Amyotrophic lateral sclerosis as a spatiotemporal mislocalization disease: location, location, location. - PDF Download Free (2024)


Amyotrophic Lateral Sclerosis as a Spatiotemporal Mislocalization Disease: Location, Location, Location Noga Gershoni-Emek1, 2, Michael Chein1, 2, Shani Gluska1, 2 and Eran Perlson1, 2, * 1

Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel 2 The Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel *Corresponding author: E-mail: [emailprotected]

Contents 1. Introduction 2. Amyotrophic Lateral Sclerosis 2.1 Models for ALS Research 3. Axonal Transport 3.1 Axonal Cytoskeleton 3.2 Motor Proteins 3.3 Mitochondrial Transport 4. RNA-Binding Proteins 4.1 Tar DNA-Binding Protein 43 4.2 Fused in Sarcoma 4.3 MicroRNA and RBP 4.4 Chromosome 9 Open Reading Frame 72 5. Neurotrophic Factors and Their Precursor Forms 6. Death Signals 6.1 p75 Neurotrophin Receptor 6.2 Death Receptor 6 6.3 Fas 6.4 Semaphorins 7. Amyloid Precursor Protein 8. Concluding Remarks Acknowledgments References

International Review of Cell and Molecular Biology, Volume 315 ISSN 1937-6448

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© 2015 Elsevier Inc. All rights reserved.




Noga Gershoni-Emek et al.

Abstract Spatiotemporal localization of signals is a fundamental feature impacting cell survival and proper function. The cell needs to respond in an accurate manner in both space and time to both intra- and intercellular environment cues. The regulation of this comprehensive process involves the cytoskeleton and the trafficking machinery, as well as local protein synthesis and ligand–receptor mechanisms. Alterations in such mechanisms can lead to cell dysfunction and disease. Motor neurons that can extend over tens of centimeters are a classic example for the importance of such events. Changes in spatiotemporal localization mechanisms are thought to play a role in motor neuron degeneration that occurs in amyotrophic lateral sclerosis (ALS). In this review we will discuss these mechanisms and argue that possible misregulated factors can lead to motor neuron degeneration in ALS.

1. INTRODUCTION Neurons are unique among cells in their highly polarized morphology and extended neurites: short, branched dendrites and long polarized axons. The morphological differences between dendrites and axons, first pointed out over a century ago by Ramon y Cajal, reveal functional differences that require many molecular distinctions. The electrical signals propagated throughout the nervous system are only one type of communication taking place within the neuron and between the neuron and its diverse microenvironments. In order to facilitate the inter- and intracellular communication essential for the neuron’s maintenance and survival, tightly regulated mechanisms of ligand–receptor specificity, local synthesis, and cellular transport are necessary. In this review we discuss the mechanisms that can regulate multifactorial spatiotemporal localization and so control cell fate and function. We emphasize these notions to suggest that alterations in these mechanisms may underlie ALS pathology. One of the early common events occurring in many neurodegenerative diseases, including ALS, is a defect in transport machinery that leads to misregulated spatiotemporal localization of various factors. While neurodegeneration could be largely explained by dysfunctional supply/clearance, recent discoveries highlight the contribution of impaired signaling to neuronal dysfunction (Fanara et al., 2012). The active intracellular transport of organelles, proteins, and RNA can ensure proper spatiotemporal localization of various factors. This process requires molecular motors that move along the “highways” of the neuron’s cytoskeleton as well as targeting

Spatiotemporal Localization in ALS


and anchoring factors to specific places at specific times. Anterograde transport, originating from the cell body, supplies the distal parts of the axon with newly synthesized RNA, proteins, and lipids as well as organelles like mitochondria, while retrograde transport clears away unwanted proteins for proper degradation in the cell body. Retrograde transport also allows for signals to be conveyed from the distal parts of the axon to the cell body, thus enabling the neuron to respond to a changing environment at distinct locations (Butowt and von Bartheld, 2009). Transport regulation is highly complex and occurs at many levels, starting from the cytoskeleton maintenance and modulation, followed by regulation of the transport vehicles, the molecular motors, cargoes, and the adaptor proteins that regulate the motors’ association either with the cargo or with the track. All these may be modulated in disease. Alterations may also be observed in mitochondria, which provide energy for cellular processes, Ca2þ buffering that can regulate protein interactions and more. Additional changes may occur in the proteins and mRNAs themselves. Dysregulation at any and all of these levels can lead to neuronal malfunction and death. Here, we will discuss these ideas in the context of ALS.

2. AMYOTROPHIC LATERAL SCLEROSIS Amyotrophic lateral sclerosis (ALS) is an adult-onset, progressive neurodegenerative disease that affects both upper and lower motor neurons. The degeneration of motor neurons leads to muscle atrophy and, eventually, death from respiratory paralysis, typically within 5 years of diagnosis. Pathological hallmarks of ALS include neuromuscular junction disruption, cytoplasmic protein aggregations, and at later stages also neuroinflammation. Most cases of ALS are sporadic (sALS), while approximately 10% are inherited, usually dominantly (familial ALSdfALS). Various genetic approaches over the past 20 years have enabled new insights into the mechanisms leading to disease initiation and progression. The first gene found to be ALS-related was superoxide dismutase 1 (SOD1), heralding the era of molecular research focusing on protein toxicity. Over 160 mutations in SOD1 have been found to link exclusively to ALS. Mutant SOD1 has been shown to have a gain-of-function toxicity that may manifest through several mechanisms, affecting mitochondrial metabolism, axonal degeneration, axonal transport, and more (Sreedharan and Brown, 2013).


Noga Gershoni-Emek et al.

The discovery of TDP-43 (Tar DNA-binding protein, tardbp) mutations in fALS cases introduced a new period in molecular ALS research. The numerous roles of TDP-43 in transcription, RNA shuttling, splicing, and translation and microRNA biogenesis pointed to RNA processing as a major mechanism underlying ALS. The discovery of ALS-linked mutations in fused in sarcoma (FUS), which shares similar functional domains to TDP-43, further enhanced this notion. Additionally, the discovery of these genes linked ALS with frontotemporal lobe dementia, creating a spectrum of proteinopathies. Most recently, the discovery of chromosome 9 open reading frame 72 (C9orf72) expansions as the most common genetic cause of ALS brought forth a new concept: repeat expansion pathology, despite a mechanism of action yet to be elucidated (Sreedharan and Brown, 2013).

2.1 Models for ALS Research The advancement of our knowledge of ALS relies largely on animal models of the disease. The diversity of ALS-related mutations has given rise to many animal models with phenotypes ranging from no effect on motor neuron function to severe neurodegeneration. The differences between these genetic models point to the uniqueness of each mutation and the complexity of the disease. While rodent models are suitable for studying motor function, many mechanistic molecular insights have been garnered from chick embryo (Tripathi et al., 2014), Drosophila melanogaster (Jaiswal et al., 2012) and Caenorhabditis elegans (Ash et al., 2010; Therrien and Parker, 2014). For many years, the gold standard for ALS research is the SOD1G93A mouse model. Despite SOD1 mutations being the underlying cause for w2% of ALS patients worldwide, the transgenic mouse recapitulates many features of ALS, including motor neuron loss, neuromuscular junction dysfunction, axonal transport defects, gliosis, and axonal degeneration. Moreover, recent exploration of ALS-related biomarkers supports the possibility of a molecular link between fALS and sALS, bolstering the benefits of this well-characterized model (Lilo et al., 2013). Mutations in SOD1 were also found in sALS cases (Babin et al., 2014; Guareschi et al., 2012). Additional SOD1 mouse models have also brought about some interesting discoveries, most notably that SOD1 toxicity stems from gain of function and the recent finding that overexpression of wild-type human SOD1 in mice can cause progressive motor neuron degeneration (Graffmo et al., 2013). A recently described low-copy transgenic SOD1G93A shows a slower progressive pathology, and may provide a more suitable model for studying early stages of the disease. SOD1 rodent models differ in age of

