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Mechanisms of Axonal Degeneration and Regeneration: Lessons Learned From Invertebrates
Abstract and Keywords
In the face of acute or chronic axonal damage, neurons and their axons undergo a number of molecular, cellular, and morphological changes. These changes facilitate two types of responses, axonal degeneration and regeneration, both of which are remarkably conserved in both vertebrates and invertebrates. Invertebrate model organisms, including Drosophila and C. elegans, have offered a powerful platform with accessible genetic tools for manipulation and amenable nervous system for visualization. Thus far, several critical components and pathways in axonal degeneration and regeneration have been identified in invertebrate studies, including Sarm and Wallenda/DLK. This article highlights important findings in Drosophila, C. elegans, and other invertebrate injury models that have shed light upon the mechanism in axonal injury response.
Keywords: axon damage, degeneration, adaptation, regeneration, Sarm, Nmnat, DLK/Wallenda, cytoskeleton, intracellular transport
Neurons and the connections that they make with each other typically need to persist for an animal's entire life, even in the face of injury. How do nervous systems, including our own, cope with and respond to damage? It would be ideal if we simply could regenerate the damaged part, like a planarian which can regenerate an entire head de novo (Owlarn & Bartscherer, 2016). However, most nervous systems across the animal kingdom lack such capacity. Instead, many nervous systems do their best to repair damaged axons and synapses. And in many cases in adult nervous systems, damaged axons and synapses are simply lost.
Axons are thought to be particularly vulnerable components of neuronal circuitry. They are often exceptionally long: Human motoneuron axons can reach lengths over 10,000 times the diameter of their cell body, and even in small invertebrates such as Drosophila an axon can be 200 times the cell body diameter. Such length can be a vulnerability: A problem occurring anywhere along the axon's length could result in lost ability to communicate with downstream cells. Furthermore, because axons connect to distant sites, it can be difficult to re-establish lost connections, especially in the adult nervous system. During development, axonal growth to specific targets is directed via a series of growth promoting and guidance cues (Tessier-Lavigne & Goodman, 1996). Many of these cues are only transiently present during nervous system development and are largely absent in the adult system.
In this chapter we review some important factors that mediate neuronal responses to axonal damage. We will emphasize here what has been learned from research using invertebrate model organisms (especially Drosophila and C. elegans), which has directed exciting discoveries of mechanisms that are conserved across the animal kingdom. (p. 576)
Overview of Acute and Chronic Models of Axonal Damage
The response(s) that neurons make to axonal damage or stress can vary according to the types of damage and neuron type (Fig. 25.1, and Table 25.1). However, a simple determinant for classification is the duration of the harmful stimulus: acute (short term, cartooned in Fig. 25.1A–C) verses chronic (long term, cartooned in Fig. 25.1D–E).
Acute axon injuries can be induced experimentally by directly transecting or crushing nerves, or by microsurgical cutting of individual axons using a high-energy laser. After such an injury, repair may be possible if the part of the axon that remains attached to the cell body (the proximal "stump") can grow again. The ability of an injured axon to reinitiate new axonal growth and eventually reconnect to its target (Fig. 25.1Bi and 25.1Ci) is commonly termed axon "regeneration." Axon regeneration has been documented and studied in many invertebrate models, including cockroach, crickets, crayfish, squid, Aplysia, Great pond snail, earthworm, leech, and more recently C. elegans and fruit flies (Table 25.1). Is the ability to regenerate axons universal for invertebrate neurons? Probably not, since failures have also been documented (Wu et al., 2007; Ayaz et al., 2008; Song et al., 2012). It is interesting that some of these failures occur after (p. 577) (p. 578) (p. 579) (p. 580) injuries in the central nervous system (CNS) (Ayaz et al., 2008; Song et al., 2012), where stalled regeneration (Fig. 25.1Bii), followed by degeneration of the proximal stump (Fig. 25.1Ciii) have been described. In the mammalian CNS, the failure to regenerate damaged axons is a major clinical impediment to recovery after brain and spinal cord injuries; hence, the possibility that some aspects of this failure are shared with invertebrates, where it can be studied in a simple model system, is interesting and potentially exciting.
