Targeting reactive astrogliosis by novel biotechnological strategies

Biotechnology Advances 30 (2012) 261–271

Contents lists available at ScienceDirect

Biotechnology Advances j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v

Research review paper

Targeting reactive astrogliosis by novel biotechnological strategies Anna Maria Colangelo b,⁎, Giovanni Cirillo a, Maria Luisa Lavitrano c, Lilia Alberghina b, Michele Papa a,⁎⁎ a b c

Laboratory of Morfology of Neural Networks, Department of Medicina Pubblica Clinica e Preventiva, Second University of Napoli, 80138 Napoli, Italy Laboratory of Neuroscience “R. Levi-Montalcini”, Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, Italy Department of Surgical Sciences and Intensive Therapy, University of Milano-Bicocca, 20052 Monza, Italy

a r t i c l e

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Available online 5 July 2011 Keywords: Neuro-glial network Nerve injury NGF-like peptide Reactive astrocytosis Synaptic homeostasis

a b s t r a c t Neuroglial cells are fundamental for control of brain homeostasis and synaptic plasticity. Decades of pathological and physiological studies have focused on neurons in neurodegenerative disorders, but it is becoming increasingly evident that glial cells play an irreplaceable part in brain homeostasis and synaptic plasticity. Animal models of brain injury and neurodegenerative diseases have largely contributed to current understanding of astrocyte-specific mechanisms participating in brain function and neurodegeneration. Specifically, gliotransmission (presence of glial neurotransmitters, and their receptors and active transporters), trophic support (release, maturation and degradation of neurotrophins) and metabolism (production of lactate and GSH components) are relevant aspects of astrocyte function in neuronal metabolism, synaptic plasticity and neuroprotection. Morphofunctional changes of astrocytes and microglial cells after traumatic or toxic insults to the central nervous system (namely, reactive gliosis) disrupt the complex neuro-glial networks underlying homeostasis and connectivity within brain circuits. Thus, neurodegenerative diseases might be primarily regarded as gliodegenerative processes, in which profound alterations of glial activation have a clear impact on progression and outcomes of neuropathological processes. This review provides an overview of current knowledge of astrocyte functions in the brain and how targeting glial-specific pathways might ultimately impact the development of therapies for clinical management of neurodegenerative disorders. © 2011 Elsevier Inc. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . 2. Astrocytes in synapse formation and plasticity . . . . . . . . 3. Glial activation and neurodegenerative pathologies . . . . . 4. Neuroglia, neuroinflammation and neurodegeneration . . . . 5. Neurotrophins, astrogliosis and neurodegenerative diseases . 6. Neurotrophins and reactive gliosis in ophthalmic pathologies 7. Neurotrophin-derived drug candidates for neuroprotection . . 8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: CNS, Central Nervous System; GFAP, Glial Fibrillary Acidic Protein; EAAT1, excitatory amino acid transporter 1; GLAST, glutamate-aspartate transporter; GLT, glutamate transporter; GSH, glutathione; i.t., intrathecal; NGF, Nerve Growth Factor; BB14®, Nerve Growth Factor-like peptide; MMPs, metalloproteinases; CCI, chronic constriction injury; SNI, Spared Nerve Injury. ⁎ Correspondence to: A.M. Colangelo, Department of Biotechnology and Biosciences, Laboratory of Neuroscience “R. Levi-Montalcini”, University of Milano-Bicocca, piazza della Scienza 4, 20126 Milano, Italy. Tel.: + 39 02 64483536; fax: + 39 0264483519. ⁎⁎ Correspondence to: M. Papa, Department of Medicina Pubblica Clinica e Preventiva, Institute of Human Anatomy, Second University of Napoli, 80100 Napoli, Italy. Tel./fax: + 39 081 296636. E-mail addresses: [email protected] (A.M. Colangelo), [email protected] (M. Papa). 0734-9750/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2011.06.016

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1. Introduction The relevance of glial cells to the central nervous system (CNS) function and plasticity started to be noticed relatively recently, about 20 years ago. Until then, in fact, astrocytes and other cells of the glial lineage, such as oligondendrocytes and microglia, were believed to be structural, electrically silent elements, lacking transmitter receptors and transporters, with the main function of holding neurons together (“brain glue”). This view of “embedded elements among neurons” was challenged by a series of in vitro and in vivo studies that clearly

