Phosphorylation of MAPs is one of the key factors that determines how they bind to microtubules (Brugg and Matus, 1991) and it has been proposed that abnormal phosphorylation could be potentially a driver of neurodegenerative disease. In this regard, hyperphosphorylation of tau and CRMP2 have been reported as an early event in AD (Cole et al., 2007; Mondragón-Rodríguez et al., 2014; Rissman et al., 2004). Phosphorylation state is controlled by balance of kinase action to add phosphate groups and phosphatase action to remove phosphate groups. While there are a number of kinases implicated in neurodegenerative disease, which act specifically on the different MAPs, phosphatase activity is primarily performed by protein phosphatase PP2A, which removes phosphate groups from MAPs (Li et al., 2002; Van Kanegan and Strack, 2009). Protein phosphatase 2A specifically binds to tau and MAP2, which are both associated with microtubule stability (Sontag et al., 2012). This makes activating PP2A an attractive target for investigating MAP phosphorylation using drugs such as sodium selenate, which activates PP2A and removes phosphate groups (Corcoran et al., 2010). Pharmacological manipulation using sodium selenate has been shown to alter microtubule assembly in both in vitro and in vivo models (Shi et al., 2013). Therefore, determining whether MAPs are altered following an excitotoxic insult and whether altering their phosphorylation affects the degeneration process may lead to mechanistic insight in the role of microtubules in excitotoxin-induced axon degeneration.
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However, the Fas receptor is expressed on multiple ret- inal cell types, including astrocytes, RGCs, Mueller cells, microglia, and retinal pigment epithelial cells [7, 31, 36, 73]. Therefore, additional studies in which the Fas receptor is deleted from specific cell types will be necessary to deter- mine which Fas receptor-positive cell(s) actually drive(s) the development of neuroinflammation in glaucoma. More- over, Fas mediates both apoptotic and inflammatory path- ways and it is not possible from the current studies to determine the extent to which Fas-mediated apoptosis and/ or Fas-mediated inflammation contributes to axon degener- ation and death of RGCs in glaucoma. Yet, previous thera- peutic approaches that specifically targeted the apoptotic pathway alone resulted in neuroprotection of the RGC soma but failed to prevent axon degeneration [11, 12], sug- gesting the robust neuroprotective effect afforded by ONL1204 is dependent upon the ability of ONL1204 to antagonize both Fas-mediated apoptosis of RGCs and Fas- mediated activation of retinal microglia and induction of neuroinflammation. Additional studies in which the Fas re- ceptor is specifically knocked out in RGCs or glial cells (microglia, astrocytes, and Mueller cells) are necessary to determine if the neuroprotective effects of ONL1204 are mainly driven by modulating the inflammatory response of retinal glial cells or preventing FasL-induced apoptosis of RGCs.
