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1.
Mol Ther ; 31(11): 3277-3289, 2023 11 01.
Article in English | MEDLINE | ID: mdl-37766430

ABSTRACT

Amyotrophic lateral sclerosis (ALS) is a uniformly lethal neurodegenerative disease characterized by progressive deterioration of motor neurons and neuromuscular denervation. Adeno-associated virus (AAV)-mediated delivery of trophic factors is being considered as a potential disease-modifying therapeutic avenue. Here we show a marked effect of AAV-mediated over-expression of neuron-derived neurotrophic factor (NDNF) on SOD1G93A ALS model mice. First, we adopt AAV-PHP.eB capsid to enable widespread expression of target proteins in the brain and spinal cord when delivered intrathecally. Then we tested the effects of AAV-NDNF on SOD1G93A mice at different stages of disease. Interestingly, AAV-NDNF markedly improved motor performance and alleviated weight loss when delivered at early post-symptomatic stage. Injection in the middle post-symptomatic stages still improved the locomotion ability, although it did not alleviate the loss of body weight. Injection in the late stage also extended the life span of SOD1G93A mice. Furthermore, NDNF expression promoted the survival of spinal motoneurons, reduced abnormal protein aggregation, and preserved the innervated neuromuscular functions. We further analyzed the signaling pathways of NDNF expression and found that it activates cell survival and growth-associated mammalian target of rapamycin signaling pathway and downregulates apoptosis-related pathways. Thus, intrathecally AAV-NDNF delivery has provided a potential strategy for the treatment of ALS.


Subject(s)
Amyotrophic Lateral Sclerosis , Neurodegenerative Diseases , Animals , Mice , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/therapy , Dependovirus/genetics , Disease Models, Animal , Disease Progression , Mice, Transgenic , Motor Neurons/metabolism , Nerve Growth Factors/metabolism , Neurodegenerative Diseases/metabolism , Spinal Cord/metabolism , Superoxide Dismutase-1/genetics , Superoxide Dismutase-1/metabolism
2.
Biochem Soc Trans ; 50(6): 1753-1762, 2022 12 16.
Article in English | MEDLINE | ID: mdl-36382964

ABSTRACT

The nervous system is composed of a variety of neurons and glial cells with different morphology and functions. In the mammalian peripheral nervous system (PNS) or the lower vertebrate central nervous system (CNS), most neurons can regenerate extensively after axotomy, while the neurons in the mammalian CNS possess only limited regenerative ability. This heterogeneity is common within and across species. The studies about the transcriptomes after nerve injury in different animal models have revealed a series of molecular and cellular events that occurred in neurons after axotomy. However, responses of various types of neurons located in different positions of individuals were different remarkably. Thus, researchers aim to find the key factors that are conducive to regeneration, so as to provide the molecular basis for solving the regeneration difficulties after CNS injury. Here we review the heterogeneity of axonal regeneration among different cell subtypes in different animal models or the same organ, emphasizing the importance of comparative studies within and across species.


Subject(s)
Axons , Nerve Regeneration , Animals , Nerve Regeneration/physiology , Axotomy , Peripheral Nervous System , Central Nervous System , Mammals
3.
Development ; 147(10)2020 05 15.
Article in English | MEDLINE | ID: mdl-32321712

ABSTRACT

Cortex development is controlled by temporal patterning of neural progenitor (NP) competence with sequential generation of deep and superficial layer neurons, but underlying mechanisms remain elusive. Here, we report a role for heterogeneous nuclear ribonucleoprotein A3 (HNRNPA3) in regulating the division of early cortical NPs that mainly give rise to deep-layer neurons via direct neurogenesis. HNRNPA3 is expressed at high levels in NPs of mouse and human cortex at early stages, with a unique peri-chromosome pattern. Intriguingly, downregulation of HNRNPA3 caused chromosome disarrangement, which hindered normal separation of chromosomes during NP division, leading to mitotic delay. Furthermore, HNRNPA3 is associated with the cohesin-core subunit SMC1A and controls its association with chromosomes, implicating a mechanism for the role of HNRNPA3 in regulating chromosome segregation in dividing NPs. Hnrnpa3-deficient mice exhibited reduced cortical thickness, especially of deep layers. Moreover, downregulation of HNRNPA3 in cultured human cerebral organoids led to marked reduction in NPs and deep-layer neurons. Thus, this study has identified a crucial role for HNRNPA3 in NP division and highlighted the relationship between mitosis progression and early neurogenesis.


Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism , Mitosis/genetics , Neural Stem Cells/metabolism , Neurogenesis/genetics , Animals , Cell Line , Cell Proliferation/genetics , Cerebral Cortex/embryology , Chromosome Segregation/genetics , Female , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/genetics , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Neurons/metabolism , Transfection , Cohesins
4.
Cell Discov ; 6: 9, 2020.
Article in English | MEDLINE | ID: mdl-32140252

ABSTRACT

During the development of mammalian neuromuscular junction (NMJ), the original supernumerary axon inputs are gradually eliminated, finally leaving each muscle fiber innervated by a single axon terminal. However, the molecular cues that mediate the elimination of redundant axon inputs remain unclear. Here we show that tumor necrosis factor-α (TNFα) expressed in postsynaptic muscle cells plays an important role in presynaptic axonal elimination at the NMJ. We found that intramuscular injection of TNFα into the levator auris longus (LAL) muscles caused disassociation of presynaptic nerve terminals from the postsynaptic acetylcholine receptor (AChR) clusters. By contrast, genetic ablation of TNFα globally or specifically in skeletal muscle cells, but not in motoneurons or Schwann cells, delayed the synaptic elimination. Moreover, ablation of TNFα in muscle cells attenuated the tendency of activity-dependent competition in a motoneuron-muscle coculture system. These results suggest a role of postsynaptic TNFα in the elimination of redundant synaptic inputs.

5.
Dev Cell ; 28(6): 670-84, 2014 Mar 31.
Article in English | MEDLINE | ID: mdl-24631402

ABSTRACT

During the development of vertebrate neuromuscular junction (NMJ), agrin stabilizes, whereas acetylcholine (ACh) destabilizes AChR clusters, leading to the refinement of synaptic connections. The intracellular mechanism underlying this counteractive interaction remains elusive. Here, we show that caspase-3, the effector protease involved in apoptosis, mediates elimination of AChR clusters. We found that caspase-3 was activated by cholinergic stimulation of cultured muscle cells without inducing cell apoptosis and that this activation was prevented by agrin. Interestingly, inhibition of caspase-3 attenuated ACh agonist-induced dispersion of AChR clusters. Furthermore, we identified Dishevelled1 (Dvl1), a Wnt signaling protein involved in AChR clustering, as the substrate of caspase-3. Blocking Dvl1 cleavage prevented induced dispersion of AChR clusters. Finally, inhibition or genetic ablation of caspase-3 or expression of a caspase-3-resistant form of Dvl1 caused stabilization of aneural AChR clusters. Thus, caspase-3 plays an important role in the elimination of postsynaptic structures during the development of NMJs.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Caspase 3/metabolism , Muscle, Skeletal/metabolism , Neuromuscular Junction/physiology , Phosphoproteins/metabolism , Synaptic Potentials/physiology , Synaptic Transmission , Acetylcholine/metabolism , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/genetics , Agrin/physiology , Animals , Cells, Cultured , Dishevelled Proteins , Electrophysiology , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Image Processing, Computer-Assisted , Immunoenzyme Techniques , Mice , Mice, Knockout , Motor Neurons/cytology , Motor Neurons/metabolism , Muscle, Skeletal/cytology , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/genetics , RNA, Small Interfering/genetics , Rats , Rats, Sprague-Dawley , Receptors, Cholinergic/metabolism , Signal Transduction
6.
J Neurosci ; 33(24): 9957-62, 2013 Jun 12.
Article in English | MEDLINE | ID: mdl-23761891

ABSTRACT

During development, mammalian neuromuscular junctions (NMJs) transit from multiple-innervation to single-innervation through axonal competition via unknown molecular mechanisms. Previously, using an in vitro model system, we demonstrated that the postsynaptic secretion of pro-brain-derived neurotrophic factor (proBDNF) stabilizes or eliminates presynaptic axon terminals, depending on its proteolytic conversion at synapses. Here, using developing mouse NMJs, we obtained in vivo evidence that proBDNF and mature BDNF (mBDNF) play roles in synapse elimination. We observed that exogenous proBDNF promoted synapse elimination, whereas mBDNF infusion substantially delayed synapse elimination. In addition, pharmacological inhibition of the proteolytic conversion of proBDNF to mBDNF accelerated synapse elimination via activation of p75 neurotrophin receptor (p75(NTR)). Furthermore, the inhibition of both p75(NTR) and sortilin signaling attenuated synapse elimination. We propose a model in which proBDNF and mBDNF serve as potential "punishment" and "reward" signals for inactive and active terminals, respectively, in vivo.