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demonstrated that astrocytes and neurons share almost the same set of ion channels, receptors and transporters, allowing to conclude that glial cells sense and respond to neuronal activity. The complexity of cellular circuitries in the CNS, an unicum in living systems, resides in the dynamic changes of neural connections (i.e. synaptic plasticity). This function, through a highly developed blood– brain barrier, is entrusted to glial cells. In particular, astrocytes provide for the micro architecture of the gray matter by forming relatively independent structural domains. Within these domains, each astrocyte covers synaptic contacts (tripartite synapse) and establishes connections with neuronal membranes and blood vessels. Through gap junctions, astrocytes of distinct domains create an astroglial syncitium, thus providing a glial information-transfer system, a pathway for rapid intercellular diffusion and long-range signaling. It is now clear that glial cells are fundamental for control of brain homeostasis and constitute an intrinsic brain defense system. In fact, they possess an evolutionary conserved program of activation in response to brain damage. Microglial cells represent the resident macrophages of the CNS. Like astrocytes, microglial cells disseminate throughout the brain and occupy well-defined territorial domains, which do not overlap with neighboring microglia. Resident microglia have small somatas and multiple fine processes constantly moving and scanning the microenvironment of their domains (Davalos et al., 2005; Nimmerjahn et al., 2005), thus providing the first line of defense as sensors of nervous system injury (Hanisch and Kettenmann, 2007; Ransohoff and Perry, 2009). Microglia and astrocytes become activated (reactive gliosis) in response to several CNS insults. Astrocytic activation occurs through mechanisms involving specific structural and functional alterations, such as hypertrophy and increased expression of glial fibrillary acidic protein (GFAP) (Pekny and Nilsson, 2005). Phenotypic changes affecting reactive astrocytes impair neuronal network function by producing or boosting neuronal degeneration as “non cell-autonomous diseases” (Lobsiger and Cleveland, 2007). Decades of pathological and physiological studies have focused on neuronal abnormalities in brain disorders, but it is becoming increasingly evident that astrocytes are also important players. Our understanding of the normative biology of astrocytes has been fostered by the development of animal models in which astrocytespecific proteins and pathways have been manipulated. Models of neurodegenerative diseases have also led to current knowledge of glial function in neurodegenerative pathologies. Thus, a comprehensive understanding of mechanisms contributed by astrocytes appears to be relevant for the development of targeted therapies for clinical management of neurodegenerative disorders. 2. Astrocytes in synapse formation and plasticity Several studies support the key role for astrocytes from synaptic formation to metabolic support and neurotransmitter release. Astrocytes represent the key elements in synaptogenesis. It has been reported that addition of astrocytes to in vitro neuronal cultures triggers a significant increase in synapse formation (Pfrieger and Barres, 1996): through production of cholesterol (Nieweg et al., 2009) and release of trophic factors, astrocytes are crucial for synapse maturation and maintenance (Pfrieger, 2009); through production and release of thrombospondins 1 and 2, they promote synaptogenesis suggesting their crucial role in post-lesion synaptic plasticity, remodeling and regeneration. Astrocytes are also essential for neuronal energy metabolism and glutathione synthesis. Astrocytic endfeet contact neighboring capillaries through perivascular processes, thus forming a functional link between neurons and blood vessels. An increase in neural activity within an astrocytic domain results in the release of vasoactive substances (arachidonic acid metabolites) (Hirase, 2005) that promote dilation in nearby arterioles (Takano et al., 2006). Metabolic support to neurons is achieved through the astrocyte–neuron lactate