Table 25.1 Axon Response to Acute Injury in Different Models
Degeneration of Distal Stump
Regeneration of Proximal Stump
Drosophila melanogaster (fruit fly)
Olfactory receptor neuron (adult)
Fragmentation starts 1 day after injury and completes by a week
Motor neuron (larva)
Crush/laser surgery
Fragmentation initiates 4 hours after injury and continues for up to a day
Forming growth cones 10–14 hours after injury, followed by axon growth
Sensory neuron (adult wing)
Transection/laser surgery
Fragmentation initiates within a day and completes by around a week
(Laser surgery): regeneration initiates by 3 days
Fang et al., 2012; Neukomm et al., 2014; Soares et al., 2014
DA sensory neuron (larva)
Laser surgery
Fragmentation starts 6 hours after injury for Class I neurons
Regeneration observed for Class IV neurons (3 days after injury) and some Class I neurons (1–3 days) but not for Class III; limited CNS regeneration versus prominent peripheral regeneration
Stone et al., 2010, 2012; Song et al., 2012
Mushroom body neuron (larva, pupa, and adult)
Disassociation (in vitro)
Neurites sprout 1 day after dissociation in culture
Marmor-Koller & Schuldiner, 2016
Motor neuron (larva)
Disassociation (in vitro)
Neurites sprout 2–3 days after dissociation in culture
sLNv neuron (adult brain explant)
Transection by microdissection device (in vitro)
Fragmentation starts 1 day after injury
Filopodia sprouts observed 2 days after injury in a portion of axons while a majority of axon do not regenerate
Periplaneta americana (cockroach)
Motor neuron
Reinnervation by 4 weeks and recovery of circuits by 7–9 weeks
Acheta domestica (cricket)
Medial giant interneuron (giant fiber system)
Crush or cut
Sprouting; crush near cell body induces dendritic branching
Sensory neuron
Transection of cerci
Sprouting; target to the same giant fiber
Procambarus clarkii (crayfish)
Motor neuron
Crush or transection
Not oberved for 3 months
Fusion occurs; a majority of injured axons establish reconnection by 30 days
Caenorhabditis elegans
Mechanosensory neuron (ALM, PLM, AVM); chemosensory neuron (ASH, ASJ); GABAergic motor neuron (DD, VD); cholinergic motor neuron (DA/DB); HSN motor neuron
Laser surgery
Axons become thin, beaded, and invisible in ALM/PLM/DD neurons 24 hours for L1 or several days for L3 after injury
Axon growth 12–24 hours after surgery; fusion observed with some mechanosensory neuron
Yanik et al., 2004; Wu et al., 2007; Gabel et al., 2008;Chen & Chisholm, 2011; Chung et al., 2016; Nichols et al., 2016; for details, see Hammarlund & Jin, 2014
GABAergic motor neuron (β-spectrin mutant)
70% axons regenerate
Aplysia californica (California sea hare)
Sensory neuron and motor neuron
Dissociated or crush (in vitro)
Degeneration does not occur for hours
Sprouting and regrow axons
Sensory neuron
Reinnervation and recovery of reflex behavior within 2–3 weeks
Dulin et al., 1995; Noel et al., 1995; Steffensen et al., 1995
Buccal motor neuron
Degeneration observed 8 weeks after injury
Reinnervation to targets observed 3 weeks after injury
Lymnaea Stagnails (Great pond snail)
Sprouting more prominent when injured closer to soma; synaptic connections restored 3–6 days after injury
Helodrilus caliginosus and Lumbricus terrestris (earthworm)
Giant axon
Fusion with high accuracy
Hirudo medicinalis (leech)
Starts around 1 month after injury
Sprouting and following the existing distal stump to innervate the target by 1 month and functional recovery by 2 months
CNS, central nervous system; NA, not applicable; PNS, peripheral nervous system.
Whereas the proximal stump can either regenerate or fail to regenerate, the common fate of the "distal stump," which is no longer connected to the cell body, is to degenerate (Fig. 25.1Biii). Most Drosophila neurons behave similarly to mammalian neurons by initiating axonal degeneration quite rapidly (within a day) after injury. This fast process, in theory, may allow for a "replacement" by new growth from a regenerating "proximal stump" (Fig. 25.1Ci). However, an injured "distal stump" has been observed in some invertebrate animals (crayfish and leech) to persist for months after injury with no signs of degeneration (Hoy et al., 1967; Frank et al., 1975). In these cases, as well as in C. elegans, a process of refusion between the two separated stumps (Fig. 25.1Cii) has been observed (Hall, 1921; Hoy et al., 1967; Birse & Bittner, 1976; Muller & Carbonetto, 1979; Neumann et al., 2011). Whether the repair is achieved by replacement (in Fig. 25.1Ci) or fusion (Fig. 25.1Cii), the two processes of axonal degeneration and regeneration need to be coordinated.
Axonal damage can also occur in other scenarios of injury and stress, which we define here as "chronic injuries." These scenarios include prolonged presence of a neurotoxin (such as taxol and colchicine, which induce axonal loss) or the presence of a mutation, which induces a persistent "stress" to the integrity and function of the axon (Fig. 25.1D). Many mutations that are known to cause inherited neurodegenerative disorders in humans, when expressed in vertebrate and invertebrate models, lead to loss of synapses (Fig. 25.1Ei) and/or axons (Fig. 25.1Eii) prior to neuronal death (Saxena & Caroni, 2007; Charng et al., 2014; Casci & Pandey, 2015).
A unifying feature of both acute and chronic models of axonal damage is the impairment of intracellular transport processes within axons. The transport of organelles and proteins in axons relies upon the action of motor proteins, which physically carry their cargo by walking upon microtubule tracts. In acute axonal injuries, the delivery of molecules from the cell body to the distal axon is irreversibly blocked due to the fact that they are no longer physically connected. In chronic models, although the connection remains, the process of axonal transport is also thought to be persistently impaired (Fig. 25.1D). This has been shown by altered movement of fluorescently tagged organelles such as mitochondria or synaptic vesicle precursors, or by accumulations of organelles in axons or cell bodies (Millecamps & Julien, 2013). Mutations that disrupt the cytoskeleton, comprised of the microtubule tracts and associated molecules, often lead to degeneration of axons and/or synapses (Pielage et al., 2005, 2008; Schaefer et al., 2007; Bounoutas et al., 2011; Hsu et al., 2014; Ma et al., 2014; Neumann & Hilliard, 2014).