Spatiotemporal Localization in ALS


onset and disease progression, depending on the mutation, transgene expression level, gender and genetic background. Both knockout and overexpression of the tardbp gene are embryonic lethal, suggesting a gain of function and/or loss of function, depending on the tissue in which it is expressed, thus contributing to neurodegeneration in both a cell autonomous and non-cell autonomous manner (Tsao et al., 2012). Either way, it is clear that TDP-43 levels are tightly regulated. Various transgenic mice have been created harboring point mutations, and these all share a behavioral phenotype that includes mild motor dysfunction, progressive gait abnormalities, and hind limb clasping together with significant weight loss. In animal models attempting to model TDP-43-ALS, mostly axonal phenotypes have been observed, with mild MN loss. The observed range of severity can be attributed to gender, level of expression, timing of expression and promoter. Nevertheless, currently there is no conserved working TDP-43-ALS rodent model. Knockout of FUS is not similarly embryonic lethal (Kuroda et al., 2000), nor does it lead to neuronal loss and motor dysfunction, suggesting that the adverse effects of FUS are not a result of loss of function. Overexpression of the human wild-type protein causes severe degeneration of spinal cord neurons in hom*ozygous mice, while hemizygous mice showed no cellular pathology or motor dysfunction. (For a complete review of rodent models in ALS, see McGoldrick et al. (2013)). With the lack of a good in vivo ALS model, besides SOD1 mice with given limitations (underlying only w2% of ALS patients), in vitro cellular models are being used to study disease mechanisms, with the advantage of higher spatiotemporal resolution. Cell culture models provide a better-defined platform with a specific ability to control and manipulate conditions in order to gain invaluable mechanistic knowledge. At the cellular level, genetic manipulations of ALS genes show various abnormalities and MN toxicity affecting transport, localization, and local synthesis events (Veyrat-Durebex et al., 2014). As noncell autonomous functions are now widely accepted in ALS, coculture systems have been established in order to study the interactions between neurons and surrounding cells, such as astrocytes, glia, and muscle (Zahavi et al.; Park et al., 2012; Southam et al., 2013; Veyrat-Durebex et al., 2014). Furthermore, recent advances in stem cell biology have begun to contribute to ALS research, bringing with them aspirations for a broader perspective and new hypotheses. Using iPSC (induced Pluripotent Stem Cells) technology, somatic cells taken from a patient can be reprogrammed to recapitulate embryonic stem cell properties, and then differentiated into motor neuron


Noga Gershoni-Emek et al.

cultures recapitulating both familial and sporadic ALS models (Boulting et al., 2011; Chestkov et al., 2014; Dimos et al., 2008). There are high hopes for reprogrammed iPS cells in testing therapeutics (Kiskinis and Eggan, 2010) along with understanding molecular mechanisms that will bring about novel drug targets.

3. AXONAL TRANSPORT 3.1 Axonal Cytoskeleton The cytoskeleton makes up the infrastructure of “highways” on which cargoes are trafficked, targeted, and anchored throughout the cell. Three types of protein filaments make up the axonal cytoskeleton: microtubules (MTs), actin and intermediate filaments. MTs are stiff, hollow tubes w25 nm in diameter with a distinct polarity that results from the directed assembly of ab-tubulin dimers. Within axons, MTs form uniformly oriented bundles, where fast-growing ends, also known as plus ends, point toward the distal part of the axon, while the slow-growing minus ends are found closer to the cell body. This orientation is crucial for the directed movement of molecular motors along the MT, and to distinguish neuronal axons from dendrites (Franker and Hoogenraad, 2013). In axons, the long, polarized MT serves as a highway for long range transport between the cell body and the growth cone/presynaptic domain facilitated by the kinesin superfamily, which mostly move toward the plus-end, and dynein, which moves toward the minus end. Actin-based transport is thought to serve for short-range trafficking, facilitated by myosin motors (Kapitein and Hoogenraad, 2011; Maeder et al., 2014). MTs are highly dynamic structures that undergo a regulated process of growth and catastrophe. MT stability can be controlled by posttranslational modifications (PTMs) like acetylation, glycosylation, and detyrosination, and by MT-associated proteins (MAPs) like tau, plus-end proteins like EB1-3, and the motor proteins themselves. It was recently suggested that histone deacetylase 6 (HDAC6), a major alpha-tubulin deacetylase, which specifically interacts with mutant SOD1, regulates SOD1 aggregation, and is found sequestered in SOD1 inclusions (Gal et al., 2013). Inhibition of HDAC6 increases MT acetylation, which in turn recruits both dynein and kinesin-1 to MTs, and increases the vesicular transport of mitochondria and brain-derived neurotrophic factor (BDNF) (Dompierre et al., 2007). Hyperacetylation of tubulin was indeed found in SOD1 mutants (Munch

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et al., 2004), while it was also shown that HDAC6 deletion extends the survival of mutant SOD1 mice by reducing motor neuron degeneration and improving neuromuscular junction (NMJ) stability, however not the onset of MN disease (Taes et al., 2013). Unlike other histone deacetylases, HDAC6 deacetylases MTs rather than histones. Doing that, it hinders fast axonal transport in Alzheimer’s disease (AD) and Huntington’s disease (HD). Indeed, inhibition of HDAC6 increased MT acetylation and recruited motor proteins to MTs, thus restoring fast axonal transport in animal models of several neurodegenerative diseases, including Charcot– Marie–Tooth, HD, and AD (Dompierre et al., 2007; Hinckelmann et al., 2013). MAPs have important roles in MT stabilization, modification, anchoring, and more. Tau is an MT-stabilizing protein, regulating activity of molecular motors. Mutations in TAU have been found in frontotemporal dementia with Parkinsonism. Moreover, in mouse models with carrying mutations in TAU, its binding to MTs is impaired, and fast axonal transport slowed (Hinckelmann et al., 2013). Interestingly, TAU degradation was found to precede MT disassembly by KIF2A, a member of the kinesin superfamily and a protein inducing MT depolymerization (Maor-Nof et al., 2013). Stabilization of MTs, using pacl*taxel, for example, was shown to reduce neuronal degeneration after trophic factor deprivation or axotomy (Maor-Nof et al., 2013). Dysregulation of structural, motor, or adaptor proteins of the MT system has a major effect on axonal transport and thus neuronal health, has been associated with several neurological disorders, including ALS (Cooper-Knock et al., 2014; Franker and Hoogenraad, 2013; Reddy et al., 2013). Actin filaments (F-actin) are built of globular actin monomers (G-actin), and also have a distinct polarity. Actin-based transport serves for short-range trafficking, facilitated by myosin motors (Kapitein and Hoogenraad, 2011; Maeder et al., 2014). The actin network is highly enriched at the cell cortex, due to dynamic actin growth close to the cell membrane, while dissociation occurs at the minus end. In the axon, actin is particularly abundant at the growth cone, a specialized structure at the growing tip of the axon. Dysregulation of actin dynamics as a result of an ALS-linked mutation in profilin-1 leads to defects in axon outgrowth and disrupted growth cones (Wu et al., 2012). Genome-wide association studies suggest actin cytoskeleton genes are altered in sporadic ALS (Kim et al., 2012). Intermediate filaments make up a large family of proteins, whose members include neurofilaments (NFs) characterized by their molecular


Noga Gershoni-Emek et al.

weight: NF-H (heavy), NF-M (medium), and NF-L (light), type-III intermediate filaments like peripherin, vimentin, and more, to form a fibrous protein w10 nm in diameter. These members are differentially expressed in the human brain. The intermediate filaments differ from actin and MTs by their lack of polarity, evolutionary conservation, and dynamic assembly and disassembly. Aggregation of NFs is an early event in ALS pathogenesis, leading to degeneration of neurites in a cell autonomous manner (Chen et al., 2014). Peripherin expression is upregulated in response to injury and inflammation, and is sufficient to cause late-onset motor neuron disease (Beaulieu et al., 1999). A mutation in the peripherin gene (PRPH) was documented in one sALS patient (Corrado et al., 2011), and peripherin has been shown to aggregate with NFs in cytoplasmic inclusions, while overexpression of the wild-type isoform slows down the transport of NF in neurites, causing cytoplasmic aggregates prior to disease onset (Millecamps et al., 2006). In dorsal root ganglions (DRGs), locally translated vimentin, whose mRNA is selectively transported to axons in response to growth stimuli (Willis et al., 2005), is key for binding phosphorylated MAP kinases to dynein and retrograde transport in response to injury (Perlson et al., 2005). Numerous cytoskeleton-related genes were found to be dysregulated in tissues derived from mSOD1 transgenic mice (Ferraiuolo et al., 2007; Guipponi et al., 2010; Maximino et al., 2014; Strey et al., 2004), some at the presymptomatic stage, indicating a possible relation to ALS pathogenesis. Mutant SOD1 causes a significant decrease in NF-L mRNA levels by directly binding to its 30 UTR, and brings about a reduction in protein levels of all three NF subunits (Chen et al., 2014). The 30 UTR of NF-L mRNA is also a target of TDP-43, which binds and stabilizes it against degradation (Strong et al., 2007), as well as of a subset of microRNAs (miRNAs) whose expression is altered in sALS spinal cords (Campos-Melo et al., 2013). NF abnormalities have been attributed to ALS, and genetic mutation of NF-related proteins may lead to neuropathies, however, may also contradict SOD1 toxicity in certain conditions (Julien and Beaulieu, 2000). NF proteins as well as antibodies identifying them are found in plasma and CSF (Cerebrospinal fluid) of ALS patients, and are considered promising biomarkers for ALS in the future (Gaiottino et al., 2013; Lu et al., 2014; Puentes et al., 2014). Another level of cytoskeleton regulation beside the PTMs and MAPs described above, are kinases that can phosphorylate cytoskeletal proteins, thus controlling their function. p38 is a kinase belonging to the mitogenactivated protein kinase family, which phosphorylates NFs, among other

Spatiotemporal Localization in ALS


proteins. In mice models of ALS, activated p38 was recently shown to mediate mutant SOD1 inhibition of fast axonal transport (Ferraiuolo et al., 2011; Morfini et al., 2013; Tortarolo et al., 2003). The Stathmin (STMN) family of proteins includes STMN1, SCLIP, SCG10, and RB3 are expressed solely in the nervous system, with the exception of STMN1 (Charbaut et al., 2001). STMN family proteins bind and sequester tubulin, thus have an important role in MT dynamics. In ALS (Maximino et al., 2014; Strey et al., 2004), as well as in other motor neuron diseases (Jablonka et al., 2014), their expression is altered, and their phosphorylation levels hindered. Interestingly, STMN1 is necessary for the maintenance of the D. melanogaster NMJ (Graf et al., 2011). Knockout of STMN1 was shown to counteract some neuromuscular defects related to spinal muscular atrophy (SMA), however, it did not prolong survival in SMA-like mice (Wen et al., 2013). In neurons cultured from a mouse model of progressive motor neuropathy, STMN binds STAT3 to mediate ciliar neurotrophic factor rescue effects, i.e., axonal elongation and maintenance (Selvaraj et al., 2012). Although highly researched in the context of its cell cycle activities related to malignancies, information regarding the role of STMN family proteins in neurodegenerative diseases is still not clear. As its MT-destabilizing properties may be relevant to novel therapeutic methods, further investigation of its effects on MT dynamics in neurons is highly promising.