shuttle. Astrocytes convert glucose to lactic acid, which is then taken up into neurons and converted to pyruvate for energy metabolism (Danbolt, 2001). Thus, astrocytes play a central role in coupling synaptic plasticity and glucose metabolism (neurometabolic coupling) through mechanisms involving the sodium-coupled glutamate re-uptake, which stimulates aerobic glycolysis and production/release of lactate (Magistretti, 2006). The metabolic function of astrocytes is supported by a large number of studies. More recently, astrocytic glycogen breakdown and astrocyte-neuron lactate transport have been demonstrated to be essential for long-term memory formation, and for the maintenance of long-term potentiation (LTP) of synaptic strength (Suzuki et al., 2011). The different metabolic requirements of astrocytes and neurons (high and low glycolytic, respectively) is based upon their different enzymatic assets. Neurons have very low activity of the glycolysispromoting enzyme PFKFB3 (6-phosphofructo-2-kinase/fructose 2,6bisphosphatase, isoform-3), which is constantly degraded by the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)– Cdh1. This mechanism appears to be ultimately linked to the neuronal need to redirect glucose metabolism to the pentose-phosphate (PPP) pathway to generate NADPH(H +), a necessary cofactor in the regeneration of reduced glutathione (GSH) (Herrero-Mendez et al., 2009; Bolaños et al., 2010). Neurons are particularly vulnerable to reactive oxygen (ROS) and nitrogen species (RNS), and also have low activity of γ-glutamyl cysteine synthetase, the limiting enzyme for GSH synthesis. Therefore, neuronal downregulation of glycolysis and glucose metabolism through the PPP pathway seem to be required to maintain their antioxidant status (Herrero-Mendez et al., 2009). Control of extracellular ion concentrations and water homeostasis is also a well recognized function of astrocytes. Extracellular K + accumulated from neural activity (Kofuji and Newman, 2004) is lowered by inward rectifying K + channels (Kir-channels) controlling ion concentration; water homeostasis occurs through aquaporin-4 type expression on perivascular endfeet and perisynaptic processes. Another key function of astrocytes is, moreover, the removal of neurotransmitters that are released by active neurons, in particular glutamate (Fig. 1A). Glutamate, the main excitatory neurotransmitter in CNS, is also the most powerful neurotoxin when it accumulates in the extracellular space. From the bulk of glutamate released during synaptic transmission, about 20% is re-captured by postsynaptic neurons, the remaining 80% is taken up by perisynaptic astrocytes (Rothstein et al., 1996). Astrocytes selectively express two glutamate transporters, EAAT1 and EAAT2 (in rodents known as GLAST and GLT1, respectively) (Rothstein et al., 1994; Chaudhry et al., 1995; Lehre et al., 1995; Milton et al., 1997). Besides their role in glutamate uptake, astrocytes are also crucial for the recovery of glutamate in presynaptic terminals through the glutamate–glutamine shuttle system. Astrocytic cytosolic glutamate is converted by the astrocytic-specific glutamine synthetase into the non toxic glutamine, that is released in the extracellular space and after entering the neuronal compartment is converted into glutamate to reconstitute the neurotransmitter pool. The cystine–glutamate antiporter system (xCT) is also functionally related to the role of astrocytes in regulating extracellular glutamate and providing intracellular cystine for neuronal GSH homeostasis. Compelling evidence suggests that altered glutamate uptake by astrocytes is directly involved in the pathogenesis of some neurological disorders. We also reported a decrease of glial glutamate transporters after peripheral nerve injury, together with a decrease of GSH levels and a functional block of the xCT system by extracellular glutamate accumulation (Cirillo et al., 2011). These data are in agreement with the increase of xCT under conditions of glutamate oxidative stress and GSH depletion (Dun et al., 2006; Lewerenz et al., 2006). The ability of astrocytes to release chemical transmitters (gliotransmitters) is also fundamental for their involvement in neuro-glial networks. Among gliotransmitters, astrocytes can release

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Fig. 1. Schematic representation of a tripartite synapse in “normal” (A) and under conditions of reactive astrocytosis (B). Excitatory aminoacids (EAs) (glutamate and glycine) are released in synaptic clefts and retrieved by neuronal and mainly glial glutamate and glycine transporters (GTs). The xCT transporter, through the cystine–glutamate antiporter exchange, supplies the cysteine for GSH synthesis. In normal conditions, the turn-over of intermediate filaments, in particular GFAP, and GTs occurs through the activity of calpain I, a Ca2+-dependent protease. B) Reactive astrocytosis determines an increase of calpain I activity and degradation of GTs, and failure of glutamate uptake. Extracellular glutamate spillover to perisynaptic spaces contributes to NMDA receptors sensitization and changes of synaptic efficacy. The increase of extracellular glutamate also contributes to excitotoxicity through a functional block of the xCT system and failure of GSH production.

glutamate (Haydon, 2001), ATP, D-serine, GABA and other molecules through a Ca 2+-dependent exocytosis or diffusion through either large pore channels [e.g., P2X7 receptors or hemichannels (Ye et al., 2003)], transporters (glutamate transporters), or the cystine– glutamate antiporter system. This function strengthens the double role of astrocytes in the tripartite synapse: astrocytes sense neuronal activity and neurotransmitter release through the expression of neurotransmitters receptor on astrocytic membrane; on the other hand, they modulate the efficacy of the synapse by releasing gliotransmitters that in turn might modulate the strength of inhibitory or excitatory synaptic transmission through activation of neuronal receptors. Moreover, each astrocyte can reach thousands of synapses simultaneously, and the release of gliotransmitters might lead to the synchronization of neuronal firing patterns (Allegrini et al., 2009). Propagation of glutamate transmission through the astrocytic syncitium occurs through two Ca 2+-mediated systems: one involving gap junctions (via connexin-43), the other involving the paracrine release of ATP (Simard and Nedergaard, 2004). Given the complexity of neuro-glial networks in CNS homeostasis and function, a systems biology approach of neurodegeneration (Alberghina and Colangelo, 2006) might be crucial to a comprehensive understanding of the glia–neuron interplay in synaptic function, and how changes of metabolic fluxes might influence the complex interactions between neuronal and glial compartments and lead to the structural/functional modifications underlying neuro-glial rearrangements during the degeneration process.