In contrast to acute injuries that completely disconnect cell bodies with their synaptic targets, neurons in a chronic injury condition have the chance to try to adapt to the stress and make their axons more resilient to degeneration (Fig. 25.1Eiii). Although adaptive changes and mechanisms are still very poorly characterized, they may potentially entail an induced expression of chaperones and transport of additional cytoskeletal components into axons. Depending upon the severity and duration of the stress, this response may or may not be enough to maintain the axon and/or to prevent cell death.
The processes of degeneration, regeneration, and adaptation are all interesting from the perspective of human health. The mechanisms that neurons engage to either delay or accelerate axonal and synaptic loss and repair after injury could be valuable therapeutic targets for treatment of traumatic injuries as well as neuropathies and even potentially neurodegenerative diseases in which axonal loss occurs.
Axon and Synapse Loss
The process of axonal degeneration after acute injury (Fig. 25.1Biii) is highly stereotyped and is termed Wallerian degeneration (WD), based on its first description by Augustus Waller in 1851. For a period of time after injury (termed the "lag phase") the distal stump of the axon remains intact and is able to propagate action potentials (Lubińska, 1977; Beirowski et al., 2005). In most cases, the lag phase is then followed by a rapid fragmentation phase, in which the axon breaks into many individual pieces, which are then phagocytosed by glia and immune cells (Bhatheja & Field, 2006). WD likely entails a cell autonomous chain of events that occur within the distal axon itself and, hence, can be considered as a "self-destruction" pathway, akin to (p. 581) apoptosis. However, WD appears to involve a molecular pathway that is quite distinct from apoptosis (Deckwerth & Johnson, 1994; Finn et al., 2000). The pathway itself and its molecular players are still at the early phases of being described, and studies in the invertebrate model organism Drosophila have led the way in this exciting area of study.
In Drosophila, multiple approaches for inducing axonal injury and studying WD have now been described (Table 25.1; also see Fang & Bonini, 2012). Combined with the powerful genetics of this model organism, such approaches provide an ideal vehicle to uncover vital molecular mediators of the WD pathway. Many of the injury approaches (such as cutting an antenna or a wing) are simple to carry out, enabling genetic screens to identify molecules whose function is required for degeneration to occur. For some of the approaches (including laser-directed transection and nerve crush injuries in peripheral neurons of larvae that are semitransparent) the process of WD is amenable to detailed study of the cellular changes during the course of WD, including changes in intracellular calcium, mitochondria, and cytoskeleton (Avery et al., 2012; Fang et al., 2012; Kitay et al., 2013; Mishra et al., 2013). Here we briefly highlight some of the discoveries made in Drosophila that have strongly influenced our understanding of WD.
Central Molecular Regulators of Wallerian Degeneration
A heroic large-scale genetic mosaic screen in Drosophila led to the discovery of dSarm (Drosophila Sterile alpha and HEAT/Armadillo motif containing), as a critical molecular player in WD (Osterloh et al., 2012). Neurons that are mutant in dsarm fail to degenerate distal axons after axonal injury. This role in promoting degeneration was then shown to be conserved in mammalian Sarm1 and in C. elegans tir-1 (Osterloh et al., 2012; Vérièpe et al., 2015). Although Sarm1/tir-1/dSarm (which we refer to here simply as Sarm) has been previously implicated in innate immunity and neuronal development (Couillault et al., 2004; Liberati et al., 2004; Chuang & Bargmann, 2005; Chen et al., 2011), a role in axonal degeneration would have never been guessed, highlighting the importance of forward genetic approaches. With Sarm's central role in degeneration revealed, current work is now focused upon its mechanism. Domain analysis suggests that dimerization of Sarm's TIR domains is sufficient to activate WD in uninjured axons, and this activation leads to a rapid rundown of intracellular metabolites NAD + and ATP (Gerdts et al., 2015; Sasaki et al., 2016; Summers et al., 2016). Much work remains to be done to understand the molecular events that lead to the activation of Sarm and its downstream actions.
In contrast to the unanticipated discovery of Sarm, a potential role for Nicotinamide mononucleotide adenylyl transferase enzyme (Nmnat) had been suspected for years, ever since a gain-of-function mutation in the Nmnat1 enzyme was fortuitously discovered in the background of a mouse strain. WD fails to occur in these mutant mice, and this effect can be recapitulated in Drosophila neurons, by overexpressing the Drosophila dNmnat enzyme (MacDonald et al., 2006).
Loss-of-function studies in both Drosophila and mice suggest that endogenous versions of Nmnat enzymes in healthy neurons play a protective role to inhibit degeneration (Zhai et al., 2006; Gilley & Coleman, 2010; Wen et al., 2011; Fang & Bonini, 2012; Hicks et al., 2012; Rallis et al., 2013; Sasaki et al., 2015). In many of these studies, depletion of Nmnat function leads to spontaneous axonal degeneration even in the absence of injury. To explain these observations, it has been proposed that Nmnat is an axonal "survival" factor. Drosophila Nmnat and mammalian Nmnat2 are continuously transported into distal axons, where the protein is then rapidly turned over (Gilley & Coleman, 2010; Xiong et al., 2012; Milde et al., 2013; Fig. 25.2). Once disconnected from the cell body, the distal stump loses the supply of Nmnat from the cell body and its essential but still poorly understood survival function, which leads to the initiation of degeneration.