3.2 Motor Proteins Motor proteins are the “vehicles” that power the movement of cargoes within the cell. Fast axonal transport requires available energy, thought to be provided solely by local mitochondria. However, recent evidence suggests that the ATP necessary for vesicle movement may be provided locally by glyceraldehyde-3-phosphate dehydrogenase anchored to the vesicular membrane by huntingtin (Zala et al., 2013). Kinesin moves predominately anterogradely to transport a variety of cargoes (Vale et al., 1985). Conventional kinesin is composed of two heavy chains and two light chains. The heavy chain, or motor polypeptide, is composed of an N-terminal motor domain, a long coiled-coil stalk and a globular tail domain (Vale, 2003). Kinesin’s highly processive motion is due to a hand-over-hand mechanism in which one motor domain (or head) moves forward while the other remains tethered to the MT track (Yildiz et al., 2004). Anterograde movement is very selective, with specificity conferred by different kinesin family members transporting specific


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cargoes, or by the use of various adaptor proteins. Few neurodegenerative diseases have been linked to mutations in kinesin (Chevalier-Larsen and Holzbaur, 2006), and targeted disruption of kinesin function is sufficient to induce neurodegeneration (Xia et al., 2003). Several gene members of the kinesin-like protein family (KIF) are dysregulated in both tissues and cell cultures derived from mSOD1 mice along disease progression (Ferraiuolo et al., 2007; Maximino et al., 2014). Two members of this family, KIF1Bb and KIF3Ab, were downregulated in cortices of sporadic ALS patients, but not in mSOD mice (fALS). Surprisingly, members of the kinesin superfamily can also participate in the regulation of MT dynamics, as KIF2A was recently shown to be a key factor in MT destabilization and axonal regression (Maor-Nof et al., 2013). Cytoplasmic dynein is the main motor protein driving retrograde transport. Dynein is a large complex, composed of two heavy chains that form the motor domains, and several intermediate and light chains. Dynein plays a part in multiple cellular processes, including mRNA localization, targeting, and anchoring. Further, an MT tethering role that facilitates synapse stabilization by interaction with adhesion molecules was recently described for dynein (Perlson et al., 2013). It will be interesting to see if these types of roles are altered in ALS leading to synapse disruption and to axon degeneration by a dying-back mechanism. Two missense mutations in the cytoplasmic heavy chain (Dnchc1) in mice result in distinct phenotypes and mild motor neuron disease. The legs at odd angles (Loa) and Cramping1 (Cra1) heterozygote mice both suffer from age-related progressive loss of locomotor ability and muscle tone, without affecting life span, while hom*ozygous mice display a more severe phenotype and die within 24 h of birth. Loa mice also exhibit axon loss in proprioceptive neurons (Ilieva et al., 2008). The Loa mutation occurs in the domain thought to be the binding site for dynein intermediate chains, while the Cra1 mutation is found in the putative hom*odimerization site. At the cellular level, the main effect of a hom*ozygous Loa mutation is a reduction in the frequency of high-speed trafficking, with an increase in stationary pauses. It is important to note that both Loa and Cra1 affect specific functions of dynein within motor neurons, without causing severe deficits in other DNCHC1 functions (Hafezparast et al., 2003). Surprisingly, crossing Loa mice with SOD1G93A resulted in a delay of disease onset and extended life span, without changing disease progression (Kieran et al., 2005). At the cellular level, the heterozygous Loa mutation significantly increased retrograde transport. Ilieva et al. (2008) suggest that mutant

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Loa-conferred improvement is due to a decrease in glutamatergic input resulting from loss of proprioceptive neurons, while El-Kadi et al. (2010) attribute the improvement to an amelioration of mitochondrial function in mSOD1 mice due to morphological and structural modifications to the mitochondria that make them less prone to associating with mutant SOD1. Cra1 heterozygote mice show no significant loss of primary motor neurons, and the behavioral phenotype has been attributed to the decrease in complexity of the neuromuscular junction (Courchesne et al., 2011). Interestingly, the Sprawling mouse model which has a short deletion in the dynein heavy chain, although presenting proprioceptive sensory neurons defects, shows no effect on motor neurons (Chen et al., 2007), and cannot mitigate mSOD1 neurodegeneration (Banks and Fisher, 2008). Many protein complexes can regulate efficient and processive dynein motility along the MT. Dynactin can serve as an activator, as well as a specific cargo adaptor protein in order to optimize dynein motility (McKenney et al., 2014). Binding of dynein to dynactin, and of dynactin to MT is necessary for efficient movement, and can be further regulated by additional proteins that are responsible for the selective binding of cargo. An example of one of these proteins is BicD2, a dimeric adaptor protein that binds dynein to Rab6 GTPase on membrane organelles. The N-terminal coiled-coil of BicD2 enables the interaction between dynein and dynactin, thus forming a stable ternary structure that moves processively and robustly along MT. Additional coiled-coil cargo-linking proteins were also show to increase processivity of dynein–dynactin complexes: Spindly on kinetochores, Hook3 on early endosomes, and Rab11-FIP3 on recycling endosomes. Lis1 operates like a “clutch” that shifts transmission between the “engine” and the track-binding domain, bringing dynein to a stop, possibly to prepare the dynein complex for transport and to allow it to move large cargoes by extending dynein’s dwell time at the MT plus-end (Huang et al., 2012). The disruptions of the dynein–dynactin complex, as well as mutations in dynactin, are sufficient in causing late-onset motor neuron disease (LaMonte et al., 2002). The p150glued subunit of dynactin interacts directly with the dynein motor, while independently binding MTs- and plus-end-binding proteins, EB1 and EB3. These interactions are mediated by the N-terminal cytoskeleton-associated protein glycine-rich (CAP-Gly) domain (Ligon et al., 2003; Moughamian and Holzbaur, 2012; Waterman-Storer et al., 1995). CAP-Gly is necessary for the initiation of retrograde transport at synaptic termini (Lloyd et al., 2012), where it recruits dynein onto MT,


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and maintains the association between the MT and the motor protein. CAP-Gly can additionally act as a brake to slow dynein motility (Ayloo et al., 2014). Mutations in the CAP-Gly domain have been linked to two neurodegenerative disorders, Perry syndrome (Farrer et al., 2009) and distal hereditary motor neuropathy 7B (Puls et al., 2003). Various mutations in the dynactin p150 subunit have also been linked to ALS, as well as to Parkinsonisms (Munch et al., 2004), some causing aberrant cellular localization such as aggregation or MT localization (Stockmann et al., 2013). Of these, the best characterized is the G59S mutation. Transgenic mice harboring the G59S mutation exhibit an MN disease phenotype. Pathological abnormalities are consistent with sporadic ALS: loss of motor neurons, ubiquitin-positive inclusions, accumulations of NFs, and astrocytic gliosis, as well as denervation and muscle atrophy prior to the onset of clinical signs. At the cellular level, dysregulation of vesicular transport leads to an accumulation of autophagosomes and dilated endoplasmic reticulum (Laird et al., 2008) (see Figure 1).

3.3 Mitochondrial Transport Mitochondrial transport in neurons serves two important purposes: trafficking mitochondria in accordance with changing metabolic needs (e.g., synaptic terminals and growth cones where energy is in high demand) and spatiotemporal signaling events that can regulate apoptosis process. Therefore mitochondrial transport is a complex, tightly regulated system (Sheng, 2014). In axons, the majority of mitochondria (w70%) is stationary, but can also move over large distances in both directions (Schwarz, 2013). Mitochondria can shift between motile and stationary states, in addition to pauses and abrupt changes in direction, pointing to a dynamic interaction with kinesins, dyneins, and adaptor proteins (Sheng, 2014). Mitochondria are trafficked along MTs and depend on the MT-based motors, especially the KIF5 of proteins (Pilling et al., 2006). In D. melanogaster, Milton recruits the kinesin heavy chain, but not the light chain, to mitochondria (Glater et al., 2006). This role is filled by the orthologous Trak1 and Trak2 in mammals (Brickley and Stephenson, 2011). Trak1 and Trak2 contain an N-terminal KIF5B-binding domain, and two dynein/ dynactin-binding domains at the N- and C-termini, thus mediating both anterograde and retrograde transport. While Milton is exclusively a mitochondria adaptor, mammalian Trak proteins can fulfill additional roles (Russo et al., 2009).


