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 GTPases, 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.
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.
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.
NADPH oxidases are important for neuronal function but detailed subcellular localization studies have not been performed. Here, we provide the first evidence for the presence of functional NADPH oxidase 2 (NOX2)‐type complex in neuronal growth cones and its bidirectional relationship with the actin cytoskeleton. NADPH oxidase inhibition resulted in reduced F‐actin content, retrograde F‐actin flow, and neurite outgrowth. Stimulation of NADPH oxidase via protein kinase C activation increased levels of hydrogen peroxide in the growth cone periphery. The main enzymatic NADPH oxidase subunit NOX2/gp91phox localized to the growth cone plasma membrane and showed little overlap with the regulatory subunit p40phox. p40phox itself exhibited colocalization with filopodial actin bundles. Differential subcellular fractionation revealed preferential association of NOX2/gp91phox and p40phox with the membrane and the cytoskeletal fraction, respectively. When neurite growth was evoked with beads coated with the cell adhesion molecule apCAM, we observed a significant increase in colocalization of p40phox with NOX2/gp91phox at apCAM adhesion sites. Together, these findings suggest a bidirectional functional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones, which contributes to the control of neurite outgrowth.
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.
Restoration of correct neural activity following central nervous system (CNS) damage requires the replacement of degenerated axons with newly outgrowing, functional axons. Unfortunately, spontaneous regeneration is largely lacking in the adult mammalian CNS. In order to establish successful regenerative therapies, an improved understanding of axonal outgrowth and the various molecules influencing it, is highly needed. Matrix metalloproteinases (MMPs) constitute a family of zinc‐dependent proteases that were sporadically reported to influence axon outgrowth. Using an ex vivo retinal explant model, we were able to show that broad‐spectrum MMP inhibition reduces axon outgrowth of mouse retinal ganglion cells (RGCs), implicating MMPs as beneficial factors in axonal regeneration. Additional studies, using more specific MMP inhibitors and MMP‐deficient mice, disclosed that both MMP‐2 and MT1‐MMP, but not MMP‐9, are involved in this process. Furthermore, administration of a novel antibody to MT1‐MMP that selectively blocks pro‐MMP‐2 activation revealed a functional co‐involvement of these proteinases in determining RGC axon outgrowth. Subsequent immunostainings showed expression of both MMP‐2 and MT1‐MMP in RGC axons and glial cells. Finally, results from combined inhibition of MMP‐2 and β1‐integrin were suggestive for a functional interaction between these molecules. Overall, our data indicate MMP‐2 and MT1‐MMP as promising axonal outgrowth‐promoting molecules.
Our previous work has suggested that traumatic noise activates Rho‐GTPase pathways in cochlear outer hair cells (OHCs), resulting in cell death and noise‐induced hearing loss (NIHL). In this study, we investigated Rho effectors, Rho‐associated kinases (ROCKs), and the targets of ROCKs, the ezrin‐radixin‐moesin (ERM) proteins, in the regulation of the cochlear actin cytoskeleton using adult CBA/J mice under conditions of noise‐induced temporary threshold shift (TTS) and permanent threshold shift (PTS) hearing loss, which result in changes to the F/G‐actin ratio. The levels of cochlear ROCK2 and p‐ERM decreased 1 h after either TTS‐ or PTS‐noise exposure. In contrast, ROCK2 and p‐ERM in OHCs decreased only after PTS‐, not after TTS‐noise exposure. Treatment with lysophosphatidic acid, an activator of the Rho pathway, resulted in significant reversal of the F/G‐actin ratio changes caused by noise exposure and attenuated OHC death and NIHL. Conversely, the down‐regulation of ROCK2 by pretreatment with ROCK2 siRNA reduced the expression of ROCK2 and p‐ERM in OHCs, exacerbated TTS to PTS, and worsened OHC loss. Additionally, pretreatment with siRNA against radixin, an ERM protein, aggravated TTS to PTS. Our results indicate that a ROCK2‐mediated ERM‐phosphorylation signaling cascade modulates noise‐induced hair cell loss and NIHL by targeting the cytoskeleton.
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.
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.
HIV‐1 infects the brain and, despite antiretroviral therapy, many infected individuals suffer from HIV‐1‐associated neurocognitive disorders (HAND). HAND is associated with dendritic simplification and synaptic loss. Prevention of synaptodendritic damage may ameliorate or forestall neurocognitive decline in latent HIV‐1 infections. The HIV‐1 transactivating protein (Tat) is produced during viral latency in the brain and may cause synaptodendritic damage. This study examined the integrity of the dendritic network after exposure to HIV‐1 Tat by labeling filamentous actin (F‐actin)‐rich structures (puncta) in primary neuronal cultures. After 24 h of treatment, HIV‐1 Tat was associated with the dendritic arbor and produced a significant reduction of F‐actin‐labeled dendritic puncta as well as loss of dendrites. Pre‐treatment with either of two plant‐derived phytoestrogen compounds (daidzein and liquiritigenin), significantly reduced synaptodendritic damage following HIV‐1 Tat treatment. In addition, 6 days after HIV‐1 Tat treatment, treatment with either daidzein, or liquiritigenin enhanced recovery, via the estrogen receptor, from HIV‐1 Tat‐induced synaptodendritic damage. These results suggest that either liquiritigenin or daidzein may not only attenuate acute synaptodendritic injury in HIV‐1 but may also promote recovery from synaptodendritic damage.