What exactly the Nmnat enzymes do to maintain axon integrity is still not clear, and this is the subject of much investigation and discussion (Ali et al., 2013). It is clear that Nmnat enzymes need to localize in the cytosol (Fig. 25.2A), and they become more potent at protecting axons from degeneration when they are targeted to axons (Avery et al., 2009; Beirowski et al., 2009; Sasaki et al., 2009). The enzymatic activity for NAD + synthesis also appears to be important (Araki et al., 2004; Jia et al., 2007; Sasaki et al., 2009). In line with this, a recent study has linked Sarm1's role in degeneration to a rapid rundown in NAD + (Gerdts et al., 2015). However, whether inhibiting NAD rundown is a direct action of the Nmnat enzymes has been difficult to address, and some studies have linked other metabolites on (p. 582) the NAD biosynthesis pathway with axonal degeneration (Conforti et al., 2014).
A quite different idea is that Nmnat performs an additional function that is separate from NAD + synthesis, by acting as a molecular chaperone, akin to the function of heat shock proteins (Ali et al., 2013). This idea builds upon observations that Nmnat transgenes that are nonfunctional for NAD synthesis activity can still have protective effects when overexpressed in Drosophila neurons (Zhai et al., 2006, 2008). Further, endogenous Nmnat isoforms become upregulated in several models of protein-folding disorders, and in these cases Nmnat protein is observed to colocalize with protein aggregates. In addition, Nmnat can facilitate the folding of denatured luciferase in vitro, possibly via its ATPase domain (Zhai et al., 2008; Ali et al., 2011, 2012, 2016). Although it is challenging to nail down a chaperone function in vivo, the idea remains attractive since other known chaperones (p. 583) (such as TBCE, CSP, and Hsp70) are required for continued axon and synapse integrity (Fernández-Chacón et al., 2004; Schaefer et al., 2007; Rallis et al., 2013).
Potential Sarm and Nmnat-Independent Pathways
Although axonal degeneration usually initiates within hours of injury in Drosophila and mammalian neurons, crayfish and leech axons have been observed to persist for months after injury (Hoy et al., 1967; Frank et al., 1975; Ballinger & Bittner, 1980). This implies the existence of mechanisms that have been adopted by some animals to maintain the integrity of distal "stump" and/or inhibit degeneration. Do these axons fail to activate Sarm? If so, how and why is this change achieved in these neurons? Or are there additional pathways involved? Interestingly, a recent study has documented WD in C. elegans, which occurs independently of any manipulation to TIR-1, the C. elegans homologue of Sarm, or Nmnat (Nichols et al., 2016). This suggests that Sarm may not universally promote degeneration in all neuron types, or it may be utilized to different degrees in different contexts. A revisit to old observations of axonal degeneration in nonmodel organisms with contemporary techniques may help to reveal the origin and evolution of the degeneration program.
Adaptive Mechanisms to Chronic Stress
Similarly to WD after injury, chronic stressors (such as cytoskeletal toxins and/or mutations) can also cause axonal degeneration (Fig. 25.1D and 25.1Eii). Because in most cases degeneration is inhibited by manipulations that increase Nmnat activity, it is thought that degeneration in these models shares a common underlying mechanism with WD (Coleman & Freeman, 2010). However, neurons in different injury models exhibit various tolerances to chronic stressors (Conforti et al., 2014). We posit that some of these differences are determined by whether the cell is able to make an adaptive response to delay the degeneration process (Fig. 25.1Eiii).
Some recent studies in Drosophila suggest that nuclear signaling pathways may become engaged in response to chronic stress and damage with an output that serves to enhance and/or maintain axon integrity. An important example is the upregulation of Nmnat expression and alternative splicing of Nmnat isoforms, which has been observed in response to proteotoxic stress, heat shock stress, and hypoxia (Zhai et al., 2008; Ali et al., 2011; Ruan et al., 2015). An additional pathway, discussed later, is the DLK signaling pathway, which becomes activated in injured axons. In Drosophila motoneuron axons, this pathway induces cellular changes that delay the process of WD, such that an axon that has been injured once becomes more resilient to degeneration after a second injury (Xiong & Collins, 2012). The manifestation of this protective response only occurs for the proximal stump but not the distal stump, likely because the process involves the expression and transport of new molecules into axons.
It is interesting that both Nmnat and the DLK kinase share commonalities in their regulatory mechanisms. First, both are transported in axons and associated with Golgi-derived vesicles (Fig. 25.2A). Palmitoylation allows for this localization and is required for the rapid turnover of Nmnat2 (Milde et al., 2013) and the function of DLK (Holland et al., 2015) in mammalian neurons. Whereas this has yet to be tested in invertebrate neurons, Drosophila Nmnat and DLK proteins contain palmitoylation consensus sequences. Second, the protein turnover of both Nmnat and DLK are regulated in both Drosophila and mammalian neurons by a conserved ubiquitin ligase complex, whose signature component is a highly conserved PHR protein, named Highwire (Hiw) in Drosophila, RPM-1 in C. elegans, and PAM in mice (Nakata et al., 2005; Collins et al., 2006; Wu et al., 2007; Xiong et al., 2010, 2012; Babetto et al., 2013; Brace et al., 2014). Hiw therefore becomes an intriguing regulator (and is perhaps a coordinator) of adaptive responses to axonal damage (Fig. 25.2).