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EB1 tau


Figure 1 Regulation of axonal transport at four levels: (A) Cytoskeleton stability and dynamics that can be modified by either posttranslational modifications like acetylation, glycosylation, detyrosilation, or by various microtubule-associated proteins. (B) Motor proteins and (C) adaptor proteins, that can control direction and processivity as well as specific cargo binding. (D) ATP availability, can be influenced, for example, by mitochondria mislocalization and dysfunction. Alterations in each of these levels may lead to axonal transport defects and to changes in the spatiotemporal localization of signaling events that are essential to the neuron health and proper function.

Mitochondria associate with Milton/Trak via Miro, an outer membrane Rho-GTPase receptor with Ca2þ-binding motifs (Fransson et al., 2006). In D. melanogaster, mutations in Miro lead to a depletion of mitochondria at distal synapses as a result of impaired anterograde transport (Guo et al., 2005), while overexpression of Miro in mammals leads to increased mitochondrial transport, probably due to the recruitment of more Trak2 and motors. It has been suggested that Miro acts as a Ca2þ sensor: when Ca2þ is absent Miro can bind Milton, and mitochondria are attached to MTs, while in the bound state, Miro cannot bind Milton and mitochondria are uncoupled from the MTs (Rice and Gelfand, 2006). Recent data, however, disprove this suggestion (Nguyen et al., 2014). Neuron-specific knockdown


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of Miro in mice severely impaired retrograde transport of mitochondria without affecting anterograde transport, and resulted in upper motor neuron disease phenotype and pathology (Nguyen et al., 2014). Syntabulin has also been shown to be a KIF5B adaptor protein, necessary for anterograde transport (Cai et al., 2005), along with several other proteins. This wealth of adaptor proteins hints to the existence of a complex regulatory system for mitochondria trafficking in response to various environmental signals (Sheng, 2014). What makes mitochondria stop once it reaches its proper destination? One possibility is dissociation from the MT track, a second is anchoring to the MTs, or linking to other cytoskeletal elements. Syntaphilin has recently been characterized as an “anchor protein” that docks mitochondria to the MT skeleton by inhibiting the ATPase activity of kinesin (Kang et al., 2008). Along the axon, stalled mitochondria correlate with sites of axonal branching, a process which requires local mRNA translation. These suspended mitochondria generate “hot spots” for active translation by coordinating the localization of mitochondria, mRNA, and protein translation machinery. Mitochondria stalled at previously formed local translation sites respond to signaling cues such as neuronal growth factor (NGF) to regulate translation both spatially and temporally (Spillane et al., 2013). Mitochondria are also important signaling hubs. For example, the A-kinase-anchoring protein 1 (AKAP1) is embedded in the outer mitochondrial membrane, and can recruit multiple proteins, among them kinases and Argonaute-2 (important for small RNA-induced silencing) and mRNAs to the mitochondria. AKAP1 knockdown results in mitochondrial fragmentation and apoptosis, while its overexpression confers neuroprotection (Merrill and Strack, 2014). Disrupted mitochondrial transport and mitochondrial pathology is a hallmark of many age-related neurodegenerative diseases, including AD and ALS. The link between mitochondria and axonal degeneration is seemingly self-explanatory: mitochondria are essential as a local energy source as well as a buffer for intracellular Ca2þ, and dysfunctional mitochondria also secrete harmful reactive oxygen species. Therefore, the disruption of mitochondria function and distribution would lead to neurodegeneration and cell death. Numerous syndromes, many with neurological effects, result from defects in the oxidative phosphorylation (OxPhos) system, but to date none have been linked to ALS (Schon and Przedborski, 2011). However, accumulating evidence points to mitochondria playing a larger role in the maintenance of neuronal health, possibly related to trafficking.

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The aberrant activity and localization of mutant SOD1 can directly affect mitochondrial health and function. The physiological role of SOD1, in the cytoplasm and in the intermembrane space (IMS), is the reduction of the superoxide anion radical to H2O2, which is then further reduced to H2O by catalase, glutathione peroxidase, or peroxiredoxins. Under physiological conditions, the IMS activity of SOD1 is suppressed by redox state control. Mutant SOD1 is able to evade this regulation. Mitochondrial localization of SOD1 depends on its unfolded state, and is enhanced in the spinal cord of ALS patients (Liu et al., 2004). This translocation leads to increased production of H2O2 under stress conditions, which is consistent with findings from rodent models and ALS patients (Goldsteins et al., 2008; Panov et al., 2011). Additional mitochondrial defects seen in mSOD1 ALS include mitochondrial permeability. Direct binding of mSOD1 to voltagedependent ion channel (VDAC1), has been shown to diminish ADP passage through the outer membrane, and to accelerate onset of paralysis (Israelson et al., 2010). SOD1 and Bcl-2 compete for VDAC1 binding at the mitochondria. An initial event of mSOD1 toxicity has been shown to be the alteration of Bcl-2 folding, creating a toxic protein, which alters its binding to VDAC1 and leads to reduced mitochondrial ADP permeability. The mSOD1/Bcl-2 complex appears to act upstream to VDAC1, and may be another mechanism by which mSOD1 promotes mitochondrial dysfunction (Pedrini et al., 2010; Tan et al., 2013). Hence, three mechanisms that may play a part in SOD1-related mitochondria toxicity are translocation into the IMS, activity control of SOD1 within the IMS (Vehvilainen et al., 2014), and altered mitochondrial permeability. Mutations in valosin-containing protein (VCP), recently found in both familial and sporadic ALS, lead to the uncoupling of mitochondrial respiration from OxPhos. This results in a decrease of mitochondrial respiration, higher respiration, and decreased ATP levels (Bartolome et al., 2013), making the cells more vulnerable to environmental insult. VCP also has a role in the translocation of outer membrane proteins to the cytosol for further proteasome-dependent degradation (Xu et al., 2011). In TDP-43 mutant mice, mitochondria accumulate in cytoplasmic inclusions at the cell body, while diminishing at nerve end terminals, resulting in an immature NMJ (Shan et al., 2010). Mitochondria in mutant TDP-43 cells also exhibit aberrant morphology and function (Stribl et al., 2014). Mitochondrial dysfunction can contribute to neurodegenerative pathologies not only by affecting its role in cellular respiration and buffering at the cellular level, but also by changing localization. The alterations in


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mitochondrial motility and distribution in ALS can promote axonal degeneration by locally affecting energy levels and Ca2þ, and can also affect the localization of additional cellular components that may rely on mitochondria as a scaffold or as a transport vehicle.

4. RNA-BINDING PROTEINS Axonal protein synthesis is another transport-related mechanism that can regulate the spatiotemporal localization of factors, and may be altered in ALS. This process enables neurons, highly polarized and compartmentalized cells, to functionally adapt to their environment in a spatially and temporally precise way. Local synthesis is regulated by mRNA transport, targeting, anchoring, and on-site translation. mRNA transport and proper localization involves RNA-binding proteins (RBPs) that bind to the 30 and/or 50 UTRs of the mRNA, regulatory RNA species, and accessory proteins that form transport RNA granules. The cellular transport “highways” and “vehicles” move and anchor the RNA, proteins, and organelles necessary for local synthesis in concert with various regulators that make the process more specific and efficient. Transport of specific mRNA species can occur as a response to extrinsic growth stimuli (Willis et al., 2005), and the local synthesis machinery and regulators transport to a specific location in response to axonal insult (Michaelevski et al., 2010). Interestingly, mutations in RBPs like TDP-43 and FUS were discovered in ALS. The properties and shuttling abilities of TDP-43 and FUS make them potential players in this essential ability.

