Recent evidence suggests that glycogen synthase kinase 3 (GSK3) proteins and their upstream and downstream regulators have key roles in many fundamental processes during neurodevelopment. Disruption ...of GSK3 signalling adversely affects brain development and is associated with several neurodevelopmental disorders. Here, we discuss the mechanisms by which GSK3 activity is regulated in the nervous system and provide an overview of the recent advances in the understanding of how GSK3 signalling controls neurogenesis, neuronal polarization and axon growth during brain development. These recent advances suggest that GSK3 is a crucial node that mediates various cellular processes that are controlled by multiple signalling molecules--for example, disrupted in schizophrenia 1 (DISC1), partitioning defective homologue 3 (PAR3), PAR6 and Wnt proteins--that regulate neurodevelopment.
Axons in the adult mammalian central nervous system fail to regenerate after injury. By contrast, spontaneous axon regeneration occurs in the peripheral nervous system (PNS) due to a supportive PNS ...environment and an increase in the intrinsic growth potential induced by injury via cooperative activation of multifaceted biological pathways. This study compared axon regeneration and injury responses in C57BL/6 male and female mice after sciatic nerve crush (SNC) injury. The extent of axon regeneration in vivo was indistinguishable in male and female mice when observed at 3 days after SNC injury, and primary dorsal root ganglion (DRG) neurons from injured, male and female mice extended axons to a similar length. Moreover, the induction of selected regeneration‐associated genes (RAGs), such as Atf3, Sprr1a, Gap43, Sox11, Jun, Gadd45a, and Smad1 were comparable in male and female DRGs when assessed by quantitative real‐time reverse transcription polymerase chain reaction. Furthermore, the RNA‐seq analysis of male and female DRGs revealed that differentially expressed genes (DEGs) in SNC groups compared to sham‐operated groups included many common genes associated with neurite outgrowth. However, we also found that a large number of genes in the DEGs were sex dependent, implicating the involvement of distinct gene regulatory network in the two sexes following peripheral nerve injury. In conclusion, we found that male and female mice mounted a comparable axon regeneration response and many RAGs were commonly induced in response to SNC. However, given that many DEGs were sex‐dependently expressed, future studies are needed to investigate whether they contribute to peripheral axon regeneration, and if so, to what extent.
In the mammalian peripheral nervous system, axon regeneration occurs spontaneously after injury. This study examined if axon regeneration after sciatic nerve crush injury was sex dependent and found that the extent of axon regeneration was indistinguishable in male and female mice. Transcriptome analysis revealed that many genes associated with neurite outgrowth were commonly induced in the two sexes, despite that a majority of genes were sex‐dependently induced by injury. Therefore, although male and female mice exhibit similar axon regeneration, the mechanisms involved might not be identical.
Oligodendrocytes are specialized cells that myelinate axons in the central nervous system. Defects in oligodendrocyte function and failure to form or maintain myelin sheaths can cause a number of ...neurological disorders. Oligodendrocytes are differentiated from oligodendrocyte progenitor cells (OPCs), which extend several processes that contact, elaborate, and eventually wrap axonal segments to form multilayered myelin sheaths. These processes require extensive changes in the cytoarchitecture and must be regulated by reorganization of the cytoskeleton. Here, we established a simple protocol to isolate and differentiate mouse OPCs, and by using this method, we investigated a role of microtubules (MTs) in oligodendrocyte differentiation. Oligodendrocytes developed a complex network of MTs during differentiation, and treatment of differentiating oligodendrocytes with nanomolar concentrations of MT-targeting agents (MTAs) markedly affected oligodendrocyte survival and differentiation. We found that acute exposure to vincristine and nocodazole at early stages of oligodendrocyte differentiation markedly increased MT arborization and enhanced differentiation, whereas taxol and epothilone B treatment produced opposing outcomes. Furthermore, treatment of myelinating co-cultures of oligodendrocytes and neurons with nanomolar concentrations of MTAs at late stages of oligodendrocyte differentiation induced dysmyelination. Together, these results suggest that MTs play an important role in the survival, differentiation, and myelination of oligodendrocytes.
In native tissues, cellular and acellular components are anisotropically organized and often aligned in specific directions, providing structural and mechanical properties for actuating biological ...functions. Thus, engineering alignment not only allows for emulation of native tissue structures but might also enable implementation of specific functionalities. However, achieving desired alignment is challenging, especially in three-dimensional constructs. By exploiting the elastomeric property of polydimethylsiloxane and fibrillogenesis kinetics of collagen, here we introduce a simple yet effective method to assemble and align fibrous structures in a multi-modular three-dimensional conglomerate. Applying this method, we have reconstructed the CA3-CA1 hippocampal neural circuit three-dimensionally in a monolithic gel, in which CA3 neurons extend parallel axons to and synapse with CA1 neurons. Furthermore, we show that alignment of the fibrous scaffold facilitates the establishment of functional connectivity. This method can be applied for reconstructing other neural circuits or tissue units where anisotropic organization in a multi-modular structure is desired.
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.