Finally, it is interesting to consider that many chronic paradigms of axonal injury can originate or manifest at presynaptic terminals. Several mutations that disrupt synaptic structure lead to axon and/or synapse degeneration (Fernández-Chacón et al., 2004; Pielage et al., 2005; Burgoyne & Morgan, 2011; Wishart et al., 2012). Also, in many neuropathies, the most terminal connections of the axon are lost first, suggesting a "dying-back" mechanism, which may be initiated by a toxic stimulus at the synapse (Yaron & Schuldiner, 2016). Because it has been suggested that a degeneration program may be initiated and/or restrained at synapses (Fig. 25.1Ei), the Hiw ubiquitin ligase complex gains even further cache, since Hiw and its homologues are known to localize to presynaptic terminals (Schaefer et al., 2000; Wan et al., 2000; Zhen et al., 2000) and can inhibit synaptic degeneration in at least one chronic paradigm (Massaro et al., 2009).
(p. 584) Axon and Synapse Repair
For a damaged axon to grow (or regrow) it needs to have a growth cone (Tom et al., 2004; Ertürk et al., 2007). Many early studies in cultured neurons from Aplysia and cockroach (whose giant axons are very amenable to imaging and recording after injury in culture) have helped to describe cellular events that direct a transformation of a severed axonal stump into a growth cone: Calcium influx triggered by the injury itself directs axon membrane resealing at the injury site (Yawo & Kuno, 1985; Strautman et al., 1990; Davenport & Kater, 1992; Spira et al., 1993; Ziv & Spira, 1995) and activation of local calcium-regulated proteases, which promote reconstructuring of neurofilaments and microtubules close to the stump ending (Gitler & Spira, 1998, 2002; Spira, 2003). These events ultimately lead to the formation of a growth cone that has dynamic lamelopodia and filopodia (Baas & Heidemann, 1986; Ertürk et al., 2007; Schaefer et al., 2008; Hellal et al., 2011).
The ability of the growth cone to direct new axonal growth is associated with transcriptional and translational changes in the cell body. These changes are induced by signaling pathways that become activated in injured axons, and this "injury signaling" appears to be mediated, at least in part, by molecules which are physically transported in axons (Hanz & Fainzilber, 2006). Early studies in Aplysia led to the identification of several proteins that are retrogradely transported specifically in injured neurons (Ambron et al., 1992; Schmied et al., 1993; Schmied & Ambron, 1997; Zhang et al., 2000; Sung et al., 2001; Sung et al., 2004). These findings inspired later studies in mammalian peripheral sciatic nerves, which have revealed critical components of "retrograde signaling" (Lindwall & Kanje, 2005; Perlson et al., 2005; Ben-Yaakov et al., 2012).
Upon this foundation of knowledge from Aplysia and other invertebrate studies (Table 25.1), our understanding of molecular pathways underlying regeneration was brought to an exciting new level in studies using C. elegans and Drosophila, which have enabled genetic screens and genetic dissection of pathways required for axonal growth after injury. (Detailed review can be found in Hammarlund & Jin, 2014; Brace & DiAntonio, 2016; and Byrne & Hammarlund, 2016). We'll focus our discussion on what is perhaps the most important discovery, the elucidation of the DLK/Wallenda signaling pathway, which detects and initiates responses to axonal damage.
DLK/Wallenda Is Essential for Axonal Regeneration
A role for the DLK kinase in axonal regeneration was first identified in a cleverly designed genetic screen in C. elegans (Hammarlund et al., 2007). The screen was built upon the observation that axons in β-spectrin mutants break spontaneously; however, in response they form new growth cones. Hammarlund and colleagues screened for mutants that failed to do so and identified a signaling cascade governed by DLK kinase, which is essential for the transformation of axonal breaks into new growth cones. Importantly, dlk mutants have no obvious phenotype in axonal outgrowth during development (Nakata et al., 2005; Collins et al., 2006; Miller et al., 2009). These findings suggest that DLK carries out a specific postdevelopmental role in regulating responses to axonal injury.
Several points emphasize the importance of DLK as a central player in regulating the ability of injured axons to regenerate. First, the requirement for DLK in axonal regeneration appears conserved across multiple neuron types in C. elegans, Drosophila, and also in mammalian PNS neurons, which regenerate (Yan et al., 2009; Xiong et al., 2010; Pinan-Lucarre et al., 2012; Shin et al., 2012). Second, DLK functions as an upstream regulator of a MAP Kinase signaling cascade. A number of observations suggest that it is transported in axons and becomes acutely activated after axonal damage. Activated DLK or its downstream targets give rise to retrograde signaling to initiate a nuclear response; hence, DLK appears to function as a regulator of signaling molecules that are retrogradely transported in axons (Yan et al., 2009; Xiong et al., 2010; Bounoutas et al., 2011; Shin et al., 2012; Watkins et al., 2013). In mammalian neurons DLK has also been implicated in other processes that seem to be quite distinct from axonal regeneration: DLK promotes neuronal death after nerve growth factor withdrawal (Ghosh et al., 2011), death of retinal ganglion cells after injury of their axons within the optic nerve (Watkins et al., 2013; Welsbie et al., 2013; Fernandes et al., 2014), and neuronal death in cellular models of excitotoxicity (Pozniak et al., 2013). A shared component of all of these processes that activate DLK is the presence of stress and/or damage to the axon/synapse. The current unified view in the field is that DLK functions as a "sensor" of axonal damage, with the downstream consequences of its activation varying depending upon context.
