Cholinergic signaling plays an important role in regulating the growth and regeneration of axons in the nervous system. The α7 nicotinic receptor (α7) can drive synaptic development and plasticity in the hippocampus. Here, we show that activation of α7 significantly reduces axon growth in hippocampal neurons by coupling to G protein‐regulated inducer of neurite outgrowth 1 (Gprin1), which targets it to the growth cone. Knockdown of Gprin1 expression using RNAi is found sufficient to abolish the localization and calcium signaling of α7 at the growth cone. In addition, an α7/Gprin1 interaction appears intimately linked to a Gαo, growth‐associated protein 43, and CDC42 cytoskeletal regulatory pathway within the developing axon. These findings demonstrate that α7 regulates axon growth in hippocampal neurons, thereby likely contributing to synaptic formation in the developing brain.
For our nervous system to function properly, each neuron must generate a single axon and elongate the axon to reach its target. It is known that actin filaments and their dynamic interaction with microtubules within growth cones play important roles in inducing axon extension. However, it remains unclear how cytoskeletal dynamics is controlled in growth cones. In this study, we report that Rufy3, a RUN domain‐containing protein, is a neuron‐specific and actin filament‐relevant protein. We find that the appropriate expression of Rufy3 in mouse hippocampal neurons is required for the development of a single axon and axon growth. Our results show that Rufy3 specifically interacts with actin filament‐binding proteins, such as Fascin, and colocalizes with Fascin in growth cones. Knockdown of Rufy3 impairs the distribution of Fascin and actin filaments, accompanied by an increased proportion of neurons with multiple axons and a decrease in the axon length. Therefore, Rufy3 may be particularly important for neuronal axon elongation by interacting with Fascin to control actin filament organization in axonal growth cones.
This review focuses on recent advances in the understanding of the organization and roles of actin filaments, and associated myosin motor proteins, in regulating the structure and function of the axon shaft. ‘Patches’ of actin filaments have emerged as a major type of actin filament organization in axons. In the distal axon, patches function as precursors to the formation of filopodia and branches. At the axon initial segment, patches locally capture membranous organelles and contribute to polarized trafficking. The trapping function of patches at the initial segment can be ascribed to interactions with myosin motors, and likely also applies to patches in the more distal axon. Finally, submembranous rings of actin filaments were recently described in axons, which form an actin‐spectrin cytoskeleton, likely contributing to the maintenance of axon integrity. Continued investigation into the roles of axonal actin filaments and myosins will shed light on fundamental aspects of the development, adult function and the repair of axons in the nervous system.
Cu/Zn‐superoxide dismutase is misfolded in familial and sporadic amyotrophic lateral sclerosis, but it is not clear how this triggers endoplasmic reticulum (ER) stress or other pathogenic processes. Here, we demonstrate that mutant SOD1 (mSOD1) is predominantly found in the cytoplasm in neuronal cells. Furthermore, we show that mSOD1 inhibits secretory protein transport from the ER to Golgi apparatus. ER‐Golgi transport is linked to ER stress, Golgi fragmentation and axonal transport and we also show that inhibition of ER‐Golgi trafficking preceded ER stress, Golgi fragmentation, protein aggregation and apoptosis in cells expressing mSOD1. Restoration of ER‐Golgi transport by over‐expression of coatomer coat protein II subunit Sar1 protected against inclusion formation and apoptosis, thus linking dysfunction in ER‐Golgi transport to cellular pathology. These findings thus link several cellular events in amyotrophic lateral sclerosis into a single mechanism occurring early in mSOD1 expressing cells.