4.1 Tar DNA-Binding Protein 43 The initial link between TDP-43 and ALS was found in 2006, when TDP43 was identified as a major component of ubiquitinated protein aggregates in ALS patients. This was followed shortly by genetic studies that uncovered mutations in both familial and sporadic ALS patients. TDP-43 contains two RNA recognition motifs (RRM1 and 2), and a glycine-rich C-terminal region, wherein most disease-related mutations have been located (LagierTourenne et al., 2010). RRM1 mediates binding of TDP-43 to RNA, while RRM2 is thought to have a role in DNA binding within the nucleus. The C-terminal region is thought to mediate interactions with various ribonucleoproteins to mediate cellular processes such as transcription,

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mRNA processing, and miRNA biogenesis (Buratti and Baralle, 2010; Gregory et al., 2004; Lagier-Tourenne et al., 2010). TDP-43 is ubiquitously expressed. While it primarily localizes to the nucleus, it can shuttle between the nucleus and the cytoplasm (Ayala et al., 2008). Within the cytoplasm, TDP-43 can be located in stress granules (SGs), which are transient sites of translational repression where mRNAs are harbored after exposure to environmental stress (Li et al., 2013). TDP-43 is not required for SG formation, but it does contribute to its dynamics. Cytoplasmic redistribution of TDP-43 is an early event in ALS (Giordana et al., 2010). In neurons, TDP-43 can also be found in distal parts of the axon and at the NMJ, a pattern of localization shown to be disrupted in cells harboring mutant forms of TDP-43 (Alami et al., 2014). TDP-43 is a common factor of ubiquitin-immunoreactive cytoplasmic inclusions in ALS-affected cells (De Marco et al., 2011), regardless of the genetic cause (Wu et al., 2012). A recent study by Alami et al. (2014) used live-cell imaging in D. melanogaster MN axons as well as in mouse cortical neuron cultures to show that TDP-43 selectively associates with mRNP granules that move anterogradely along the axon. This movement is MT dependent, and disrupted by ALS-linked mutations in TDP-43, possibly explaining its impaired localization at distal parts of the axon and the NMJ. This new data suggest that TDP-43 has a functional role in mRNP trafficking that may regulate essential local synthesis events (Alami et al., 2014). TDP-43 was also shown to localize to mitochondria in motor neuron axons and dendrites. This localization was enhanced by ALS-associated mutations (Wang et al., 2013). Mutant TDP-43 was shown to affect mitochondria morphology in dendrites and soma of motor neurons, but not in axons. This effect was due to a shift toward more fission events, tipping the balance between fission and fusion events in mitochondria. When the effect of TDP-43 on mitochondria transport was queried, overexpression of TDP-43 showed a reduction in bidirectional transport and an increase in stationary mitochondria in both axons and neurons. This effect was further enhanced by ALS-linked mutations in TDP-43. Knockdown of TDP-43 showed similar effects, thus alterations in spatiotemporal localization of TDP-43 and mitochondria link to ALS toxicity in a mechanism yet to be discovered. The contribution of TDP-43 to axonal degeneration may also derive from its direct effect on the cytoskeleton. By binding to NFL mRNA, TDP-43 contributes to its stability, but also sequesters it to SGs, thus disrupting the proper function and localization of both protein and mRNA


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necessary for maintaining the axonal cytoskeleton (Tripathi et al., 2014). Among the mRNAs directly bound and regulated by TDP-43 is TDP-43 itself (Ayala et al., 2011), suggesting a feedback loop type of regulation.

4.2 Fused in Sarcoma Another interesting RBP, mutated in 4% of fALS patients is FUS. FUS, originally discovered as a fusion oncogene (Crozat et al., 1993; Rabbitts et al., 1993), shares many of the characteristics of TDP-43, including the cytoplasmic inclusions that have become a hallmark of the disease (Kwiatkowski et al., 2009; Vance et al., 2009). It is an RBP and DNA-binding protein with many mRNA targets, able to shuttle between the nucleus and cytoplasm. ALS-linked mutations shift this balance toward the cytoplasm (Kwiatkowski et al., 2009). Mutant FUS binds and sequesters wild-type FUS into SGs, and delays their formation in response to stress, while accelerating their dissociation (Baron et al., 2013). This localization is attributed to dysfunctional nuclear import as a result of ALS-linked mutations in the nuclear localization sequence. FUS recruits and incorporates mRNA into SGs, where it surprisingly promotes protein translation in distinct cellular compartments (Yasuda et al., 2013). Accordingly, RNA-binding incompetent FUS does not incorporate into SGs, and predominately localizes to the nucleus (Daigle et al., 2013). Nuclear aggregation of FUS has also been suggested to cause loss of function (Schwartz et al., 2014). In hippocampal neurons, FUS has been shown to associate with PSD-95 in dendritic spines (Aoki et al., 2012; Fujii et al., 2005), but was not detected in axons. Its transport in dendrites depends both on MTs and actin, as it can associate with both kinesin (Kanai et al., 2004) and myosin (Yoshimura et al., 2006). FUS knockout mice exhibit abnormal dendritic spine morphology, possibly due to disrupted transport of the Nd-1L mRNA by FUS (Dormann and Haass, 2013; Fujii and Takumi, 2005). The most compelling insight linking FUS-mediated neurodegeneration and ALS comes from D. melanogaster, where overexpression of FUS causes a significant reduction in the levels of both the postsynaptic scaffolding protein Dlg (hom*ologous to the mammalian PSD-95), and the presynaptic scaffold Bruchpilot (Brp), resulting in a disrupted NMJ, both structurally and functionally (Machamer et al., 2014).

4.3 MicroRNA and RBP miRNAs have been found to be involved in neuronal development and maintenance, while their absence has been shown to lead to

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neurodegeneration (Haramati et al., 2010). The relative enrichment of specific miRNAs in axons (Maximino et al., 2014) points to a selective transport mechanism. The many putative targets for miRNA recognition at the distal axon suggest multiple functions. Upon their identification, both TDP-43 and FUS were thought to be involved in microRNA biogenesis and/or regulation. This was due to their association with Drosha, the nuclear endonuclease that executes the first cleavage step in microRNA processing (Gregory et al., 2004), and to their RNA-binding properties. Indeed, both TDP-43 and FUS were recently implicated in miRNA-based regulation. TDP-43 has the ability to regulate levels of specific miRNAs, possibly by direct binding to the miRNA or precursor elements (Buratti et al., 2010). A direct physical interaction with the miR-1 family, which consists of miR-1 and miR-206, was recently described. Both miRs are enriched in cardiac and skeletal myoblasts, and miR-206 has been directly linked to ALS (Williams et al., 2009). While depletion of miR-206 does not affect muscle development and function, it does prevent the efficient regeneration of NMJs. In SOD1 mice, the presence of miR-206 slows down disease progression, even though miR-206 is not expressed in motor neurons. The physical interaction between TDP-43 and miR-206 impairs its ability to bind to Ago2, the major component of the RNA-induced silencing complex. Accordingly, miR-206 targets HDAC4, an inhibitor of muscle regeneration, which was found to be upregulated in TDP-43 mutant mice. This finding correlates with data from human patients that show upregulation of HDAC4 in ALS patients, despite no known alterations in miR-1 or miR-206 expression (Bruneteau et al., 2013; King et al., 2014). An additional target of miR-206 is BDNF, shown to be downregulated in AD, corresponding with an increase in miR-206 levels (Lee et al., 2012). Downregulation of BDNF by miR-206 was also shown to impair innervation of airway smooth muscle in a mouse model (Radzikinas et al., 2011). Interestingly, injection of CSF collected from ALS patients caused an increase in miR-206 expression in mouse gastrocnemius muscle (Sumitha et al., 2014), while miR-206 was found to be elevated in serum samples of ALS patients and in blood plasma of SOD1G93A mice, suggesting further research into the role of miR-206 as an ALS biomarker (Toivonen et al., 2014) Moreover, as miR-206 has putative targets in the neuron, but is not expressed there, the question arises of a possible mechanism of miRNA transfer between muscle and neuron, perhaps by use of exosomes.


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A rare mutation in the FUS 30 UTR inhibited miR-200a and miR-141 binding, and partially accounted for an increase in FUS protein levels, pointing to an autoregulatory mechanism of FUS expression. FUS interacts in vitro with both pri-miR-141 and pri-miR-200a (Dini Modigliani et al., 2014), as well as with the chromosomal loci encoding them (Morlando et al., 2012). Hence, FUS is directly involved in the biogenesis of specific miRNAs that participate in its own regulation (Dini Modigliani et al., 2014). miR-200a has previously been linked to AD (Ding et al., 2012) and HD (Jin et al., 2012) and its targets participate in the regulation of synaptic function, development, and neuronal survival. The possibility of a convergence between the TDP-43 and FUS downstream pathways has long been discussed. There is currently evidence for the interaction of TDP-43 and FUS in regulation of HDAC6 by direct binding to its mRNA, possibly by competing for common binding sites. As simultaneous knockdown of both genes did not show an additive effect, it is possible that they work in a complex or sequentially to process HDAC6 mRNA (Kim et al., 2010). In addition to its role in MT stabilization described previously, HDAC6 may also play a part in control of motorbased transport or cargo selection due to its interaction with the p150 subunit of dynactin (Hubbert et al., 2002), as well as in protein degradation and clearance (Valenzuela-Fernandez et al., 2008) and (Rodriguez-Gonzalez et al., 2008).