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The link between mitochondria and the Wallerian pathway is par- ticularly intriguing. Mitochondrial dysfunction is a common theme in a wide group of neurodegenerative disorders in which axon degeneration is central, including Parkinson's disease (PD), Charcot-Marie-Tooth disease, hereditary spastic paraplegia and Friedrich's ataxia (Court and Coleman, 2012). We and others have previously shown that mi- tochondria contribute to the later stages of Wallerian degeneration, where the axotomy itself activates the Wallerian pathway (Barrientos et al., 2011; Loreto et al., 2015). However, mitochondrial depolarisa- tion, caused by the mitochondrial uncoupler Carbonyl cyanide m- chlorophenyl hydrazone (CCCP), also leads to degeneration of unin- jured axons (Loreto et al., 2015), which is rescued by Sarm1 deletion (Summers et al., 2014). Additional studies, both in vitro and in vivo, link the Wallerian pathway to mitochondrial impairment. Wld S mice are
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RPM-1 function including: the microtubule binding pro- tein RNA Export 1 (RAE-1) , the PPM/PP2C family phosphatase PPM-2 , and the Nesprin ANC-1  (Fig. 1a). Importantly, Rae1 was simultaneously isolated in a proteomic screen for Highwire binding proteins . In yeast, RAE-1 regulates nuclear export of RNA via binding to the nucleoporin Nup98 [74, 75]. In metazoans, RAE-1 regulates microtubule stability and spindle assembly in mitotic cells, and has a more limited or no role in RNA export [76–80]. A single domain in RPM-1 and Phr1 is sufficient to mediate binding to RAE-1, and this inter- action requires a small conserved motif in the PHR pro- teins . Mutation of this motif reduces binding of RAE- 1 to RPM-1 in vivo in neurons. Genetic findings in flies and worms show that rae-1 functions in the same pathway as highwire and rpm-1 to regulate synapse formation and axon termination [72, 73]. This is consistent with RAE-1 colocalizing with RPM-1 at the presynaptic terminal. Des- pite these similar genetic and biochemical findings, differ- ences exist in the relationship between PHR proteins and RAE-1. In the mechanosensory neurons of worms, RAE-1 regulates axon termination by functioning downstream of RPM-1 . In contrast, Rae1 protects Drosophila High- wire from degradation by autophagy suggesting Rae1 can function upstream of Highwire . These differences might be explained by autophagy mediating feedback between Rae1 and the PHR proteins. Alternatively, the relationship between PHR proteins and Rae1 might differ with neuronal context. Nonetheless, a combination of biochemical and genetic results across species indicates that RAE-1 is a conserved mediator of PHR protein function.
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Vertebrate peripheral nerves can regenerate, enabling severed axons to reconnect with their original synaptic targets. The interactions between injured nerves with cells in their environment, as well as the functional significance of these interactions, have not been determined in vivo and in real time. Here we provide the first minute-by-minute account of cellular interactions between laser transected motor nerves, macrophages, and Schwann cells in live intact zebrafish using transgenic lines that label each cell type in vivo. We find that axon fragmentation triggers macrophage invasion into the nerve to engulf axonal debris, and that delaying nerve fragmentation in a Wlds model does not alter macrophage recruitment but induces a previously unknown `nerve scanning' behavior, suggesting that macrophage recruitment and subsequent nerve invasion are controlled by separate mechanisms, both independent of Schwann cells. A major challenge for regenerating peripheral axons is to identify their original trajectory; Schwann cells are known to provide regenerating axons with factors that stimulate outgrowth, and an adhesive substrate that axons preferentially extend along during regeneration, yet their role in guiding regenerating axons onto their original trajectory is less clear. We show in mutants lacking Schwann cells that axonal growth cones sprout and extend at rates comparable to wild type, but fail to identify their original path, and instead extend along aberrant trajectories. To determine whether Schwann cells function primarily as an adhesive substrate we tested whether a Schwann cell-less axonal scaffold is sufficient to direct axonal growth. These substrates failed to compensate for the absence of Schwann cells, providing evidence that Schwann cells direct regenerating axons towards their original trajectory. To identify signals that guide regenerating motor axons in vivo, we examined mutants lacking the guidance receptor DCC. We find that in DCC mutants a significant fraction of regenerating axons extend along aberrant trajectories. Collectively, this work details the dynamic activities of macrophages and Schwann cells during axon degeneration and early regeneration, while axons are regrowing and selecting their trajectory, and we propose that Schwann cells and DCC mediated guidance are critical early during regeneration, enabling growth cones to navigate towards their original axonal trajectories.
