mTOR are critical for controlling the regenerative capacity of mouse corticospinal neurons. After development, the regrowth potential of CST axons was lost and this was accompanied by a downregulation of mTOR activity in corticospinal neurons. Axonal injury further diminished neuronal mTOR activity in these neurons. Forced upregulation of mTOR activity in corticospinal neurons by conditional deletion of Pten, a negative regulator of mTOR, enhanced compensatory sprouting of uninjured CST axons and enabled successful regeneration of a cohort of injured CST axons past a spinal cord lesion. Furthermore, these regenerating CST axons possessed the ability to reform synapses in spinal segments distal to the injury. Thus, modulating neuronal intrinsic PTEN/mTOR activity represents a potential therapeutic strategy for promoting axon regeneration and functional repair after adult spinal cord injury.
Unlike neurons in the central nervous system (CNS), injured neurons in the peripheral nervous system (PNS) can regenerate their axons and reinnervate their targets. However, functional recovery in the PNS often remains suboptimal, especially in cases of severe damage. The lack of regenerative ability of CNS neurons has been linked to down-regulation of the mTOR (mammalian target of rapamycin) pathway. We report here that PNS dorsal root ganglial neurons (DRGs) activate mTOR following damage and that this activity enhances axonal growth capacity. Furthermore, genetic up-regulation of mTOR activity by deletion of tuberous sclerosis complex 2 (TSC2) in DRGs is sufficient to enhance axonal growth capacity in vitro and in vivo. We further show that mTOR activity is linked to the expression of GAP-43, a crucial component of axonal outgrowth. However, although TSC2 deletion in DRGs facilitates axonal regrowth, it leads to defects in target innervation. Thus, whereas manipulation of mTOR activity could provide new strategies to stimulate nerve regeneration in the PNS, fine control of mTOR activity is required for proper target innervation.
In vivo regeneration of peripheral neurons is constrained and rarely complete, and unfortunately patients with major nerve trunk transections experience only limited recovery. Intracellular inhibition of neuronal growth signals may be among these constraints. In this work, we investigated the role of PTEN (phosphatase and tensin homolog deleted on chromosome 10) during regeneration of peripheral neurons in adult Sprague Dawley rats. PTEN inhibits phosphoinositide 3-kinase (PI3-K)/Akt signaling, a common and central outgrowth and survival pathway downstream of neuronal growth factors. While PI3-K and Akt outgrowth signals were expressed and activated within adult peripheral neurons during regeneration, PTEN was similarly expressed and poised to inhibit their support. PTEN was expressed in neuron perikaryal cytoplasm, nuclei, regenerating axons, and Schwann cells. Adult sensory neurons in vitro responded to both graded pharmacological inhibition of PTEN and its mRNA knockdown using siRNA. Both approaches were associated with robust rises in the plasticity of neurite outgrowth that were independent of the mTOR (mammalian target of rapamycin) pathway. Importantly, this accelerated outgrowth was in addition to the increased outgrowth generated in neurons that had undergone a preconditioning lesion. Moreover, following severe nerve transection injuries, local pharmacological inhibition of PTEN or siRNA knockdown of PTEN at the injury site accelerated axon outgrowth in vivo. The findings indicated a remarkable impact on peripheral neuron plasticity through PTEN inhibition, even within a complex regenerative milieu. Overall, these findings identify a novel route to propagate intrinsic regeneration pathways within axons to benefit nerve repair.
Mutations in doublecortin (DCX) are associated with intractable epilepsy in humans, due to a severe disorganization of the neocortex and hippocampus known as classical lissencephaly. However, the basis of the epilepsy in lissencephaly remains unclear. To address potential functional redundancy with murin Dcx, we targeted one of the closest homologues, doublecortin-like kinase 2 (Dclk2). Here, we report that Dcx; Dclk2-null mice display frequent spontaneous seizures that originate in the hippocampus, with most animals dying in the first few months of life. Elevated hippocampal expression of c-fos and loss of somatostatin-positive interneurons were identified, both known to correlate with epilepsy. Dcx and Dclk2 are coexpressed in developing hippocampus, and, in their absence, there is dosage-dependent disrupted hippocampal lamination associated with a cell-autonomous simplification of pyramidal dendritic arborizations leading to reduced inhibitory synaptic tone. These data suggest that hippocampal dysmaturation and insufficient receptive field for inhibitory input may underlie the epilepsy in lissencephaly, and suggest potential therapeutic strategies for controlling epilepsy in these patients.
Regeneration of injured neurons can restore function, but most neurons regenerate poorly or not at all. The failure to regenerate
in some cases is due to a lack of activation of cell-intrinsic regeneration pathways. These pathways might be targeted for
the development of therapies that can restore neuron function after injury or disease. Here, we show that the DLK-1 mitogen-activated
protein (MAP) kinase pathway is essential for regeneration in Caenorhabditis elegans motor neurons. Loss of this pathway eliminates regeneration, whereas activating it improves regeneration. Further, these
proteins also regulate the later step of growth cone migration. We conclude that after axon injury, activation of this MAP
kinase cascade is required to switch the mature neuron from an aplastic state to a state capable of growth.
The failure of axons to regenerate is a major obstacle for functional recovery after central nervous system (CNS) injury.
Removing extracellular inhibitory molecules results in limited axon regeneration in vivo. To test for the role of intrinsic
impediments to axon regrowth, we analyzed cell growth control genes using a virus-assisted in vivo conditional knockout approach.
Deletion of PTEN (phosphatase and tensin homolog), a negative regulator of the mammalian target of rapamycin (mTOR) pathway,
in adult retinal ganglion cells (RGCs) promotes robust axon regeneration after optic nerve injury. In wild-type adult mice,
the mTOR activity was suppressed and new protein synthesis was impaired in axotomized RGCs, which may contribute to the regeneration
failure. Reactivating this pathway by conditional knockout of tuberous sclerosis complex 1, another negative regulator of
the mTOR pathway, also leads to axon regeneration. Thus, our results suggest the manipulation of intrinsic growth control
pathways as a therapeutic approach to promote axon regeneration after CNS injury.