Localized translation of axonal mRNAs contributes to developmental and regenerative axon growth. Although untranslated regions (UTRs) of many different axonal mRNAs appear to drive their localization, there has been no consensus RNA structure responsible for this localization. We recently showed that limited expression of ZBP1 protein restricts axonal localization of both β‐actin and GAP‐43 mRNAs. β‐actin 3′UTR has a defined element for interaction with ZBP1, but GAP‐43 mRNA shows no homology to this RNA sequence. Here, we show that an AU‐rich regulatory element (ARE) in GAP‐43′s 3′UTR is necessary and sufficient for its axonal localization. Axonal GAP‐43 mRNA levels increase after in vivo injury, and GAP‐43 mRNA shows an increased half‐life in regenerating axons. GAP‐43 mRNA interacts with both HuD and ZBP1, and HuD and ZBP1 co‐immunoprecipitate in an RNA‐dependent fashion. Reporter mRNA with the GAP‐43 ARE competes with endogenous β‐actin mRNA for axonal localization and decreases axon length and branching similar to the β‐actin 3′UTR competing with endogenous GAP‐43 mRNA. Conversely, over‐expressing GAP‐43 coding sequence with its 3′UTR ARE increases axonal elongation and this effect is lost when just the ARE is deleted from GAP‐43′s 3′UTR.
A lesion to the rat rubrospinal tract is a model for traumatic spinal cord lesions and results in atrophy of the red nucleus neurons, axonal dieback, and locomotor deficits. In this study, we used adeno‐associated virus (AAV)‐mediated over‐expression of BAG1 and ROCK2‐shRNA in the red nucleus to trace [by co‐expression of enhanced green fluorescent protein (EGFP)] and treat the rubrospinal tract after unilateral dorsal hemisection. We investigated the effects of targeted gene therapy on neuronal survival, axonal sprouting of the rubrospinal tract, and motor recovery 12 weeks after unilateral dorsal hemisection at Th8 in rats. In addition to the evaluation of BAG1 and ROCK2 as therapeutic targets in spinal cord injury, we aimed to demonstrate the feasibility and the limits of an AAV‐mediated protein over‐expression versus AAV.shRNA‐mediated down‐regulation in this traumatic CNS lesion model. Our results demonstrate that BAG1 and ROCK2‐shRNA both promote neuronal survival of red nucleus neurons and enhance axonal sprouting proximal to the lesion.
Astrocytes are a target for regenerative neurobiology because in brain injury their phenotype arbitrates brain integrity, neuronal death and subsequent repair and reconstruction. We explored the ability of 3D scaffolds to direct astrocytes into phenotypes with the potential to support neuronal survival. Poly‐ε‐caprolactone scaffolds were electrospun with random and aligned fibre orientations on which murine astrocytes were sub‐cultured and analysed at 4 and 12 DIV. Astrocytes survived, proliferated and migrated into scaffolds adopting 3D morphologies, mimicking in vivo stellated phenotypes. Cells on random poly‐ε‐caprolactone scaffolds grew as circular colonies extending processes deep within sub‐micron fibres, whereas astrocytes on aligned scaffolds exhibited rectangular colonies with processes following not only the direction of fibre alignment but also penetrating the scaffold. Cell viability was maintained over 12 DIV, and cytochemistry for F‐/G‐actin showed fewer stress fibres on bioscaffolds relative to 2D astrocytes. Reduced cytoskeletal stress was confirmed by the decreased expression of glial fibrillary acidic protein. PCR demonstrated up‐regulation of genes (excitatory amino acid transporter 2, brain‐derived neurotrophic factor and anti‐oxidant) reflecting healthy biologies of mature astrocytes in our extended culture protocol. This study illustrates the therapeutic potential of bioengineering strategies using 3D electrospun scaffolds which direct astrocytes into phenotypes supporting brain repair.
The orphan nuclear receptor estrogen‐related receptor gamma (ERRγ) is highly expressed in the nervous system during embryogenesis and in adult brains, but its physiological role in neuronal development remains unknown. In this study, we evaluated the relevance of ERRγ in regulating dopaminergic (DAergic) phenotype and the corresponding signaling pathway. We used retinoic acid (RA) to differentiate human neuroblastoma SH‐SY5Y cells. RA induced neurite outgrowth of SH‐SY5Y cells with an increase in DAergic neuron‐like properties, including up‐regulation of tyrosine hydroxylase, dopamine transporter, and vesicular monoamine transporter 2. ERRγ, but not ERRα, was up‐regulated by RA, and participated in RA effect on SH‐SY5Y cells. ERRγ over‐expression enhanced mature DAergic neuronal phenotype with neurite outgrowth as with RA treatment; and RA‐induced increase in DAergic phenotype was attenuated by silencing ERRγ expression. ERRγ appears to have a crucial role in morphological and functional regulation of cells that is selective for DAergic neurons. Polo‐like kinase 2 was up‐regulated in ERRγ‐over‐expressing SH‐SY5Y cells, which was involved in phosphorylation of glycogen synthase kinase 3β and resulting downstream activation of nuclear factor of activated T cells. The likely involvement of ERRγ in regulating the DAergic neuronal phenotype makes this orphan nuclear receptor a novel target for understanding DAergic neuronal differentiation.