3. Glial activation and neurodegenerative pathologies Glial cells play a crucial role in neurological diseases by determining the progression and outcome of the neuropathological process. Insults to the nervous system trigger a complex and multistage activation of microglia which results in both phenotypic and functional changes (activated microglia). Under pathological situations, these cells migrate to and surround damaged or dead cells, clearing cellular debris from the area, similarly to the macrophages of the peripheral immune system (Fig. 2). Activated microglia undergo fundamental morphological changes from a ramified phenotype to activated amoeboid cells, and up-regulate a variety of surface receptors (including MHC and complement receptors) and the release of pro-inflammatory factors (cytokines, reactive oxygen species — ROS, nitric oxide — NO), which contribute to neuronal dysfunction and cell death, ultimately creating a vicious cycle. Current knowledge indicates that early stages of neurodegenerative processes are associated with neuroinflammation, involving microglial cells and subsequent activation of astrocytes (Fig. 3). Astrocytes seem to be involved in several mental disorders (Narita et al., 2006; Machado-Vieira et al., 2009; Musholt et al., 2009), as well as in neurodegenerative diseases (Maragakis and Rothstein, 2006; Lobsiger and Cleveland, 2007) including Alzheimer's disease (AD) (Verkhratsky et al., 2010), amyotrophic lateral sclerosis (ALS) (Sheldon and Robinson, 2007), spinocerebellar ataxia type 1 (SCA1) (Giovannoni et al., 2007), Parkinson's disease (PD) (Saijo et al., 2009)

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Subsequent changes in neurotransmitter uptake represent the basis of morphological and functional changes that sustain central plasticity (Cavaliere et al., 2007). Moreover, glial activation involves changes in cell phenotype and gene expression that might trigger glial-induced neuronal death (Bal-Price and Brown, 2001). Glialmediated inflammatory responses appear to play a key role in the pathophysiology of several neurodegenerative disorders that involve early activation of microglia and astrocytes (Mrak and Griffin, 2005). In general, over-expression of cytokines appears several years before pathological changes are evident (Griffin et al., 2006). The time needed for glial activation might explain the mid-life onset of many neurodegenerative disorders, even those that are genetically determined, before the patient displays the full clinical symptoms of the disease (Unger, 1998). However, it was unknown whether these structural changes represent a reaction to injury/degeneration process, in which astrocytes perform a supportive function in an attempt to prevent further damage, or whether astrocytes are providing detrimental signals that contribute to the disorder. Fig. 2. Section of spinal cord after SNI lesion of the right sciatic nerve, showing the increase of microglial cells (Iba-1 staining) on the right side of the spinal cord, sparing the left side.

and Huntington's disease (HD) (Cirillo et al., 2010b; Faideau et al., 2010). Toxic or traumatic injury to the peripheral or central nervous system activates quiescent microglial cells, a process that can trigger and sustain the astrocytic activation through the production and release of inflammatory mediators that in turn act on other glial cells and neurons, thus sensitizing neural cells and facilitating the neurodegenerative process. Morphological and functional changes of reactive glial cells induce a reshaping of the structure and protein expression, including aminoacid transporters, ion channels, that underlie new roles and functions to the system. This response leads to disruptions of synaptic connectivity, imbalance of neurotransmitter homeostasis, and neuronal death through increased excitotoxicity (Fig. 1B).

4. Neuroglia, neuroinflammation and neurodegeneration Neurodegeneration is a chronic process that results in progressive loss of function, structure and number of neural cells, leading to generalized atrophy. Neurodegenerative processes affect the connectivity of neural networks that is critical for the information processing and cognitive power (Knight and Verkhratsky, 2010). Our knowledge of events occurring at the onset of neurodegenerative diseases is rudimentary, and yet, we may safely suggest that it all begins with synaptic weakness, imbalance of neurotransmission and functional disturbance of the information flow through the neural networks. These functional abnormalities grow deeper with the disease progression leading to loss of synapses, alteration of cellular structure and, eventually, to cell death. Brain atrophy resulting from massive death of neuronal cells represents the final, irreversible stage of the neurodegenerative process, when the volume of the nervous tissue shrinks and neurological functions are severely impaired. Cellular and

Fig. 3. Schematic representation of glial cells in condition of rest (left), and under conditions of reactive gliosis in early (middle) and late (right) phases. In early phases, toxic or traumatic injury to the peripheral or central nervous system activates quiescent microglial cells, a process that can trigger and sustain the astrocytic activation through the production and release of inflammatory mediators (glutamate, ATP, Fractalkine, IL-1β, TNFα) that in turn act on other glial cells and neurons, thus sensitizing neural cells and facilitating neurodegenerative processes. In late phases, microglial cells appear to be quiescent while astrocytic reaction persists. Changes of protein expression (aminoacid transporters, ion channels, etc.) underlie synaptic reshaping, disruption of synaptic connectivity, imbalance of neurotransmitter homeostasis and excitotoxic neuronal death.