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critical for axonal function (Funfschilling et al., 2012). Perturbations to the oligodendrocyte-axon lactate shunt via downregulation of the monocarboxylate transporter 1 (MCT1) in mice results in axon degeneration and neuronal loss (Lee et al., 2012). Furthermore, MCT1 is reduced in the motor cortex of ALS patients (Lee et al., 2012). Interestingly a similar lactate-dependent mechanism may be mediated via astrocytes (Cassina et al., 2008; Ferraiuolo et al., 2011). Oligodendrocytes are progressively lost in ALS (Kang et al., 2013), however their role in ALS remain uncertain, in particular, their role in determining disease onset (Yamanaka et al., 2008a; Kang et al., 2013). Of particular interest to this thesis, is that oligodendrocytes are vulnerable to glutamate excitotoxicity, a known pathogenic process in ALS (refer to section 1.3). So-termed white-matter excitotoxicity, oligodendrocyte vulnerability to glutamate is likely to perturb protective interactions between oligodendrocytes and axons (both cortical and motor). Furthermore, evidence indicates that glutamate receptor subunits are present along myelinated axons (internodal regions). Whilst the function of these receptors is unknown, their presence could mean that excitotoxicity may be mediated directly via the axon (Matute et al., 2007; Matute and Ransom, 2012). These findings raise interesting questions about how excitotoxicity affects the neurons within the cortex and spinal cord, with relevance to ALS, FTLD and other neurodegenerative diseases.
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The faster removal of myelin debris in SIRPα−/− mice compared with wild-type mice (Fig. 3) could have resulted not only from the more efficient phagocytic capacity of SIRPα−/− macrophages compared with wild- type macrophages (Fig. 2) but also from differences be- tween the two mice strains with respect to the number of macrophages present in Wallerian degeneration. We addressed this issue by quantifying the number of cells that express CR3 since, as we previously documented; CR3 mediated much of the phagocytosis of myelin deb- ris in macrophages and microglia [18–20]. For this pur- pose, we sampled the same intact and freeze-crushed nerves in which we studied axon degeneration and re- generation (Fig. 5), thus 8 to 10 mm distal to lesion sites that were excluded. CR3 expressing cells were visualized by detecting the immunoreactivity to CD11b/αM sub- unit of CR3. Immunoreactivity to CR3 was infrequently observed in intact nerves from the two mice strains, which agrees with the detection of 1.2 macrophages per 100 μm 2 in intact nerves . The number of CR3 ex- pressing cells increased progressively and to similar levels in the two mice strains from days 2 and 3 after surgery, the earliest that we tested, continuing to days 4 and 7 after surgery, the later post-injury days that we tested (Fig. 6c, d). These observations agree with our previous findings that the number of macrophages (i.e., cells expressing the macrophage specific F4/80 antigen) increased progressively from 2.5 to 7 days after surgery [17, 35–37]. Noteworthy, CR3 expressing cells could be both macrophages and neutrophils [37, 38]. However, most were macrophages since macrophages outnumber neutrophils through the entire period of myelin debris removal (see the “Discussion” section).
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Substantial evidence implicates glutamate excitotoxicity in the pathogenesis of ALS. However, the mechanism by which excitotoxicity results in axon degeneration is not well understood. This thesis has utilised primary cell culture techniques and immunocytochemistry to investigate the effect of targeted excitotoxin exposure to cortical neurons. Excitotoxicity in the somatodendritic compartment resulted in degeneration of the untreated distal axon and extensive degeneration of neuronal structures in the treated compartment. However, targeted excitotoxicity to the distal axon also resulted in degeneration of the axon, in the absence of degenerative changes to the untreated somatodendritic compartment. Immunocytochemical and western blot analysis indicated distally mediated excitotoxicity likely occurred via the AMPA receptor. In addition, distally triggered degeneration occurred in a caspase-dependent manner.