4.4 Chromosome 9 Open Reading Frame 72 The hexanucleotide repeated expansion GGGGCC in the first intron located between exons 1 and 2 of C9ORF72 has been found to be the most prevalent genetic cause of ALS (DeJesus-Hernandez et al., 2011; Renton et al., 2011). Although the physiological function of C9ORF72 is still largely unknown, it may play a role in endosomal trafficking and autophagy. Thus reduced expression and loss of function may lead also to alteration in spatiotemporal localization of essential factors. Other toxic possibility being consider, include RNA aggregation (Heutink et al., 2014). Although it is not an RBP, its toxic effects appear to be through aberrant RNA localization and function, hence its appearance in this section. Similarly to TDP-43 and FUS, C9ORF72 is expressed in the nucleus and cytoplasm, as well as being secreted into the CSF (Farg et al., 2014). Both wild-type and mutant C9ORF72 associate with hnRNPs that shuttle between the nucleus and cytoplasm and aggregate in cytoplasmic SGs (Farg et al., 2014; Mori et al., 2013). A hallmark of mutant C9ORF72 is the

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presence of RNA foci (DeJesus-Hernandez et al., 2011), aggregations of RNA and RBPs that disrupt proper mRNA splicing, and possibly additional RNA-based cellular mechanisms. These RNA foci can be found in the CNS, and most abundantly in motor neurons (Cooper-Knock et al., 2014; DeJesus-Hernandez et al., 2011). Binding and sequestration of RBPs to RNA foci, thus keeping them from their proper localization and function, has been suggested as a possible mechanism of toxicity (Donnelly et al., 2013; Reddy et al., 2013). Equally important as mRNA trafficking is mRNA anchoring at distinct locations in the cell. mRNA anchoring is evolutionarily conserved throughout Eukarya, in different cell types, and at different developmental time points, most notably in the oocyte (Meignin and Davis, 2010). Different mRNA species have distinct mechanisms of transport; similarly mRNA anchoring depends on distinct mechanisms. Well characterized is the interaction between ZBP1 and b-actin, abundant in migrating fibroblasts and developing neurons. ZBP1 is necessary for b-actin mRNA transport toward the leading edge while maintaining a translationally repressed state (Martin and Ephrussi, 2009). Once there, mRNA anchoring depends on interaction with the actin cytoskeleton, mediated by the translation elongation factor, EF1a. Staufen, is an RBP which recognizes a stem-loop structure, and required for the MT-dependent transport of neuronal RNAs, and RNA anchoring in the oocyte. Staufen has been shown to target, for example, CamKII mRNA to dendrites, as well as the mRNA of the cytoplasmic scaffolding protein coracle to the NMJ (Gardiol and St Johnston, 2014). Another distinct mechanism was recently described, in which the tumor suppressor gene adenomatous polyposis coli participates in the anchoring of RNAs in granules to detyrosinated MTs (Glu-MTs). Glu-MTs are preferentially used by the KIF5c isoform (Mili and Macara, 2009). Diffusion-based localization and capture has been described in the oocyte (Meignin and Davis, 2010), but is less likely to occur in neurons (see Figure 2).

5. NEUROTROPHIC FACTORS AND THEIR PRECURSOR FORMS The spatiotemporal localization of survival and death signals is yet another level of regulation serving cell function and survival. The neurotrophic hypothesis marked by the discovery of NGF, states that the survival of neurons during development depends on factors secreted from the neuron’s


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Figure 2 Local protein synthesis as an additional level of regulation to the spatiotemporal localization of signaling events. Local synthesis occurs along the axon, probably at distinct sites that are enriched with mitochondria, which may supply ATP, and regulating factors like Ca2þ buffering or others. This highly regulated process involves, among others, RNA-induced silencing by specifically localized microRNAs.

target cell. Early studies established that dying neurons during development initiate programmed cell death (PCD), and that neurotrophic factors (NTFs) have the ability to inhibit this process (Gould and Oppenheim, 2011). It was this robust activity that soon led to the theory that NTFs may assist in the treatment of neurodegenerative disorders in adults (Henriques et al., 2010; Nagahara and Tuszynski, 2011). The neurotrophin (NT) protein family consists of four structurally and functionally related factors NGF, BDNF, NT-3, and NT-4 that can induce neurite growth and differentiation while preventing neuronal apoptosis. NTs are initially synthesized as precursor proteins (pro-NTs) and later cleaved by intra- or extracellular proteases into the mature form (Chao, 2003). NTs are bound by the neurotrophin receptor (NTR) family of receptors, which include tropomyosin-related kinase (Trk) A, B, and C receptors, which exhibit high affinity to their respective ligands NGF, BDNF/NT-4 and NT-3. Another member of the family, p75 neurotrophin receptor (p75NTR), has a lower affinity to all these ligands in their mature form, however, has high affinity to their proforms (Lu et al., 2005). Trk activation following ligand binding was shown to induce antiapoptotic signaling whereas p75NTR activation promotes apoptosis (Friedman and Greene, 1999). Hence, in mature form, NTs act as prosurvival signals,

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while, surprisingly, the unprocessed premature form of these factors is thought to activate death signaling (Lu et al., 2005). The spatial localization of NT signaling in the neuron plays a key role in its function (Kaplan and Miller, 2000; Segal, 2003); to this end axonal transport is crucial. The signaling endosome hypothesis suggests that following ligand-binding, NT’s receptors undergo endocytosis and travel in vesicles together with their ligand from axon tips toward the neuron cell body using the retrograde transport machinery and the dynein motor protein (Howe and Mobley, 2004). This mechanism was introduced, as signals diffusion cannot explain the aspects of the long distance NT signaling (Riccio et al., 1997). An intriguing speculation is the existence of specialized prosurvival endosomes along with proapoptotic ones that have distinguished characteristics like size, rate of transport, and molecular markers of specialized adaptors, i.e., Rabs, receptors, etc. Intriguingly, the importance of proper localization is demonstrated as application of BDNF to motor neuron axons, but not cell bodies, is sufficient to induce TrkB-mediated toxicity in an in vitro mSOD1 transgenic model, in a noncanonical process yet to be understood (Jeong et al., 2011; Lowry et al., 2001). Furthermore, membrane-bound receptors and endocytosed receptors initiate different signaling pathways upon activation (von Zastrow and Sorkin, 2007); hence, control of signaling location by axonal transport is of utmost importance in both health and disease. Axonal retraction or elongation is thought to be mediated by local signaling mechanisms that do not require retrograde transport. The neuronal survival response, on the other hand, involves the cell body and consists of changes in gene expression mediated by the retrograde transport of the signaling endosomes, which upon reaching the neuronal cell body activate transcription factors (Campenot and MacInnis, 2004). Lack of trophic support as a result of either transport or signaling deficiency can lead to neurodegeneration (Hinckelmann et al., 2013), but is it sufficient to cause disease? Other mechanisms of signal transduction may contribute to signal propagation, for example, NGF-induced TrkA phosphorylation of receptors free of bound ligand (Senger and Campenot, 1997). Senger and Campenot (Senger and Campenot, 1997) have suggested that TrkA retrograde signaling occurs via a “domino-like” effect, with one receptor activating the adjacent one. This suggestion came about upon observation of phosphorylated TrkA in the cell body in response to ligand binding at time points shorter than would be expected using motor-driven retrograde transport. However,


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this hypothesis requires a very large number of receptors to be aligned along the axon, and fails to explain the unidirectionality of the signal (Ginty and Segal, 2002). Regardless, the spatiotemporal localization of activated signals is of key importance. Microfluidic compartmental chambers are gaining popularity as a method for studying these spatiotemporal localization processes, as it enables the isolated control of microenvironments in discrete compartments, for example, soma, axon, and growth cones. Using mouse DRG explants in microfluidic chambers, we have shown that rabies virus is transported faster when bound to p75NTR than with NGF (Gluska et al., 2014). Previous work has shown that BDNF dissociates from p75NTR and binds TrkB during transport in sympathetic retinal neurons, indicating they are located in the same vesicle and cannot therefore travel at different rates (Butowt and von Bartheld, 2009). A contradictory finding showed p75NTR and TrkA to internalize into different compartments and transport via segregated mechanisms, but at similar rates in rat sympathetic neurons (Hibbert et al., 2006). If indeed p75 is retrogradely transported faster than Trks, it could imply a mechanism by which death signals arrive at the cell body faster than survival signals.

6. DEATH SIGNALS The neuron’s environment contains not only prosurvival factors, but death signals as well. Whether a neuron lives or dies depends on the delicate balance of signals that induce cell survival, and those that induce selfprogrammed death received by the neuron. This is determined not only by the signaling ligands, but also by the milieu of receptors expressed on the cell membrane. These are crucial for forming a healthy nervous system during development, as well as disposing of damaged cells. Death receptors are transmembranal proteins related to the tumor necrosis factor receptor (TNFR) superfamily, and include Fas, TNF receptor-1 (TNFR1), p75NTR, death receptors 4–6 (DR4, DR5, DR6), and others. They contain an intracellular death domain, and act to mediate cell death, an important process in the development of the nervous system. Fas, p75NTR, and DR6 all bind CaM in a Ca2þ-dependent manner, to later modulate apoptosis (Cao et al., 2014). Though once thought to be expressed normally only in the developing nervous system and downregulated early in postnatal life, such death receptors are expressed in the adult nervous system as well, and are

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upregulated after injury or in neurological disorders (Jiang et al., 2007; Lowry et al., 2001; Seeburger et al., 1993; Vargas and Johnson, 2010). It is important to note that these receptors act also to induce growth, differentiation, and proliferation. Changes in the delicate balance between these receptors, coreceptors, and ligands may lead to cell death and neurodegeneration either by inducing neuronal apoptosis directly, or by mediating effects arising from surrounding cells. For example, astrocyte activation leads to the secretion of NGF, which, upon binding of TrkA, has trophic properties. In the absence of TrkA, NGF signaling via p75NTR can induce neuronal death (Vargas and Johnson, 2010). Manipulation of these receptors and ligands may provide novel therapeutic possibilities (signaling by death receptors in the nervous system (Haase et al., 2008)) (see Figure 3).