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molecular mechanisms involved in the development of neurodegenerative processes are many. Conceptually, the neurodegeneration can be viewed as a chronic and progressive failure of the brain homeostasis that finally assumes toxic proportions leading to massive cell demise. For many years, neurodegenerative diseases were considered to be a specific pathology of neurons. Recent years, however, have challenged the neurocentric views and evidence demonstrating the pathological potential of neuroglia began to accumulate (Rossi and Volterra, 2009; Heneka et al., 2010). Astroglial changes in the progression of neurodegenerative diseases are complex. It was generally believed that neurodegeneration triggers reactive microand macro-gliosis; reactive astrocytes in turn contribute to neuroinflammation, which is considered to be an important component of neurodegenerative diseases (Lobsiger and Cleveland, 2007). Moreover, morphological and functional changes in astroglia occurring at the early stages of neurodegenerative processes, may result in fading astroglial support that can affect synaptic transmission. Neurodegenerative diseases represent major unmet challenges for therapeutic interventions. Characterization and targeting of the processes that initiate specific disease pathologies and act primarily at the level of neurons are clearly important areas for continued investigation. The emerging evidence for both protective and pathogenic roles of microglia and astrocytes, and the activation of common inflammation pathways in these cells in several neurodegenerative diseases, supports the concept that glia-induced inflammation is an amplifier of the pathology (Glass et al., 2010; Bajramovic, 2011). Although inhibition of neuroinflammation may not alter the underlying cause of disease, it may reduce the production of factors that contribute to neurotoxicity, thereby resulting in clinical benefit. Further knowledge of the inducers, sensors, transducers, and effectors of neuroinflammation can make possible the attainment of this goal. Virchow's seminal descriptions of activated glial cells anticipated by more than a century the current interest in roles of the innate and adaptive immune systems in diverse forms of neurodegenerative disease. Direct evidence for inflammatory responses in AD was described nearly 20 years ago and subsequent studies have documented inflammatory components in PD (Tansey and Goldberg, 2010; Chung et al., 2010), ALS, multiple sclerosis (MS) (Glass et al., 2010; van Noort et al., 2011), as well as in a growing number of other pathologies of the nervous system. Although inflammation may not typically represent an initiating factor in neurodegenerative disease, there is emerging evidence in animal models that sustained inflammatory responses involving microglia and astrocytes contribute to disease progression. A major unresolved question is whether inhibition of these responses will be a safe and effective means for reversing or slowing the course of disease. To effectively address this question, it will be necessary to learn more about how inflammatory responses are induced within the CNS and the mechanisms by which these responses ultimately contribute to the pathology. Recently, our groups have focused on different experimental models of reactive gliosis in rodents to better understand early morphological and functional glial changes underlying the response of the CNS to injury and neurodegeneration. We have evidence of microglial and astrocytic activation as early signs of the pathology in a mouse model of SCA1, in a rat model of toxic-induced HD, as well as in the central response to peripheral nerve injury. SCA1 is a dominantly inherited, progressive, neurodegenerative disorder clinically characterized by ataxia and cranial motor neuropathies due to expansion of a CAG repeat in the coding region of the SCA1 gene (Schols et al., 2004; Orr and Zoghbi, 2007), encoding for ataxin-1 protein. Ataxin-1 is expressed throughout the brain, in Purkinje cells in cerebellum but also in non-neuronal cells, mainly radial Bergmann glia (Servadio et al., 1995). We generated a conditional mouse model using the tetracycline-responsive gene (TET) system to control the expression of ataxin-1 (Giovannoni et al.,