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exhibit a marked decrease in large-caliber (>8 µm) axons and ultrastructural changes consistent with primary motor axon degeneration, secondary Schwann cell reactions, and axonal regeneration. These findings resemble those found in studies of affected nerves of patients with acute porphyria and thus provide strong evidence that PBGD deficiency causes degeneration of motor axons without signs of primary demyelination, thereby resolving a long-standing controversy. Interestingly, the neuropathy in PBGD –/– mice developed chronically and progressively and in the presence of normal or only slight-
SWI/SNF knockdown may have resulted in decreased cell viability of the MB γ neurons and the initiation of cell death, ultimately leading to loss of the MB γ neurons. A previous study in Drosophila found that null mutations in brm result in decreased cell viability and cause defects in peripheral nervous system development (Elfring et al., 1998). In line with these findings, I observed a significant reduction in relative signal intensity of the γ-lobe following knockdown of brm using two independent RNAi lines. Future studies should investigate this possibility by immunostaining for the active form of caspase-9, an active marker of apoptosis (McIlwain et al., 2013). In addition, it is also possible that the observed decrease in relative signal intensity may have resulted from defects in axon guidance or re-extension. I have demonstrated that knockdown of several SWI/SNF components resulted in defects in axon morphogenesis and γ -neuron remodelling. The remodell ing of the MB γ neurons involves degeneration of the larval specific axons, followed by re-extension of the adult specific axonal projections. Loss of SWI/SNF function in the MB may have caused defects in axon re-extension, such that a proportion of the MB γ neurons failed to re -extend their axons past the peduncle. As a result, a reduced number of γ neurons would be pres ent in the MB γ lobe of the adult. Notably, the results of my study found that knockdown of several SWI/SNF components resulted in the appearance of small and stunted γ -lobes. Due to the severity of the stunted γ-lobe phenotype observed following knockdown of Act5C 42651
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image quality of two sample slices before and after the preprocessing. 2.3 Centerline Extraction in 3D Axon Images. We have discussed the dynamic programming (DP) technique for 2D centerline extraction in Zhang et al. (2007). Dynamic programming (Bellman & Dreyfus, 1962) is an opti- mization procedure designed to efficiently search for the global optimum of an energy function. In the field of computer vision, dynamic program- ming has been widely used to optimize the continuous problem and to find stable and convergent solutions for the variational problems (Amini, Wey- mouth, & Jain, 1990). Many boundary detection algorithms (Geiger, Gupta, Costa, & Vlontzos, 1995; Mortensen, Morse, Barrett, & Udupa, 1992) use dynamic programming as the shortest or minimum cost path graph search- ing method. The DP technique is able to extract centerlines accurately and smoothly if both starting and ending points are specified. However, direct use of conventional DP technique is not suitable for the problem of extract- ing centerlines in 3D axon image volumes. First, given the complexity and dimensionality of a 3D image volume, it is usually difficult for users to select the correct pair of starting-ending points. Second, due to the limita- tion of imaging resolution, some axon objects are seen overlapping (i.e., the boundaries separating them are barely visible from the slices), even though in reality they belong to different axon structures. If the DP search is ap- plied on such image data, it will follow the path that leads to the strongest area, which may result in missing axon objects. Third, the DP technique is computationally expensive if the search is for all the optimal paths for each voxel in the 3D image volume.
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Fig. 2B shows examples of peak distribution for representative target mRNAs encoding proteins with known functions in axon development: Ncam1, Clasp1, Dcx and Robo2. Over 90% of the identified binding peaks were located in mRNA 3 ′ UTRs (Fig. 2C), as is typical of many other regulatory RNA-binding proteins (Holt and Schuman, 2013). A consensus sequence was identified for IMP2 interaction (Fig. 2D, Fig. S2). This relatively short 7-8 nt sequence was found in only a fraction of targets (Fig. 2D, Fig. S2). These features are comparable to motifs associated with many other RNA-binding proteins (Anko and Neugebauer, 2012), and suggest that the motif is unlikely to be an obligate IMP2 binding consensus and might instead be associated with RNA secondary structure or the binding of accessory factors. Previously, IMP2 was used among a panel of several RNA-binding proteins to validate that the PAR- CLIP technique can detect protein-RNA interactions, resulting in identification of a shorter and highly degenerate 3-4 nt motif (Hafner et al., 2010); that a different motif was observed could reflect the use of overexpressed recombinant IMP2 in HEK 293 cells (Bell et al., 2013). The HITS-CLIP approach used here examined RNAs associated with endogenously expressed IMP2 in the native context of developing brain. We next used bioinformatic tools to assess the functions of the identified targets.