Figure 3 Neuron’s fate determined by the spatiotemporal localization of ligand–receptor mechanism. External signals can be sensed by a milieu of receptors and coreceptors to promote either survival or cell death. Signals can either be propagated along the axon to the soma using the cellular transport machinery to activate transcription events, or act locally by altering the cytoskeleton or regulating local synthesis events. Trk, tropomyosin-related kinase; pro-NT, pro-neutrophin; DR6, death receptor 6.


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6.1 p75 Neurotrophin Receptor p75NTR contains a type II death domain in its cytoplasmic C-terminal section (Bronfman and Fainzilber, 2004). p75-induced trophic effects are mediated via Trk family receptors. Binding of pro-NTs, on the other hand, involves sortilin and has deleterious effects on neuronal cells (Ibanez and Simi, 2012). p75NTR is highly expressed in the developing nervous system, and its expression is reduced in the adult. Expression is reestablished, however, in the case of nerve injury and in the spinal cords of both ALS patients (Lowry et al., 2001; Seeburger et al., 1993) and SOD1 mice (Lowry et al., 2001; Shepheard et al., 2014). A switch in retrograde signaling occurs in ALS, where retrograde transport of survival factors is disrupted, yet apoptotic signals such as p75NTR-mediated caspase-8 is increased (Perlson et al., 2009). Liu et al. recently reported that when they observed limb muscles from ALS patients, which are more prone to damage throughout the disease, p75NTR was found at the innervating nerve fiber but not at the neuromuscular junction (Liu et al., 2013). Although this phenomenon is not fully understood, it suggests a deleterious effect starting at the neuromuscular junction that is propagated retrogradely to the motor axon. Antisense treatment against p75NTR reduced death signaling and delayed the locomotor symptoms of SOD1 mice (Turner et al., 2003). A different study that succeeded in reducing NGF-mediated death in motor neuron cell lines using an antibody against p75NTR, could show only a short delay of disease progression only in female SOD1G93A mice (Turner et al., 2004). Since motor neurons do not express TrkA, a receptor for NGF, involvement of NGF in motor neuron death and ALS is most likely due to the p75NTR. Still, studies with double transgenic mice strengthen the notion that altered p75NTR signaling is not sufficient to cause substantial MN damage, as genetic downregulation of p75NTR in mSOD1 mice prolonged the survival of female mice only (Kust et al., 2003). Both groups suggest that the effect of p75NTR reduced signaling affects astrocyte activation rather than motor neuron survival. Cleavage of p75NTR is a fundamental event in cellular apoptosis. In sympathetic neurons, cleavage of p75NTR by g-secretase results in ubiquitination and translocation of the intracellular domain to the nucleus to induce PCD (Kenchappa et al., 2006). Following cleavage, the extracellular domain of p75NTR can be found in the urine of SOD1 mice prior to symptom onset (Shepheard et al., 2014). Therefore, p75NTR has also been proposed as an ALS biomarker.

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6.2 Death Receptor 6 Another member of the TNF receptor family, DR6, is highly similar to p75NTR and plays a role in the regulation of immune response and development of the nervous system, and is highly expressed in lymphoid tissue, heart, pancreas, and the brain, as well as on the surface of several tumors (Benschop et al., 2009). Its expression and activation are associated with apoptosis, via JNK and NF-kB pathways, Bax, and caspases (Hu et al., 2014; Nikolaev et al., 2009). DR6 was shown to bind both amyloid precursor protein (APP) (Nikolaev et al., 2009) and, in a complex with p75NTR, b-amyloid (Hu et al., 2013). In both cases, DR6 induces apoptosis of CNS neurons, pointing to its involvement in the pathology of neurodegenerative disorders, such as AD. On the other hand, Kallop et al., have recently reported that, although DR6 has a role in regulation of synaptic density in the adult CNS, its genetic deletion in AD mouse models has no effect on disease pathology (Kallop et al., 2014). The possible role of DR6 in ALS pathology is less studied, however, Huang et al., have recently shown an elevation in the levels of DR6 mRNA and protein both in spinal cords of mSOD1 mice, and in cultured MN derived from ALS patients (Huang et al., 2013). Moreover, the treatment of NTF-deprived or otherwise stressed neurons with an antibody against the receptor increased their survival and axon length. Presymptomatic mSOD1 mice treated with the antibody showed reduced NMJ disruption, MN death, sciatic degeneration, and astrogliosis, as well as reduced plasma NFH protein and had increased hind limb strength. Neuronal cultures taken from DR6 null mice had reduced caspase-3 cleavage activity and increased survival. Hence, manipulation of DR6 expression may present novel therapeutic methods in the treatment of ALS.

6.3 Fas Fas (APO-1/CD95) induces apoptosis by binding Fas ligand (FasL) and activating caspase pathways. Fas regulates T-cell activation in the immune system, and was shown to play a role in spinal cord neuron death as a result of ischemia (Matsush*ta et al., 2000). Furthermore, NTF-deprived cells express FasL, and it was shown to have a selective apoptotic pathway involving nitric oxide synthase (NOS) in MNs (Raoul et al., 2002). NOS produce nitric oxide, an important cellular signaling molecule. In the same study, mSOD1-derived motor neurons showed greater sensitivity to Fas-mediated death. The same group later reported that this process involves


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a reduction of the calcium-binding ER-chaperone calreticulin (BernardMarissal et al., 2012). A very recent study shows that axotomy in the mSOD1 mouse model of ALS causes an abnormal reaction of surrounding glia cells in the CNS. These cannot support MN survival, leading to increased facial motor neuron death after target disconnection, via Fas signaling (Haulcomb et al., 2014). Fas activation has an additive effect to IFNg, which induces selective MN death (Aebischer et al., 2011). In an attempt to block Fas-induced MN death in ALS, Petri et al., crossed mSOD1 mice with a mutation in FasL, and showed a reduction in MN loss and prolonged survival (Petri et al., 2006). Furthermore, silencing of Fas by RNAi promotes the survival of both wild-type and mSOD1 motor neurons in vitro, while treatment of SOD1 animals with the same RNAi improved their motor function and survival (Locatelli et al., 2007). Interestingly, a new study shows that vitamin D may have a protective effect against Fas-mediated neuron death, and that its deficiency in ALS patients hastens their deterioration (Camu et al., 2014).

6.4 Semaphorins Semaphorins serve as guidance cue molecules and play critical roles in embryonic development and in pathologies such as neurodegenerative diseases, cancer, and injury. Most members of the semaphorin family of proteins function as axon chemorepellents and have also been shown to induce neuronal cell death (Nakamura et al., 2000). Semaphorininduced cell death can occur independently of axon guidance via binding to plexins (Ben-Zvi et al., 2008), the canonical semaphorin receptors. One of the best-characterized molecules in this family is Sema3A. Members of the Sema3 family cannot bind plexin directly and therefore require members of the neuropilin (Npn) family as coreceptors (Tamagnone et al., 1999). During development, Sema3A functions as a chemorepellent and death factor to a number of neuronal populations in vitro and in vivo (Nakamura et al., 2000). In the adult, Sema3A expression is very low; however, it is upregulated in many human neurodegenerative diseases such as AD (Good et al., 2004) and multiple sclerosis (Williams et al., 2007), ischemia (Fujita et al., 2001), and inflammation (Majed et al., 2006), as well as in response to various environmental stressors (Kaneko et al., 2006; Shirvan et al., 1999). Importantly, Sema3A is also upregulated in terminal Schwann cells in animal models of ALS (De Winter et al., 2006). Additional type 3 semaphorins have also been implicated in neurodegenerative conditions (Blalock et al., 2004).

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Sema3A signaling has been shown to facilitate both retrograde and anterograde transport in DRG cultures, however, regulation by Npn1 was restricted to the growth cone (Goshima et al., 1999). Further studies introduced a novel mechanism of Sema3A-induced axonal transport by local calcium signaling, a signal which is then propagated to other regions of the cell (Ko et al., 2012). Intracellular signaling can additionally be disrupted by Sema3A interference in the polymerization of both actin and tubulin. In NMJ disruption, Sema3A signaling through Npn1 is thought to be an early event, an effect that can be mitigated by selectively blocking Npn1 in SOD1G93A mice (Venkova et al., 2014). Thus, reactivation of embryonic guidance signaling pathways like the semaphoring, in adults can serve as a possible mechanism for synapse elimination, axon degeneration, and initiations of neurodegenerative diseases like ALS.