Subject(s)
Brain-Derived Neurotrophic Factor/physiology , Gene Expression Regulation, Developmental/genetics , Neuromuscular Junction/metabolism , Protein Precursors/physiology , Signal Transduction/physiology , Analysis of Variance , Animals , Animals, Newborn , Axons/metabolism , Brain-Derived Neurotrophic Factor/deficiency , Female , Gene Expression Regulation, Developmental/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nerve Tissue Proteins/metabolism , Neuromuscular Junction/drug effects , Neuromuscular Junction/growth & development , Neurons/cytology , Neurons/drug effects , Neurons/metabolism , Presynaptic Terminals/metabolism , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Receptor, trkB/genetics , Receptor, trkB/metabolism , Receptors, Nerve Growth Factor/deficiency , Signal Transduction/drug effects , Spinal Cord/cytology
7.
Dev Cell ; 21(3): 431-44, 2011 Sep 13.
Article in English | MEDLINE | ID: mdl-21856246

ABSTRACT

Directed membrane trafficking is believed to be crucial for axon development during neuronal morphogenesis. However, the underlying mechanisms are poorly understood. Here, we report a role of Lgl1, the mammalian homolog of Drosophila tumor suppressor Lethal giant larvae, in controlling membrane trafficking underlying axonal growth. We find that Lgl1 is associated with plasmalemmal precursor vesicles and enriched in developing axons. Lgl1 upregulation promoted axonal growth, whereas downregulation attenuated it as well as directional membrane insertion. Interestingly, Lgl1 interacted with and activated Rab10, a small GTPase that mediates membrane protein trafficking, by releasing GDP dissociation inhibitor (GDI) from Rab10. Furthermore, Rab10 lies downstream of Lgl1 in axon development and directional membrane insertion. Finally, both Lgl1 and Rab10 are required for neocortical neuronal polarization in vivo. Thus, the Lgl1 regulation of Rab10 stimulates the trafficking of membrane precursor vesicles, whose fusion with the plasmalemma is crucial for axonal growth.


Subject(s)
Axons/metabolism , Proteins/metabolism , rab GTP-Binding Proteins/metabolism , Animals , Cell Line , Cell Membrane/metabolism , Cell Polarity , Cells, Cultured , Down-Regulation , Guanine Nucleotide Dissociation Inhibitors/metabolism , Hippocampus/growth & development , Hippocampus/metabolism , Humans , Protein Transport , Rats , Up-Regulation
8.
J Neurosci ; 30(33): 11104-13, 2010 Aug 18.
Article in English | MEDLINE | ID: mdl-20720118

ABSTRACT

At the vertebrate neuromuscular junction (NMJ), acetylcholine receptor (AChR) clustering is stimulated by motor neuron-derived glycoprotein Agrin and requires a number of intracellular signal or structural proteins, including AChR-associated scaffold protein Rapsyn. Here, we report a role of nuclear factor kappaB (NF-kappaB), a well known transcription factor involved in a variety of immune responses, in regulating AChR clustering at the NMJ. We found that downregulating the expression of RelA/p65 subunit of NF-kappaB or inhibiting NF-kappaB activity by overexpression of mutated form of IkappaB (inhibitor kappaB), which is resistant to proteolytic degradation and thus constitutively keeps NF-kappaB inactive in the cytoplasma, impeded the formation of AChR clusters in cultured C2C12 muscle cells stimulated by Agrin. In contrast, overexpression of RelA/p65 promoted AChR clustering. Furthermore, we investigated the mechanism by which NF-kappaB regulates AChR clustering. Interestingly, we found that downregulating the expression of RelA/p65 caused a marked reduction in the protein and mRNA level of Rapsyn and upregulation of RelA/p65 enhanced Rapsyn promoter activity. Mutation of NF-kappaB binding site on Rapsyn promoter prevented responsiveness to RelA/p65 regulation. Moreover, forced expression of Rapsyn in RelA/p65 downregulated muscle cells partially rescued AChR clusters, suggesting that NF-kappaB regulates AChR clustering, at least partially through the transcriptional regulation of Rapsyn. In line with this notion, genetic ablation of RelA/p65 selectively in the skeletal muscle caused a reduction of AChR density at the NMJ and a decrease in the level of Rapsyn. Thus, NF-kappaB signaling controls AChR clustering through transcriptional regulation of synaptic protein Rapsyn.