We have found that the evolutionarily conserved cytoplasmic tyrosine phosphatase Ptpmeg contributes to the establishment and the maintenance of axonal projections in the Drosophila central brain. ptpmeg is required for the proper establishment of axon projections in the ellipsoid body (EB), where formation of the EB axon ring is not completed in the absence of ptpmeg. ptpmeg is also required for the formation of normal patterns of axonal projections in the adult mushroom body (MB), but in this case ptpmeg is required to stabilize MB axon projection patterns that have already formed. In the MB, ptpmeg promotes the retention of the dorsally directed ␣ and ␣⬘ axon branches and inhibits the overgrowth of the medially-directed ␤ and ␤⬘ axon branches. The FERM domain of Ptpmeg is required for MB dorsal branch retention, but is dispensable for preventing medial branch overgrowth, suggesting ptpmeg functions via distinct molecular pathways in dorsal and medial MB axon branch stabilization. Members of the Ptpmeg family of tyrosine phosphatase are neuronally expressed in animals from worms to flies to mice. The present work provides the first evidence that a member of the Ptpmeg family is important for neuronal connectivity.
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The vertebrate retinotectal projection provides an excellent model in which to explore the molecular and cellular mechanisms of axon growth and topographic mapping (Thanos and Mey, 2001). These processes rely on the recognition by growth cones of environmental cues including cell adhesion molecules (CAMs), matrix molecules, and axon guidance molecules (for review, see Tessier Lavigne and Goodman, 1996; Chisholm and Tessier- Lavigne, 1999). The functions of these cues can be broadly classified as being permissive and attractive to axons, or inhibi- tory and repulsive. Topographic mapping of axons requires the recognition and integration of both types of signal (Muller et al., 1996; Tessier Lavigne and Goodman, 1996; Dingwell et al., 2000). The ephrin protein family and the repulsive guidance molecule are examples of repulsive cues in the optic tectum, inducing characteristic collapse of growth cones (Muller et al., 1996; Mon- schau et al., 1997; Frisen et al., 1998). With ephrins, these repul- sive signals are transmitted through Eph receptor protein ty- rosine kinases (RPTKs) (Drescher et al., 1995; O’Leary and Wilkinson, 1999). Our understanding of the signals that instead promote retinal axon growth within the optic tectum is more rudimentary. Maintenance of retinal axon growth in the retina
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This new category of morphogens as potential guidance mol- ecules will need to be explored in the context of SAG neurite outgrowth. Our lab has preliminary data showing that there are transcripts for many different Wnt ligands, Wnt receptors and Wnt inhibitors expressed in the embryonic chicken otocyst, the SAG or the nerve during the key stages of neurite outgrowth (Sienknecht and Fekete, unpublished observations). Mouse mutants have been used to show that both Wnts and Shh are required for otic morphogenesis, particularly in the context of dorsal-ventral ligand gradients originating from tissues outside the ear (Bok et al., 2007, Riccomagno et al., 2002, Riccomagno et al., 2005). The presumed concentration gradients of Wnt (high dorsally) and Shh (high ventrally) raise the possibility that SAG neurons may subsquently use the same gradients to establish tonotopic projec- tions along the cochlea or for guiding vestibular afferents. BMP ligands have long been known to be present in otic sensory primordia from their earliest appearance; perhaps we need to consider their potential as chemoattractants directing SAG axons toward these target sites. What is now needed are functional studies designed to dissociate the known morphogenetic impact of Wnts, BMPs and Shh on the inner ear from their possible effects on axon guidance.