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2007) using a brain specific promoter to induce ataxin-1 throughout brain tissues. Our results indicate that expression of mutated ataxin-1 was associated with the onset and progression of cerebellar motor deficits, together with a peculiar clustering of the glutamate transporter in the cerebellar cortex and reactive astrocytosis (GFAP upregulation). These phenotypic and morpho-molecular changes appeared to be related to cerebellar dysfunction of both Purkinje cells and Bergmann glia. In this regard, neuronal degeneration might be the ultimate step of the neuropathology, when rescue from the disease is no longer possible. Our findings provide evidence that this mouse model exhibits features of inflammatory pathology and that it might be useful for studying the role of inflammatory pathways in CAG triplet diseases. As observed in the cerebellum of SCA1 mouse, astrocytic activation in the striatum is a common reaction after toxic, ischemic or degenerative processes and seems to play a role in the development of HD (Hickey et al., 2008). HD is a neurodegenerative autosomic dominant inherited disorder, characterized, in humans, by involuntary choreiform movements and cognitive impairment caused by a CAG expansion in the coding region of the gene huntingtin (Landles and Bates, 2004). HD is characterized by marked striatal atrophy with neuronal loss, astrocytic activation and intraneuronal protein aggregates (Paulson and Fischbeck, 1996; Paulsen et al., 2006). Mechanisms underlying the selective neuronal death of the GABAergic medium spiny neurons (MSN) in the striatum still remains unknown. One hypothesis suggests that the genetic defect may cause an impairment of energy metabolism that subsequently makes these neurons more vulnerable to excitotoxic degeneration (Brouillet et al., 2005). 3-Nitropropionic acid (3NP), a suicide inhibitor of mitochondrial complex II succinate dehydrogenase has been shown to cause a HD-like syndrome in rodents (Borlongan et al., 1997; Cirillo et al., 2010b) and primates (Brouillet et al., 2005). We administered 3NP intra-peritoneally on a subchronic schedule and after treatment we found an intense reactive astrocytosis in the striatum, and altered expression of the glial glutamate transporters, without clear sign of neuronal death. Our results support the hypothesis that the onset and progression of motor deficits are linked to the reactive astrocytosis and changes of glial glutamate transporter expression, thus altering the glutamate uptake system and exposing neurons to glutamate excitotoxicity. These could represent early events leading to neuronal death, which occurs only in the late phase of neurodegenerative diseases. Our recent studies also indicate that glial activation after peripheral nerve injury is an important component of the neuropathic pain-like syndrome characterized by allodynia and thermal hyperalgesia (Cavaliere et al., 2007; Cirillo et al., 2010a). After peripheral nerve injury, a rapid glial response occurs within the CNS, as revealed by microglial activation and increased expression of GFAP in astrocytes in the spinal cord (Fig. 4) (Watkins et al., 2001; Raghavendra et al., 2002; Wieseler-Frank et al., 2004). Activation of quiescent microglial cells in the spinal cord sustains the astrocytic activation through the release of pro-inflammatory mediators (Watkins and Maier, 2003; Zhuang et al., 2005), such as interleukin1β (IL-1 β) (Hashizume et al., 2000) and cyclooxygenase 2 (COX-2) (McMahon et al., 2005), which sensitize dorsal horn neurons (Ji and Strichartz, 2004) and facilitate pain transmission (Watkins and Maier, 2003; Fellin and Carmignoto, 2004). The massive cytoskeletal rearrangement due to reactive astrocytosis seems to be sustained by calpain I-dependent processes (Lee et al., 2000; Gray et al., 2006), a Ca 2+-dependent protease that is highly active following nerve injury and has several substrates, including membrane glutamate and glycine transporters (Li et al., 1996; Serbest et al., 2007) (Fig. 1). Since glial cells through glutamate/aspartate transporter EAAT1 (also known as GLAST) and EAAT2 (also known as GLT1) are responsible for the majority of extracellular glutamate uptake, we investigated changes of glial glutamate transporters (GTs) expression following reactive astrocytosis-induced nerve injury. Both neuronal and glial aminoacid transporter systems actively participate to synaptic plasticity

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Fig. 4. Immunohistochemical analysis of glial markers in the dorsal horn of lumbar spinal cord sections. A strong increase of GFAP staining and Iba1 levels is evident in vehicle-treated animals 3 days after SNI surgery, as compared to the sham-operated rat.

by regulating the duration and intensity of activation of glutamate receptors during signal transduction, thus preventing overstimulation of glutamate receptors and limiting spillover activation of NMDA receptors (Mennerick et al., 1999; Lievens et al., 2000; Trotti et al., 2001). Thus, spinal GTs might change after peripheral nerve injury and contribute to the pathogenesis of neuropathic pain, a pathological condition that involves glutamate transmission in the CNS. Our results are consistent with an early activation of microglial cells after nerve injury, that precedes a sustained and massive astrocytic reaction. The release of microglial pro-inflammatory factors (IL-1 β, COX-2) and activation of P38-MAPK follows the time course of microglial activation, sustaining the inflammatory process. This response is paralleled by reduction of GTs and activation of calpain I, that seems to be involved in degradation of the cytoskeletal structural proteins (mainly GFAP) and astrocytic excitatory aminoacid transporters (Fig. 1B). Taken together, these results support the hypothesis that gliosis and associated changes in the expression and function of GTs cause the increase of glutamate and glycine levels in the lumbar spinal cord, which results in overstimulation of NMDA receptors. This mechanism might have a key role in determining the persistent neuropathic state. Based on current knowledge of the critical role of reactive astrogliosis in most neurodegenerative diseases, molecules that antagonize glial activation may represent a new prospect for disease-modifying therapies. To this end, a number of molecules have been tested in experimental and clinical studies for their potential to reduce neuroinflammatory processes. Besides the clinical trial of indomethacin in AD (Rogers et al., 1993), the efficacy of minocycline has been recently evaluated in experimental models of cerebral ischemia, spinal cord injury (Festoff et al., 2006), HIVdementia (Kuang et al., 2009), PD and ALS (Keller et al., 2011), although the efficacy in neuroprotection was not confirmed in clinical trials (Gordon et al., 2007). Interestingly, we recently reported a novel, crucial role of intrathecal administration of Nerve Growth Factor (NGF) as a strong