Subject(s)
NF-kappa B/metabolism , Neuromuscular Junction/metabolism , Receptors, Cholinergic/metabolism , Agrin/metabolism , Animals , Cell Line , Cytoplasm/metabolism , Gene Expression Regulation , I-kappa B Proteins/genetics , I-kappa B Proteins/metabolism , Mice , Mice, Knockout , Mice, Transgenic , Muscle Proteins/genetics , Muscle Proteins/metabolism , Muscle, Skeletal/growth & development , Muscle, Skeletal/metabolism , Mutation , Myoblasts/metabolism , NF-kappa B/antagonists & inhibitors , NF-kappa B/genetics , Neuromuscular Junction/growth & development , Promoter Regions, Genetic , RNA, Messenger/metabolism , Transcription Factor RelA/genetics , Transcription Factor RelA/metabolism , Transcription, Genetic
9.
Cell Biol Int ; 33(5): 578-85, 2009 May.
Article in English | MEDLINE | ID: mdl-19254772

ABSTRACT

Myosin X (Myo X), an unconventional myosin with a tail homology 4-band 4.1/ezrin/radixin/moesin (MyTH4-FERM) tail, is expressed ubiquitously in various mammalian tissues. In addition to the full-length Myo X (Myo X FL), a headless form is synthesized in the brain. So far, little is known about the function of this motor-less Myo X. In this study, the role of the headless Myo X was investigated in immortalized gonadotropin-releasing hormone (GnRH) neuronal cells, NLT. NLT cells overexpressing the headless Myo X formed fewer focal adhesions and spread more slowly than the wild-type NLT cells and GFP-expressing NLT cells. In chemomigration assays, the NLT cells overexpressing the headless Myo X migrated shorter distances and had fewer migratory cells compared with the control NLT cells.


Subject(s)
Cell Movement/physiology , Gonadotropin-Releasing Hormone/metabolism , Myosins/metabolism , Animals , Cell Adhesion/physiology , Cell Line , Mice , Myosins/genetics , Neurons/cytology , Neurons/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
10.
Proc Natl Acad Sci U S A ; 105(44): 17181-6, 2008 Nov 04.
Article in English | MEDLINE | ID: mdl-18957540

ABSTRACT

Dendrite morphogenesis is regulated by neuronal activity or neurotrophins, which may function by activating intrinsic signaling proteins, including Rho family GTPases. Here we report that activity- and brain-derived neurotrophic factor (BDNF)-dependent dendritic morphogenesis requires activation of geranylgeranyltransferase I (GGT), a prenyltransferase that mediates lipid modification of Rho GTPases. Dendritic arborization in cultured hippocampal neurons was promoted by over-expression of GGT, and reduced by inhibition or down-regulation of GGT. Furthermore, GGT was activated by neuronal depolarization or BDNF, both of which promote dendritic arborization, in cultured hippocampal neurons. Moreover, exploration of a novel environment caused activation of GGT in the mice hippocampus, suggesting that neural activity activates GGT in vivo. Interestingly, GGT was physically associated with tropomyosin-related kinase B (TrkB), the receptor for BDNF, and this association was enhanced by depolarization. Disrupting the GGT-TrkB interaction or down-regulating GGT activity attenuated depolarization- or BDNF-induced dendrite development. Finally, the GGT effect on dendrite arborization was prevented by over-expressing Rac1 with the prenylation site deleted or mutated. Thus depolarization- or BDNF-dependent dendrite development may be mediated by GGT-induced prenylation of Rho GTPases.


Subject(s)
Alkyl and Aryl Transferases/metabolism , Dendrites/enzymology , Dendrites/ultrastructure , Morphogenesis/physiology , Receptor, trkB/metabolism , Alkyl and Aryl Transferases/genetics , Animals , Brain-Derived Neurotrophic Factor/metabolism , Cells, Cultured , Humans , Mice , Mice, Inbred ICR , Neurons/metabolism , Prenylation , Rats , Rats, Sprague-Dawley , Transfection , rac1 GTP-Binding Protein/metabolism
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