7. AMYLOID PRECURSOR PROTEIN The physiological function of amyloid beta (Ab), notorious as the plaque-forming protein in AD, is yet unclear (Pearson and Peers, 2006). Ab has been linked to ALS pathogenesis: it was found to colocalize with SOD1 aggregates in SOD1G93A cell cultures, and was also shown to bind with greater affinity to mutant SOD1 than to wild type and inhibit its antioxidant enzymatic activity (Yoon et al., 2009). Ab additionally triggers ALS-associated RBP TDP-43 aggregations in cortical sections of mouse AD models (Herman et al., 2011). It is not clear whether axonal transport dysregulation is a cause or an effect of Ab pathology. On the one hand, deficiencies in axonal transport were associated with formation of Ab aggregates early in AD progression (Stokin et al., 2005). On the other hand, transgenic mice overexpressing Ab display axonal transport defects (Millecamps and Julien, 2013). APP levels were found to be elevated in spinal cord and muscle fibers of SOD1G93A mice, and genetic ablation of APP delayed disease progression in ALS transgenic mice (Bryson et al., 2012). Soluble APP was found to be decreased in CSF of ALS patients as measured by both western blotting (Sennvik et al., 2000) and ELISA (Steinacker et al., 2011, 2009). This decrease is attributed to loss of functional neurons (Sennvik et al., 2000). We would like to propose the possibility that this decrease is actually due to transport and localization deficiencies. Studies of processed Ab levels in CSF are less consistent, although there does seem to be a change in ratio between the different Ab isomers, hinting at an alteration in alpha, beta,


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or gamma secretase function. Of note, gamma secretase also cleaves the intracellular death domain of p75NTR (Youker et al., 2013). Ab42 accumulation was observed in the anterior horn of spinal cords from ALS patients (Calingasan et al., 2005), and interestingly, an increase in Ab was shown in ALS patient skin samples (Tamaoka et al., 2000). Serine–threonine kinase, glycogen synthase kinase 3 (GSK-3) was shown to have a major role in AD (Hooper et al., 2008), and is suspected to phosphorylate kinesin light chain, thus inhibiting kinesin and affecting Ab transport (Weaver et al., 2013). Ab immunopercipitation showed it binds kinesin-1 light chain, leading to the hypothesis that Ab normally functions as a membrane receptor for kinesin-1-driven transport (Kamal et al., 2000), although other studies have failed to show the connection between these two proteins, leaving Ab’s role in transport somewhat controversial. The suggestion that Ab normally functions as a membrane receptor for kinesin-1-driven transport is also disputed (Lazarov et al., 2005). An elevation in GSK-3 levels was observed in the spinal cord and cortex of ALS patients, as well as in animal models. Furthermore, inhibition of GSK-3 was shown to delay disease onset and progression in mSOD1 transgenic mice (Koh et al., 2011). Yet another hypothesis suggests that APP processing occurs during transport and is effected by transport deficits, although data supporting this is not consistent (Millecamps and Julien, 2013).

8. CONCLUDING REMARKS The various genetic causes for ALS, together with the evidence for differences in cellular and behavioral pathologies, enforce the notion that ALS is not a single disease, but rather a spectrum with underlying genetic and environmental causes. Data from the mSOD1 mouse model point to transport defects being an early event in ALS, preceding symptom onset (Bilsland et al., 2010; Williamson and Cleveland, 1999). The ALS hallmark of cytoplasmic inclusions or aggregates spans the various models, but the question whether these are the underlying cause or simply a symptom remains unanswered. It is possible that these inclusions, that sequester both RNA and proteins, promote neurodegeneration, but are not the initiating event in neurodegeneration. The identity of the initiating event or events in motor neuron degeneration remains the “holy grail” of ALS research. One possibility is that the initiating event(s) is unique to

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the underlying genetic cause, but propagation of degeneration is executed via common mechanisms. Such mechanisms may include alterations in spatiotemporal localization of essential factors, signaling molecules that can be regulated by axonal transport and the cytoskeleton infrastructure. Motor neuron degeneration can be linked to mutations in the major components of trafficking, the cytoskeleton and motor proteins, but also to the proteins playing smaller roles in this process, i.e., the proteins conferring specificity to cargodbe it organelles, proteins, mRNA, or miRNA. The mutations and events we have detailed all enforce the concept that we introduced in the opening lines of this review: spatiotemporal localization is imperative. Axonal transport and local protein synthesis may work together to form a forward-feeding loop that advances neurodegeneration. Even minor alterations in such a highly regulated process can over time seriously damage the neuron’s proper function, eventually leading to its death. For example, failure of an mRNA or regulator to arrive at the right place at the right time leads to a failure to synthesize new proteins, among them cytoskeleton components and possibly receptors, which enhance the dysfunction of the transport machinery and impede signal transduction. As ice buckets are being spilled over heads worldwide (Steel, 2014), it is clear that the quest for uncovering the initiating events in neurodegeneration should be continued while searching for effective treatments and biomarkers. This raises the question of can we interfere with transport and localization, and, if so, will it help slow the advancement of neurodegeneration? To date, interventions in axonal transport have focused on the “highways.” In mutant SOD1 mice, MTs are highly dynamic and unstable (Fanara et al., 2007), impairing traffic. Modulation of MT dynamics pharmacologically recovered axonal transport, increased motor neuron survival, and prolonged the life of SOD1 mice (Fanara et al., 2007; Jouroukhin et al., 2013). Downregulation of the MT deacetylase HDAC6, either by genetic ablation or pharmacologically, also had a positive effect on disease progression in SOD1 mice (Taes et al., 2013). However, a general acceleration of axonal transport is an oversimplified solution that does not take into account the complexity of trafficking mechanisms in the cell. Changes in the cargo of motor proteins have also been observed in ALS models, shifting the balance between retrogradely transported survival and death factors (Perlson et al., 2009). In this case, acceleration of defective transport will only serve to exacerbate


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neurodegeneration. Additionally, as previously mentioned, “rest stops” are equally as important as transport for the localization of signaling and local synthesis events. ALS therapeutics are generally divided into two groups: causal, targeting the effects of the original genetic mutation, and modifying, targeting pathological mechanisms that enhance disease progression. The treatments detailed here are only a fraction of those in ongoing preclinical and clinical trials. A number of attempts at modulating mitochondrial function have failed in clinical trials. A new approach, inhibition of the caspase-1/cytochrome c/caspase-3 death pathway via administration of melatonin has had promising results in disrupting the formation of toxic complexes formed by SOD1 binding to mitochondrial proteins and improving calcium buffering capabilities (Poppe et al., 2014). The administration of NTFs to promote neuronal health, prolong life span, and alleviate symptoms seems like an obvious therapeutic strategy. The reality is much more complex. Delivery methods, short half-lives, and safety concerns significantly hinder the use of NTFs as therapeutics. Two clinical trials performed with BDNF were deemed failures, not showing significant improvement, while surmounting data has pointed to BDNF as an exacerbating factor in ALS (Henriques et al., 2010). The discovery of vascular endothelial growth factor (VEGF) as an ALS modifier was surprising, given its well-documented role in blood vessel formation in the embryo and under hypoxic conditions (Poppe et al., 2014). VEGF appears to have multiple effects on motor neuron health including the reduction of astrogliosis in the spinal cord and stabilization of the NMJ (Zheng et al., 2007). Moreover, enhancing the expression levels of VEGF by direct injection (Storkebaum et al., 2005), transgene delivery (Dodge et al., 2010) or the introduction of stem cells secreting VEGF (Hwang et al., 2009) had beneficial effects on the phenotype of SOD1 rodent models. A novel approach introduces a “co*cktail” of NTFs that aim to inhibit stress signals along with the beneficial effects of NTFs. A recent study transplanted muscle progenitor cells bioengineered to excrete BDNF, GDNF (glial cell derived neurotrophic factor), IGF-1 (insulin-like growth factor), and VEGF into SOD1G93A mice and showed significant improvement in motor function and life span (Dadon-Nachum et al., 2014). From our perspective, the heterogeneity of ALS as a disease makes it ideal for personal medication approaches, but difficult to find a treatment that will be beneficial to the majority of patients. Therefore, the

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development of treatments that target modifying processes holds more promise. As impaired transport is common in all ALS disease research platforms, we find it to be an encouraging prospect for intervention. Nevertheless, we are fully aware of the inherent difficulties in this strategy, which include the specific targeting and delivery to MNs, and the limitation of adverse effects. Our knowledge of the genetics and pathologies of ALS has greatly expanded in recent years, revealing a highly complex, multifactorial disease. The many genetic causes underlying ALS, as well as the collective mechanisms involved in degeneration provide broad scope for therapeutic interference. It is with high hopes that we view the future of ALS research, with the belief that the coming years will provide us with a better mechanistic understanding of the disease, providing additional treatment targets and options.

ACKNOWLEDGMENTS Our own work was supported by the Israel Science Foundation and European Research Council Grants 561/11 and 309377, respectively.

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