A set of specific precursor microRNAs (pre‐miRNAs) are reported to localize into neuronal dendrites, where they could be processed locally to control synaptic protein synthesis and plasticity. However, it is not clear whether specific pre‐miRNAs are also transported into distal axons to autonomously regulate intra‐axonal protein synthesis. Here, we show that a subset of pre‐miRNAs, whose mature miRNAs are enriched in axonal compartment of sympathetic neurons, are present in axons of neurons both in vivo and in vitro by quantitative PCR and by in situ hybridization. Some pre‐miRNAs (let 7c‐a and pre‐miRs‐16, 23a, 25, 125b‐1, 433, and 541) showed elevated axonal levels, while others (pre‐miRs‐138‐2, 185, and 221) were decreased in axonal levels following injury. Dicer and KSRP proteins are also present in distal axons, but Drosha is found restricted to the cell body. These findings suggest that specific pre‐miRNAs are selected for localization into distal axons of sensory neurons and are presumably processed to mature miRNAs in response to extracellular stimuli. This study supports the notion that local miRNA biogenesis effectively provides another level of temporal control for local protein synthesis in axons.
The principal motor tract involved in mammalian locomotor activities is known as the corticospinal tract (CST), which starts in the brain motor cortex (upper motor neuron), extends its axons across the brain to brainstem and finally reaches different regions of spinal cord, contacting the lower motor neurons. Visualization of the CST is essential to carry out studies in different kinds of pathologies such as spinal cord injury or multiple sclerosis. At present, most studies of axon structure and/or integrity that involve histological tissue sectioning present the problem of finding the region where the CST is predominant. To solve this problem, one could use a novel technique to make the tissues transparent and observe them directly without histological sectioning. However, the disadvantage of this procedure is the need of costly and non‐conventional equipment, such as two‐photon fluorescence microscopy or ultramicroscopy to perform the image acquisition. Here, we show that labeling the CST with FluoroRuby in the motor cortex and then performing the clearing technique, the z‐acquisition of the entire CST in unsectioned tissue followed by three‐dimensional reconstruction can be carried out by standard one‐photon confocal microscopy, with yields similar to those obtained by two‐photon microscopy. In addition, we present an example of the application of this method in a spinal cord injury model, where the disruption of CST is shown at the lesion site.
Hydrogen sulfide (H2S) functions as a physiological gas transmitter in both normal and pathophysiological cellular events. H2S is produced from substances by three enzymes: cystathionine β‐synthase (CBS), cystathionine γ‐lyase (CSE), and 3‐mercaptopyruvate sulfurtransferase (MST). In human tissues, these enzymes are involved in tissue‐specific biochemical pathways for H2S production. For example, CBS and cysteine aminotransferase/MST are present in the brain, but CSE is not. Thus, we examined the expression of H2S production‐related enzymes in peripheral nerves. Here, we found that CSE and MST/cysteine aminotransferase, but not CBS, were present in normal peripheral nerves. In addition, injured sciatic nerves in vivo up‐regulated CSE in Schwann cells during Wallerian degeneration (WD); however, CSE was not up‐regulated in peripheral axons. Using an ex vivo sciatic nerve explant culture, we found that the inhibition of H2S production broadly prevented the process of nerve degeneration, including myelin fragmentation, axonal degradation, Schwann cell dedifferentiation, and Schwann cell proliferation in vitro and in vivo. Thus, these results indicate that H2S signaling is essential for Schwann cell responses to peripheral nerve injury.