modulator of plasticity in neuronal–glial networks by reducing reactive astrocytosis and restoring glial glutamate transporter expression and GSH levels (Colangelo et al., 2008; Cirillo et al., 2010a, 2011). As detailed in the next section, our data are consistent with a key role of NGF in maintaining synaptic homeostasis and neuroprotection. 5. Neurotrophins, astrogliosis and neurodegenerative diseases Following the discovery of NGF by Rita Levi-Montalcini (LeviMontalcini, 1952), and the identification of the other neurotrophin family members (BDNF, NT-3 and NT-4/5 in mammalians), the activity of neurotrophic factors in development and function of the nervous system is now well-recognized (Lu et al., 2005; Reichardt, 2006). Neurotrophins act on selected neuronal populations through interaction with two subtypes of receptors: the Trk tyrosine kinase family members (TrkA, TrkB and TrkC, specific for NGF, BDNF/NT-4 and NT-3, respectively) and the common p75NTR receptor (Fig. 5) (Skaper, 2008). Signaling pathways activated by neurotrophins through Trk receptors (Ras-MAPK, PI3K-Akt, and PLCγ-PKC) result in many neuronal functions, including survival, differentiation, axonal growth, synapse formation and plasticity (Reichardt, 2006; Kuczewski et al., 2010). The p75NTR receptor, instead, is believed to be important for ligand selectivity of Trk activation (Chao, 2003; Chao et al., 2006). Moreover, p75 can also activate pro-apoptotic responses and other cellular mechanisms, depending upon the presence of Trk and/or other coreceptors (e.g., sortilin) and ligands availability (mature neurotrophins or their precursors) (Casaccia-Bonnefil et al., 1996; Friedman, 2000; Bentley and Lee, 2000; Beattie et al., 2002; Chao and Bothwell, 2002). The complexity of p75 signaling arises from the presence of an intracellular death domain (like TNF receptors) and the capability to interact with different co-receptors (sortilin, Nogo receptor, LINGO-1) and adaptor proteins (TRAF-6, RhoGDI, NRIF, NRAGE, NADE and SC-1), which underlies its pleiotropic roles on neuronal and non-neuronal cells

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Fig. 5. Schematic diagram of neurotrophins, receptors, key signaling pathways and activities.

(Wong et al., 2002; Chao, 2003; Domeniconi et al., 2007; Jansen et al., 2007; Cragnolini and Friedman, 2008). Besides its activity on glial cells (astrocytes, microglia, Schwann cells and oligodendrocytes) and its role in myelination, migration and proliferation (Bentley and Lee, 2000; Cosgaya et al., 2002; Vilar et al., 2006; Cragnolini et al., 2009), particularly relevant to the pathophysiology of neurodegenerative conditions is its role in pro-neurotrophin-induced neuronal death (Lee et al., 2001; Harrington et al., 2004; Teng et al., 2005; Domeniconi et al., 2007; Yano et al., 2009). Neurotrophins are secreted as pro-neurotrophins (pro-NGF, proBDNF, pro-NT-3) and proteolytically cleaved to the mature proteins in the extracellular space (Fahnestock et al., 2001; Bruno and Cuello, 2006). Therefore, the neurotrophic activity of NGF is currently believed to be the result of a dynamic balance between conversion of pro-NGF to mature NGF and its degradation by the tissue plasminogen activator (tPA)–plasmin–matrix metalloproteinase-9 (MMP-9) system (Fig. 6) (Bruno and Cuello, 2006; Cuello and Bruno, 2007). Pro-neurotrophins are high-affinity ligands of p75NTR in complex with its co-receptor sortilin (Lee et al., 2001; Teng et al., 2005; Domeniconi et al., 2007; Yano et al., 2009). Thus, the biological functions of these receptors in the adult brain appear far more complex, as differential activation of p75 by mature neurotrophins or their precursors can have distinct and sometimes opposing actions in regulating cell death/survival and in modulating synaptic plasticity (Chao and Bothwell, 2002). Alteration of NGF maturation/degradation processes and increase of proNGF levels in the brains of AD patients are currently believed to underlie the vulnerability and atrophy of NGF-dependent cholinergic neurons in AD, as well as in PD and age-related neurodegeneration (Fahnestock et al., 2001; Cuello and Bruno, 2007; Al-Shawi et al., 2007; Chen et al., 2008). Similar mechanisms of cell death have been described for motor and sympathetic neurons in response to proNGF, proBDNF and proNT-3, and suggested to play a crucial role in several neurodegenerative pathologies, such as ALS (Teng et al., 2005; Domeniconi et al., 2007; Yano et al., 2009). This hypothesis is in agreement with the evidence of increased p75NTR expression in several brain disorders (AD and motor diseases), as well as following CNS injury (Masoudi et al., 2009). These findings provide a challenging interpretation of the complex interplay between neuronal survival and neurotrophin processing/signaling in health and under pathological conditions and appear strictly linked to mechanisms of reactive gliosis (Fig. 7). As extensively described above, astrocytes play a crucial role in maintaining neuronal homeostasis during development and in adulthood (Heales et al., 2004; Benarroch, 2005). Glial function in neuroprotection is known to be also due to their role in production and secretion of neurotrophic factors for neuronal survival. In particular, astrocytes have been shown to up-regulate neurotrophin production and secretion in response to glial stimulation by inflammatory mediators and pro-inflammatory cytokines (IL-1β, TNF-α, INFγ, LPS), as well as by neurotransmitters (glutamate and