(p. 585) The amenability of Drosophila and C. elegans to combining mutagenesis and genetic interaction analysis has provided insight into the cellular pathways and processes that appear to regulate DLK's signaling functions in neurons. In C. elegans, a mechanism for direct regulation by intracellular calcium has been described (Yan & Jin, 2012): An isoform of DLK binds to and inhibits the full-length form of DLK, and this inhibitory binding is released in conditions that elevate intracellular calcium. However, the sequences that mediate these interactions are not conserved for mammalian or Drosophila DLK, so there are likely additional important mechanisms for its regulation. Indeed, a recent study has identified the cAMP effector kinase PKA as an important upstream activator of DLK in Drosophila and mammalian neurons (Hao et al., 2016).
Interestingly, studies in all model organisms have noted DLK's relationship with microtubule and actin cytoskeleton. Induced cytoskeletal stresses, such as treatment with taxol or cholchicine, or mutations in cytoskeletal components (tubulin) or regulators (microtubule associated protein), lead to changes of structure and expression in neurons. Interestingly, many of these changes are suppressed when DLK is mutated (Bounoutas et al., 2011; Valakh et al., 2013; Chen et al., 2014; Marcette et al., 2014; Richardson et al., 2014). These findings imply that DLK acts downstream to these manipulations to cytoskeleton, and indeed, DLK signaling becomes activated in mammalian neurons that are treated with cytoskeletal destabilizing agents (Valakh et al., 2013, 2015). Complementary to this point, it appears that a downstream effect of DLK signaling is the induction of alterations in cytoskeleton organization (Lewcock et al., 2007; Hendricks & Jesuthasan, 2009; Eto et al., 2010; Feltrin et al., 2012; Klinedinst et al., 2013) and tubulin expression (Nadeau et al., 2005). These findings place DLK as both a sensor and effector to regulate cytoskeleton dynamics.
Is Regeneration "Programmed"?
During development, axons respond to specifically placed cues to direct their growth correctly, often over a long path that involves many intermediate targets, to find their appropriate synaptic targets. Is the same developmental process re-engaged for regeneration? The answer is likely both Yes and No. Extracellular factors are important for both development and regeneration. However, in mammalian PNS regeneration, where motor neurons can reinnervate their targets accurately after crush (Nguyen et al., 2002), nerve growth factors are released by Schwann cells and microphages rather than targets, which are the main source during development. Schwann cell "tubes" can physically confine axon outgrowth during regeneration, but not during development (Bhatheja & Field, 2006; Scheib & Höke, 2013). In invertebrates, little is known about the mechanisms that govern pathfinding and reinnervation. However it is striking that reconnection to original targets after axonal injury has been observed in many species, including cockroaches, crickets, leech, crayfish, Aplysia, and snails (Bodenstein, 1957; Case, 1957; Edwards & Sahota, 1967; Hoy et al., 1967; Muller & Carbonetto, 1979; Allison & Benjamin, 1985; Benjamin & Allison, 1985). In instances where regeneration has been studied on a molecular level (in C. elegans and Drosophila), pathways that are distinctly required for regeneration and not for development have been most notable and well characterized. These include the DLK/Wallenda and PTEN/PI3K signaling pathways. In C. elegans, DLK is dispensable for axon outgrowth during development (Hammarlund et al., 2009). Likewise, in the Drosophila mushroom body, neither PI3K nor DLK is required for axon outgrowth during development (Marmor-Kollet & Schuldiner, 2016). On the other hand, unc-40/DCC is required for axon initial outgrowth during development (Chan et al., 1996; Keino-Masu et al., 1996), but not axon regeneration (Gabel et al., 2008). These "regeneration-specific" pathways suggest that axon outgrowth and innervation after injury are intrinsically and uniquely programmed.
The functional endpoint for axonal regeneration is to re-establish a functional circuit. Unless refusion occurs (as in Fig. 25.1Cii), this requires a newly formed axon to reform lost synaptic contacts (Fig. 25.1Ci). Although mechanisms that promote the growth of injured axons have been the topic of much investigation, there are very few studies to characterize whether and how synapses can be formed by regenerating axons. Studies of mammalian NMJ regeneration have provided insights into roles of extracellular matrix proteins (Skouras et al., 2011). But little is known about the intracellular pathways in neurons that promote regeneration of synapses. Synapse regeneration may share similarities in "programming" with axon regeneration, with mechanisms that are both shared and distinct from developmental pathways. The field simply needs more studies and more information (p. 586) on this topic. Invertebrate model systems in which functional regeneration can occur (which can result in readily screenable behavioral phenotypes) have important contributions to make for these important future questions.
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BEIJING (REUTERS) - Police in China on Thursday (Sept 29) arrested a man suspected of killing 16 people, including three children, found dead in a remote south-western village, state media said.
Mass killings are rare in China and the incident dominated discussion on social media platforms. It was not immediately clear how the victims were killed, or what the motive was.