The STriatal‐Enriched protein tyrosine Phosphatase 61 (STEP61) inhibits the activity of the tyrosine kinase Fyn and dephosphorylates the GluN2B subunit of the NMDA receptor, whereas the protein kinase A phosphorylation of STEP61 inhibits the activity of the phosphatase (Pharmacol. Rev., 64, 2012 , p. 65). Previously, we found that ethanol activates Fyn in the dorsomedial striatum (DMS) leading to GluN2B phosphorylation, which, in turn, underlies the development of ethanol intake (J. Neurosci., 30, 2010 , p. 10187). Here, we tested the hypothesis that inhibition of STEP61 by ethanol is upstream of Fyn/GluN2B. We show that exposure of mice to ethanol increased STEP61 phosphorylation in the DMS, which was maintained after withdrawal and was not observed in other striatal regions. Specific knockdown of STEP61 in the DMS of mice enhanced ethanol‐mediated Fyn activation and GluN2B phosphorylation, and increased ethanol intake without altering the level of water, saccharine, quinine consumption or spontaneous locomotor activity. Together, our data suggest that blockade of STEP61 activity in response to ethanol is sufficient for the activation of the Fyn/GluN2B pathway in the DMS. Being upstream of Fyn and GluN2B, inactive STEP61 in the DMS primes the induction of ethanol intake.
Huntington's disease (HD) is one of many neurodegenerative diseases with reported alterations in brain iron homeostasis that may contribute to neuropathogenesis. Iron accumulation in the specific brain areas of neurodegeneration in HD has been proposed based on observations in post‐mortem tissue and magnetic resonance imaging studies. Altered magnetic resonance imaging signal within specific brain regions undergoing neurodegeneration has been consistently reported and interpreted as altered levels of brain iron. Biochemical studies using various techniques to measure iron species in human samples, mouse tissue, or in vitro has generated equivocal data to support such an association. Whether elevated brain iron occurs in HD, plays a significant contributing role in HD pathogenesis, or is a secondary effect remains currently unclear.
The subcellular compartmentalization of kinase activity allows for regulation of distinct cellular processes involved in cell differentiation or survival. The PTEN‐induced kinase 1 (PINK1), which is linked to Parkinson's disease, is a neuroprotective kinase localized to cytosolic and mitochondrial compartments. While mitochondrial targeting of PINK1 is important for its activities regulating mitochondrial homeostasis, the physiological role of the cytosolic pool of PINK1 remains unknown. Here, we demonstrate a novel role for cytosolic PINK1 in neuronal differentiation/neurite maintenance. Over‐expression of wild‐type PINK1, but not a catalytically inactive form of PINK1(K219M), promoted neurite outgrowth in SH‐SY5Y cells and increased dendritic lengths in primary cortical and midbrain dopaminergic neurons. To identify the subcellular pools of PINK1 involved in promoting neurite outgrowth, we transiently transfected cells with PINK1 constructs designed to target PINK1 to the outer mitochondrial membrane (OMM‐PINK1) or restrict PINK1 to the cytosol (ΔN111‐PINK1). Both constructs blocked cell death associated with loss of endogenous PINK1. However, transient expression of ΔN111‐PINK1, but not of OMM‐PINK1 or ΔN111‐PINK1(K219M), promoted dendrite outgrowth in primary neurons, and rescued the decreased dendritic arborization of PINK1‐deficient neurons. Mechanistically, the cytosolic pool of PINK1 regulated neurite morphology through enhanced anterograde transport of dendritic mitochondria and amplification of protein kinase A‐related signaling pathways. Our data support a novel role for PINK1 in regulating dendritic morphogenesis.
It has been suggested that propofol can modulate microglial activity and hence may have potential roles against neuroinflammation following brain ischemic insult. However, whether and how propofol can inhibit post‐cardiac arrest brain injury via inhibition of microglia activation remains unclear. A rat model of asphyxia cardiac arrest (CA) was created followed by cardiopulmonary resuscitation. CA induced marked microglial activation in the hippocampal CA1 region, revealed by increased OX42 and P2 class of purinoceptor 7 (P2X7R) expression, as well as p38 MAPK phosphorylation. Morris water maze showed that learning and memory deficits following CA could be inhibited or alleviated by pre‐treatment with the microglial inhibitor minocycline or propofol. Microglial activation was significantly suppressed likely via the P2X7R/p‐p38 pathway by propofol. Moreover, hippocampal neuronal injuries after CA were remarkably attenuated by propofol. In vitro experiment showed that propofol pre‐treatment inhibited ATP‐induced microglial activation and release of tumor necrosis factor‐α and interleukin‐1β. In addition, propofol protected neurons from injury when co‐culturing with ATP‐treated microglia. Our data suggest that propofol pre‐treatment inhibits CA‐induced microglial activation and neuronal injury in the hippocampus and ultimately improves cognitive function.