Studies in flies and vertebrates have also suggested that netrin1 has an additional guidance activity establishing boundaries [12, 76, 77]. In the vertebrate spinal cord, netrin1 appears to encourage axon growth specifically around a netrin1 + domain . This boundary activity was called a “hederal” boundary, from the analogy of a wall supporting the growth of ivy (genus: hedera) that is not itself penetrated by the ivy. Commissural axons always respect the edge of the NPC-netrin1 + domain, to grow around the VZ, and then adjacent to netrin1 + cells in the FP. When a small region of netrin1 expression was extinguished in the intermedi- ate spinal cord, axons deviated from their normal trajectories to follow the new boundaries in netrin1 expression . At later stages in spinal development, new domains of netrin1 expression emerge adjacent to the dorsal root entry zone, which also serve as boundar- ies for spinal axon growth. Thus, netrin1 may supply both an adhesive substrate along which axons can grow in a fasciculated manner, while also providing a border to delineate axon tract formation. The mechanism that mediates the hederal boundary is not known, although it may require the deposition of netrin1 on commissural axons, since only netrin1 − axons are observed to stray into the VZ .
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The growth cone is both a sensory structure that receives directional cones from the environment and a motor structure whose activity leads to axon elon- gation during development. The growth cones are actively growing tips of all branches of axons and dendrites .
How, then, do cone signals reach the horizontal cell axon terminal? Axon terminals make synaptic contacts with rod pho- toreceptors in the wild-type retina. However, retinal neurons in transgenic mice with photoreceptor deficiencies have been shown to reorganize their synaptic contacts (Strettoi et al., 2002; Dick et al., 2003). To rule out the possibility that axon terminals in Cx36-deficient mice contact cone pedicles, we incubated reti- nas containing injected axon terminals with peanut agglutinin, which labels the base of the cone pedicles (Hack and Peichl, 1999; Haverkamp et al., 2001). Rotations of single scans revealed no overlap between axon terminal and cone pedicle labeling (Fig. 7) (n ⫽ 3). In addition, axon terminal morphology appeared nor- mal; no evidence of sprouting or reorganization was seen. Thus, our data suggest that axon terminals receive cone inputs from the horizontal cell soma by way of the axon.
Fmi helps to form synapse between these neurons and their postsynaptic targets. When Fmi is absent, C4da axon terminals fail to stabilize in the target area and do not form correct synapses. Bao et al. showed that fmi mutation causes a significant increase in the number of ectopic synapses on muscles . In the C4da system, loss of fmi may cause the axon termi- nals of V neuron to form ectopic synapses, resulting in a dorsal shift of these axon terminals to the middle of C4da neuropil. Supporting this idea, we found that loss of fmi led to an increase in the C4da axon ter- minal branch number, which may reflect ectopic syn- apse formation. Overexpression of d Trim9 and
The time course of myelin removal is determined by the kinetics of macrophage recruitment and the kinetics of the activation of macrophages and Schwann cells to scavenge degenerated myelin. Bone-marrow derived macrophages, which are scarce in intact PNS nerves of normal and Wld s mice, accumulate at injury sites within hours after the trauma through ruptured vasculature and secondary to the rapid local production of cytokines and chemokines that attract macrophages to these sites; [61-63] and Figure 1B. The recruitment of macrophages during normal Wallerian degeneration is by diapedesis through vasculature that is structurally intact since it does not encounter physical trauma directly. It begins 2 to 3 days after a cut injury and it peaks at about 7 days [16,42,43,64,65]. In contrast, macrophage recruitment is delayed considerably in Wld s mice during slow Wallerian degeneration. However, Wld s macrophages invade freeze-damaged Wld s PNS nerves promptly , suggesting that Wld s macrophages can respond to chemotactic signals that freeze-damaged nerves produce, and further, that chemotactic signals are not upregulated during slow Wallerian degeneration, as indeed it was later shown  (see also below). The exact molecu- lar mechanisms that link between the physical impact at lesion sites and macrophage recruitment to distal nerve segments during normal Wallerian degeneration are not fully understood. Yet, cytokines and chemokines that attract macrophages [61-63,66-68], MMPs (matrix metal- loproteinases) [69-72], and complement [73-75] play roles (see below).
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