The suspect was arrested in Kunming, capital of Yunnan province, about 200km from Yema, the site of the murders, state news agency Xinhua said, adding that the victims were members of six families.
The bodies were found on Thursday at different locations in the village, a state-backed news website, ThePaper.cn, said, adding that the suspect had given authorities some details of the incident.
The public security bureau in the nearby city of Qujing told Reuters it was investigating the incident, but declined to provide details.

subset: commoncrawl


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Husband wanted a poster for his golf club event. A job for me!
I was inspired by a poster I saw online.
Studio Double-D
Hand Scripted Talk Brushes and Stamps No. 01
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Musically Inclined Brushes and Stamps
Research Laboratory Brushes and Stamps
Watching You Layered Template (The TV graphic)
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Room 2 tickets and 2020 tour dates
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Formed out of a mutual love of good music, as well as a distaste for the Indie scene, Room 2 have been together since mid-2007. Combining their love of Grunge, Metal and slightly more interesting genres, they are making a name for themselves around Dundee and beyond.
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Title: Melosira
Description: (Aulacoseira). Filament widths 5-10 Micrometre.
Photographer: Hilda Canter-Lund
Curator: Freshwater Biological Association
Copyright Holder: Freshwater Biological Association
Publisher: Freshwater Biological Association
Note (original item): Record creation date:2008-11-10 15:55:44
Taxonomic Keywords: Bacillariophyta; Aulacoseira
Taxonomic Depictions: Melosira
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Out in Wisconsin...
... at the Wisconsin Farm Technology.
If you are up in Marshfield, Wisconsin, be sure to stop by the D&B Sternweis Farm! That is where this years Wisconsin Farm Technology Days is being held. We have a wide variety of products on display from the amazing AMAZONE Catros, to the FarmBuilt CR 5.0 chisel ripper and even some nice trailers that you can tow with your ATV!
The past couple of days have been wonderful with lots of discussions and people interested in learning more about our products! If you get a chance, stop buy and say hi to Matt! Tell him we sent you!
The Royal Treatment!
Yesterday a local farmer got the Royal treatment with our 13 ft Catros minimum tillage tool!
Royal, Illinois is just 6 miles north of our headquarters in Ogden, Illinois. After stopping in last week and asking about some of our different tillage options, we decided to give him a demonstration of what the AMAZONE Catros could do for him on his 6 acre plot of fallow land. His plan is to enroll this land into CRP conservation using flowers, oats and barley. Needless to say, he was very impressed with the final product!
On the road and talking about quality farm equipment and different farming practices... that is my passion!
July 2018


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We offer installation service with qualified installers
Get the unique look of natural stone while keeping things subdued with the light colours available from travertine. A hard material, travertine can resist scratches, chips, and cracks. One of the oldest building materials, add a classic and prestigious look to your floor. Earthy tones combined with natural stone can work for almost any design whether you're tiling your front entrance or your laundry room!
Why Choose Travertine?
Check out the benefits of selecting travertine tiles:
Browse our gallery to select the most appealing style for your kitchen and bathroom.
Now that you've read about it, come see our beautiful products for yourself.


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iHeartMedia's Bobby Bones and the cast of The Bobby Bones Show will do a live broadcast from the middle of CMA Music Fest on June 8 during the inaugural The Bobby Bones Show, Live From the Ryman. For the first time ever for one day only, Bones is inviting fans to join in for a live broadcast of the show at Nashville's Ryman Auditorium during CMA Fest, which will feature Chris Janson, Kassi Ashton, American Idol 2018 winner Maddie Poppe and American Idol runner-up Caleb Lee Hutchinson.
There will be a special pop-up shop onsite where fans will be able to pre-order Bones' new book, FAIL UNTIL YOU DON'T: FIGHT. GRIND. REPEAT via Parnassus Books as well as other exclusive items. The live broadcast begins at 7 a.m. Friday, June 8, with doors opening at 6 a.m. Seating is limited and available on a first come, first served basis.
Fans will also have a chance to catch Bones in cities across the country in support of the upcoming new book, set to hit shelves June 19. Launching June 15 in Charleston, South Carolina, Bones will visit Washington D.C.; Boston; Tampa, Florida; Ft. Pierce, Florida; Austin, Texas and Wichita, Kansas, for special pre-order, signing and live-stream events, including a stop at the iHeart Theater in New York, NY, and starting this Friday, (June 1) fans can sign up to attend the event at BobbyBones.com/NYC.
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About the AuthorHollabaugh, a staff writer at MusicRow magazine, has written for publications including American Profile, CMA Close Up, Nashville Arts And Entertainment, The Boot and Country Weekly. She has a Broadcast Journalism and Speech Communication degree from Texas Christian University, (go Horned Frogs), and welcomes your feedback or story ideas at [email protected]
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Yesterday I shared that I was officially on summer vacation and that this means I finally have time to share some of the things that went on in my classroom these past few months. Today I'd like to share some of my 2017-2018 end of the year confessions in the hopes that you will realize I'm a normal, everyday teacher just like you!
- I had to get a second job this spring to help pay the bills. I was fortunate to get a good job in the activities department of a nursing home. Although things like watching movies and doing puzzles with the elderly isn't that physically demanding, it's exhausting being out of the house for 12+ hours each day Monday through Friday.