The membrane trafficking and actin cytoskeleton remodeling mediated by ADP ribosylation factor 6 (Arf6) are functionally linked to various neuronal processes including neurite formation and maintenance, neurotransmitter release, and receptor internalization. EFA6A is an Arf6‐specific guanine nucleotide exchange factor that is abundantly expressed in the brain. In this study, we identified sorting nexin‐1 (SNX1), a retromer component that is implicated in endosomal sorting and trafficking, as a novel interacting partner for EFA6A by yeast two‐hybrid screening. The interaction was mediated by the C‐terminal region of EFA6A and a BAR domain of SNX1, and further confirmed by pull‐down assay and immunoprecipitation from mouse brain lysates. In situ hybridization analysis demonstrated the widespread expression of SNX1 in the mouse brain, which overlapped with the expression of EFA6A in the forebrain. Immunofluorescent analysis revealed the partial colocalization of EFA6A and SNX1 in the dendritic fields of the hippocampus. Immunoelectron microscopic analysis revealed the overlapping subcellular localization of EFA6A and SNX1 at the post‐synaptic density and endosomes in dendritic spines. In Neuro‐2a neuroblastoma cells, expression of either EFA6A or SNX1 induced neurite outgrowth, which was further enhanced by co‐expression of EFA6A and SNX1. The present findings suggest a novel mechanism by which EFA6A regulates Arf6‐mediated neurite formation through the interaction with SNX1.
Activity‐dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) endocytosis mode in central nerve terminals during intense neuronal activity. By definition this mode is triggered by neuronal activity; however, key questions regarding its mechanism of activation remain unaddressed. To determine the basic requirements for ADBE triggering in central nerve terminals, we decoupled SV fusion events from activity‐dependent calcium influx using either clostridial neurotoxins or buffering of intracellular calcium. ADBE was monitored both optically and morphologically by observing uptake of the fluid phase markers tetramethylrhodamine‐dextran and horse radish peroxidase respectively. Ablation of SV fusion with tetanus toxin resulted in the arrest of ADBE, but had no effect on other calcium‐dependent events such as activity‐dependent dynamin I dephosphorylation, indicating that SV exocytosis is necessary for triggering. Furthermore, the calcium chelator EGTA abolished ADBE while leaving SV exocytosis intact, demonstrating that ADBE is triggered by intracellular free calcium increases outside the active zone. Activity‐dependent dynamin I dephosphorylation was also arrested in EGTA‐treated neurons, consistent with its proposed role in triggering ADBE. Thus, SV fusion and increased cytoplasmic free calcium are both necessary but not sufficient individually to trigger ADBE.
Alzheimer's disease (AD) is a neurodegenerative disorder that represents the most common type of dementia among elderly people. Amyloid beta (Aβ) peptides in extracellular Aβ plaques, produced from the amyloid precursor protein (APP) via sequential processing by β‐ and γ‐secretases, impair hippocampal synaptic plasticity, and cause cognitive dysfunction in AD patients. Here, we report that Aβ peptides also impair another form of synaptic plasticity; cerebellar long‐term depression (LTD). In the cerebellum of commonly used AD mouse model, APPswe/PS1dE9 mice, Aβ plaques were detected from 8 months and profound accumulation of Aβ plaques was observed at 18 months of age. Biochemical analysis revealed relatively high levels of APP protein and Aβ in the cerebellum of APPswe/PS1dE9 mice. At pre‐Aβ accumulation stage, LTD induction, and motor coordination are disturbed. These results indicate that soluble Aβ oligomers disturb LTD induction and cerebellar function in AD mouse model.
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