Girdin, an actin‐binding protein, possesses versatile functions in a multitude of cellular processes. Although several studies have shown that Girdin is involved in the cell DNA synthesis, actin cytoskeleton rearrangement, and cell motility, the molecular mechanisms of Girdin in tumor development and progression remain elusive. In this study, through over‐expression and siRNA experiments, we found that Girdin increased migration of LN229 human glioblastoma cells. On the other hand, reducing Girdin impaired F‐actin polymerization, which is essential for cell morphogenesis and motility. Matrix metalloproteinase 2, critical in human glioma migration and invasion, was down‐regulated upon Girdin reduction and led to decreased invasion in vitro and in vivo. In addition, silencing Girdin expression impaired the phosphorylation of two important adhesion molecules, integrin β1 and focal adhesion kinase, resulting in cell adhesion defects. Our immunohistochemical study on human gliomas tissue sections indicated that Girdin expression was positively related with glioma malignancy, supporting the in vitro and in vivo results from cell lines. Collectively, our findings suggest a critical role for Girdin in glioma infiltration.
The 19‐transmembrane, multisubunit γ‐secretase complex generates the amyloid β‐peptide (Aβ) of Alzheimer's disease (AD) by an unusual intramembrane proteolysis of the β‐amyloid precursor protein. The complex, which similarly processes many other type 1 transmembrane substrates, is composed of presenilin, Aph1, nicastrin, and presenilin enhancer (Pen‐2), all of which are necessary for proper complex maturation and enzymatic activity. Obtaining a high‐resolution atomic structure of the intact complex would greatly aid the rational design of compounds to modulate activity but is a very difficult task. A complementary method is to generate structures for each individual subunit to allow one to build a model of the entire complex. Here, we describe a method by which recombinant human Pen‐2 can be purified from bacteria to > 95% purity at milligram quantities per liter, utilizing a maltose binding protein tag to both increase solubility and facilitate purification. Expressing the same construct in mammalian cells, we show that the large N‐terminal maltose binding protein tag on Pen‐2 still permits incorporation into the complex and subsequent presenilin‐1 endoproteolysis, nicastrin glycosylation and proteolytic activity. These new methods provide valuable tools to study the structure and function of Pen‐2 and the γ‐secretase complex.
Vaccination therapies constitute potential treatment options in neurodegenerative disorders such as Alzheimer disease or Parkinson disease. While a lot of research has been performed on vaccination against extracellular amyloid β, the focus recently shifted toward vaccination against the intracellular proteins tau and α‐synuclein, with promising results in terms of protein accumulation reduction. In this review, we briefly summarize lessons to be learned from clinical vaccination trials in Alzheimer disease that target amyloid β. We then focus on tau and α‐synuclein. For both proteins, we provide important data on protein immunogenicity, and put them into context with data available from both animals and human vaccination trials targeted at tau and α‐synuclein. Together, we give a comprehensive overview about current clinical data, and discuss associated problems.
Microglia are the resident macrophages of the central nervous system that survey the microenvironment for signals of injury or infection. The response to such signals induces an inflammatory response involving macrophages derived from both resident microglia and recruited circulating monocytes. Although implicated as contributors to autoimmune‐mediated injury, microglia/ macrophages have recently been shown to be critical for the important central nervous system regenerative process of remyelination. This functional dichotomy may reflect their ability to be polarized along a continuum of activation states including the well‐characterized cytotoxic M1 and regenerative M2 phenotypes. Here, we review the roles of microglia, monocytes and the macrophages which they give rise to in creating lesion environments favourable to remyelination, highlighting the specific roles of M1 and M2 phenotypes and how the pro‐regenerative role of the innate immune system is altered by ageing.
The administration of pan histone deacetylase (HDAC) inhibitors reduces ischemic damage to the CNS, both in vitro and in animal models of stroke, via mechanisms which we are beginning to understand. The acetylation of p53 is regulated by Class I HDACs and, because p53 appears to play a role in ischemic pathology, the purpose of this study was to discover, using an in vitro white matter ischemia model and an in vivo cerebral ischemia model, if neuroprotection mediated by HDAC inhibition depended on p53 expression. Optic nerves were excised from wild‐type and p53‐deficient mice, and then subjected to oxygen–glucose deprivation in the presence and absence of a specific inhibitor of Class I HDACs (MS‐275, entinostat) while compound action potentials were recorded. Furthermore, transient focal ischemia was imposed on wild‐type and p53‐deficient mice, which were subsequently treated with MS‐275. Interestingly, and in both scenarios, the beneficial effects of MS‐275 were most pronounced when p53 was absent. These results suggest that modulation of p53 activity is not responsible for MS‐275‐mediated neuroprotection, and further illustrate how HDAC inhibitors variably influence p53 and associated apoptotic pathways.
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.