- I never told my Spanish 2 Quizbowl team that we were bumped down from 3rd place to 4th place due to a miscalculation of points the day of the competition. They felt so awesome for being the only team from our school to place and I couldn't take that away from them!
- I assigned a project the last 9 days of class because I just couldn't mentally invest anymore. A day or two into the project, I realized it was way too much time butI didn't mind as long as students were working, met each day's goal, and not in my hair.
- In addition to an activity tied to students reading clubs, I made students also do an activity from Martina Bex's Independent Textivities packets for Los Baker van a Perú so I could use some of their activities (timelines, crossword puzzles, etc.) as main activities in class before moving on to the next chapter. I'd call students up by the activity they chose and then choose the best after glancing it over to go make photocopies in the teacher's lounge.
- I applied for a handful of full time Spanish teaching jobs around the area and only even received a phone call from one. Although I really want to be a full time teacher again, I'm happy to be returning to such a positive and progressive school!
- I had a student failing up until yesterday even though I use CI and I am standards-based (meaning the student can do retakes). The student had issues in another class which caused him to mentally tune out the month or so that we read Los Baker van a Perú. After retaking some assessments twice (or even three times!), the student was still failing. My deduction is that the student didn't do their part to pay attention (one of the biggest indicators of TPRS/CI classes in my honest opinion) nor did they prepare on their own before retakes.
- The last four days of class (and the weekend in-between) I didn't have any hot water in my house nor AC! Rough to feel your best when you haven't had a hot shower in several days and you're sweating in your sleep! Before these issues started, we had called the fire department the week before because we had smoke in our basement from water build up.
- I left most of my wall decor up on the walls as I walked out… even though some of it's already falling down due to humidity levels. My colleagues tell me there's no official check list like I'm used to in the past so I moved my breakables to the counter and I got out of there!
- This was probably my second most emotional ending to a school year. My mother, who lives 8 hours away in my home state of Michigan, has some health issues going on and my husband and I recently decided to rehome one of our dogs (something I thought I'd never do ever!). Why is this only the second most emotional? Because the worst were all tied to problems at school while these are in my private life.
Don't let this list fool you – there were positive things about my end of the year, but sometimes it's important to admit we're all human!


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To build a Mabon altar, you can use:
- Yellow Candles
- Bronze or Copper coins
- A plate of apples, berries, nuts and other harvest fruits
- Soft blue crystals, such as Blue lace agate
- or Rose Quartz
- Symbolic salmon, representing the salmon of knowledge
- A green or blue cloth
- Small personal treasures that you no longer use, to sacrifice as a symbol of what no longer serves you.
- A blue dish of water.
Set up altar facing the West.
Once you have built your altar, you can design any number or rituals.
Suggested themes: abundance, mending quarrels, forgiving transgressions, gratitude, fruition of long term goals, harvest, sharing the wealth/abundance


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Out on Cumberland Island, Georgia there is herd of about 200 wild horses. They have been on this island, which is the size of Manhattan, since the 1700's. They are a mixture of many breeds and they do not have an easy life. However, they have outlasted the plantation owners and the steel barons that once built their mansions there.
Monthly Archives: September 2014
Sapelo Island Afternoon
It was a hot afternoon on Sapelo Island when I saw this dog lounging in the back of the pickup truck ( no license plate or tailgate). His master had apparently gone fishing and left him here in the shade. I took a snapshot and forgot about it for a year. Last week I pulled it out and did this painting.


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Cedar Waxwing Bird Plush Stuffed Animal Toy with Sounds – National Bird Day
January 5 is National Bird Day. A special way to celebrate this day is in admiring and learning about a special unique looking bird called the Cedar Waxwing. With it's multi-colored plumage it is totally alone in it's beautiful appearance. Like the colors of a woodsy rainbow the Cedar Waxwing has black, red, green, silver, gold and white colors extending out it's wings, tail, torso and head. The Cedar Waxwing Plush Stuffed Animal Toy also features a sound chip which has authentic Cedar Waxwing sounds in affiliation with the National Audubon Society. Many other bird plush stuffed animals with sounds are available from Anwo.com Animal World including Blue Jay, Cardinal, Crow, Puffin, Chickadee, Kingfisher, Falcon, Red Tail Hawk and Loon. Bird Plush Stuffed Animals Toys with Sounds can serve so many purposes from Teachers aids, to Christmas Tree ornaments to business giveaways to clients to help them remember a specific trait like a certain insurance company has used a noisy white duck to help customers remember a specific insurance company. Birds bring so much beauty and color to our wildlife world. The Cedar Waxwing Plush Stuffed Animal Toy with Sounds as well as all the Bird Plush Stuffed Animals with authentic bird sounds are all a perfect way to educate, entertain and enjoy the bird world.
Posted on January 5, 2011, in Animal and Pet Lover Gifts, animal gift, Animal Gifts, Anwo.com Animal World, bird plush stuffed animals with sounds, cedar waxwing plush stuffed animal toy, National Bird Day January 5 and tagged animal gifts, animal world, anwo, bird plush stuffed animals with sounds, cedar waxwing plush stuffed animal toy, January 5 National Bird Day, wildlife plush stuffed animals. Bookmark the permalink. Leave a comment.