Katanin was the first microtubule (MT)-severing enzyme discovered, but how katanin executes MT severing remains poorly understood. Here, we report X-ray crystal structures of the apo and ATPγS-bound ...states of the catalytic AAA domain of human katanin p60 at 3.0 and 2.9 Å resolution, respectively. Comparison of the two structures reveals conformational changes induced by ATP binding and how such changes ensure hexamer stability. Moreover, we uncover structural details of pore loops (PLs) and show that Arg283, a residue unique to katanin among MT-severing enzymes, protrudes from PL1 and lines the entry of the catalytic pore. Functional studies suggest that PL1 and Arg283 play essential roles in the recognition and remodeling of the glutamylated, C-terminal tubulin tail and regulation of axon growth. In addition, domain-swapping experiments in katanin and spastin suggest that the non-homologous N-terminal region, which contains the MT-interacting and trafficking domain and a linker, confers specificity to the severing process.
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•Structures of the apo and ATPγS-bound AAA domain of katanin p60 are determined•The YRG motif of pore loop 1, unique to katanin, is crucial for katanin activity•Katanin remodels glutamylated microtubules and controls axon growth•Katanin and spastin recognize and remodel microtubules in distinct ways
Shin et al. report structures of the apo and ATPγS-bound AAA domain of katanin p60. Structural modeling and functional studies suggest that Arg283 in the YRG motif, unique to katanin, crowns the entry of the catalytic core and is essential for remodeling of glutamylated microtubules and regulation of axon growth.
Schwann cells (SCs), the primary glia in the peripheral nervous system (PNS), display remarkable plasticity in that fully mature SCs undergo dedifferentiation and convert to repair SCs upon nerve ...injury. Dedifferentiated SCs provide essential support for PNS regeneration by producing signals that enhance the survival and axon regrowth of damaged neurons, but the identities of neurotrophic factors remain incompletely understood. Here we show that SCs express and secrete progranulin (PGRN), depending on the differentiation status of SCs. PGRN expression and secretion markedly increased as primary SCs underwent dedifferentiation, while PGRN secretion was prevented by administration of cAMP, which induced SC differentiation. We also found that sciatic nerve injury, a physiological trigger of SC dedifferentiation, induced PGRN expression in SCs in vivo. These results suggest that dedifferentiated SCs express and secrete PGRN that functions as a paracrine factor to support the survival and axon growth of neighboring neurons after injury.
Main Points
Schwann Cells (SC) induce the expression of PGRN after sciatic nerve injury, a physiological process that triggers SC dedifferentiation.
Parkinson's disease (PD) is a heterogeneous neurodegenerative disease characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta and the widespread occurrence ...of proteinaceous inclusions known as Lewy bodies and Lewy neurites. The etiology of PD is still far from clear, but aging has been considered as the highest risk factor influencing the clinical presentations and the progression of PD. Accumulating evidence suggests that aging and PD induce common changes in multiple cellular functions, including redox imbalance, mitochondria dysfunction, and impaired proteostasis. Age-dependent deteriorations in cellular dysfunction may predispose individuals to PD, and cellular damages caused by genetic and/or environmental risk factors of PD may be exaggerated by aging. Mutations in the
gene cause late-onset, autosomal dominant PD and comprise the most common genetic causes of both familial and sporadic PD. LRRK2-linked PD patients show clinical and pathological features indistinguishable from idiopathic PD patients. Here, we review cellular dysfunctions shared by aging and PD-associated
mutations and discuss how the interplay between the two might play a role in PD pathologies.
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and sporadic Parkinson's disease (PD). Elevated kinase activity is associated with LRRK2 toxicity, but the ...substrates that mediate neurodegeneration remain poorly defined. Given the increasing evidence suggesting a role of LRRK2 in membrane and vesicle trafficking, here we systemically screened Rab GTPases, core regulators of vesicular dynamics, as potential substrates of LRRK2 and investigated the functional consequence of such phosphorylation in cells and in vivo.
In vitro LRRK2 kinase assay with forty-five purified human Rab GTPases was performed to identify Rab family proteins as substrates of LRRK2. We identified the phosphorylation site by tandem mass-spectrometry and confirmed it by assessing phosphorylation in the in vitro LRRK2 kinase assay and in cells. Effects of Rab phosphorylation on neurodegeneration were examined in primary cultures and in vivo by intracranial injection of adeno-associated viral vectors (AAV) expressing wild-type or phosphomutants of Rab35.
Our screening revealed that LRRK2 phosphorylated several Rab GTPases at a conserved threonine residue in the switch II region, and by using the kinase-inactive LRRK2-D1994A and the pathogenic LRRK2-G2019S along with Rab proteins in which the LRRK2 site was mutated, we verified that a subset of Rab proteins, including Rab35, were authentic substrates of LRRK2 both in vitro and in cells. We also showed that phosphorylation of Rab regulated GDP/GTP-binding property in cells. Moreover, in primary cortical neurons, mutation of the LRRK2 site in several Rabs caused neurotoxicity, which was most severely induced by phosphomutants of Rab35. Furthermore, intracranial injection of the AAV-Rab35 -T72A or AAV-Rab35-T72D into the substantia nigra substantially induced degeneration of dopaminergic neurons in vivo.
Here we show that a subset of Rab GTPases are authentic substrates of LRRK2 both in vitro and in cells. We also provide evidence that dysregulation of Rab phosphorylation in the LRRK2 site induces neurotoxicity in primary neurons and degeneration of dopaminergic neurons in vivo. Our study suggests that Rab GTPases might mediate LRRK2 toxicity in the progression of PD.
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