beta-adrenergic stimulation) and nitric oxide donors (Friedman et al., 1996; Galve-Roperh et al., 1997; Colangelo et al., 1998; Mocchetti and Colangelo, 2001; Domeniconi et al., 2007). Increased production of pro-NGF and pro-BDNF by activated microglia and astrocytes has been shown to occur following CNS and spinal cord injury and trigger p75mediated death of cortical and motor neurons (Harrington et al., 2004; Pehar et al., 2004; Domeniconi et al., 2007). We also found that following peripheral nerve lesion by chronic constriction injury (CCI) or spared nerve injury (SNI), glial activation and changes of synaptic homeostasis were paralleled by i) increase of MMPs activity, ii) decrease of mature NGF levels, iii) and increase of both pro-NGF and p75 receptor expression (Cirillo et al., 2010a, 2011). All these responses were restored by i.t. administration of NGF or GM6001, a generic MMP inhibitor (Cirillo et al., 2011). These findings are consistent with a model in which NGF is essential in maintaining synaptic homeostasis (through modulation of glutamatergic and GABAergic components) and neuroprotection (through control of synaptic glutamate levels and providing neurons with GSH) (Cirillo

Fig. 6. Scheme of the tPA/plasminogen/plasmin/metalloproteinases (MMPs) system on NGF maturation and degradation. The tPA/plasmin system promotes maturation of proNGF to mature NGF, as well as cleavage of proMMPs to their mature active forms (MMPs). NGF degradation by MMPs can be blocked by GM6001, a generic MMPs inhibitor.

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Fig. 7. Schematic representation of neuro-glial dysfunction linked to decreased NGF activity. Reactive gliosis determines the reduction of glial glutamate transporters and glutamate uptake. Glutamate spillover sensitizes NMDA and non-NMDA (mGlu, AMPA) glutamate receptors. The increase of extracellular glutamate also impairs the cystine–glutamate antiporter system, thus decreasing GSH synthesis and neuroprotection. These changes are prevented by i.t. administration of NGF or by increasing endogenous NGF levels by MMPs inhibition.

et al., 2011). Thus, by establishing a strict correlation between glial dysfunction and aberrant NGF metabolisms and signaling, our data provide additional evidence of the detrimental role of pro-NGF following abnormal NGF metabolism and the relevance of NGFmediated function of astrocytes in neuroprotection. 6. Neurotrophins and reactive gliosis in ophthalmic pathologies The activity of neurotrophins, in particular NGF, and the detrimental effect of altered NGF metabolism following glial activation may be also relevant for pathologies of the visual system. The neural retina contains Muller (radial) glial cells, which span through the entire retina. Muller cells interact with all neurons and constitute the functional link between retinal neurons and the other compartments (blood vessels, vitreous chamber and subretinal space) for the exchange of trophic substances and metabolic waste products. Muller glia also plays a critical role in the regulation of extracellular space volume, ion and water homeostasis in the retina, as well as in the maintenance of the blood–retinal barrier and the retinal blood flow. Reactive gliosis plays a critical role also in ophthalmic neurodegenerative pathologies (de Melo Reis et al., 2008; Bringmann et al., 2009). Besides their role in trophic support and metabolism, Muller glia modulates neuronal excitability and transmission. Like astrocytes, Muller cells participate in neurotransmission through: i) release of gliotransmitters; ii) uptake and metabolism of aminoacid neurotransmitters (glutamate, GABA and glycine, as well as the purinergic receptor agonists ATP and adenosine); and iii) release of precursors of neurotransmitters to the neurons (de Melo Reis et al., 2008; Loiola and Ventura, 2011). Glutamate is the most prominent excitatory neurotransmitter in the retina, and is used in retinal transmission of visual signals by photoreceptors, bipolar cells and ganglion cells. The removal of glutamate from extracellular sites involves all the excitatory amino acid transporters (EAATs): Muller cells and

astrocytes express EAAT1-5 transporters. Glutamate is metabolized to glutamine by Muller cells, released and taken up by neurons as a precursor for the synthesis of glutamate and GABA (glutamate– glutamine cycle). The metabolism of glutamate in Muller cells also produces various substrates for the oxidative metabolism of photoreceptors (glutamine, lactate, alanine, and α-ketoglutarate). Muller cells (but not neurons) of the rat retina also express a chloridedependent cystine–glutamate antiporter (system Xc-) that mediates the uptake of cystine in exchange with glutamate, both used for the production of glutathione (Mysona et al., 2009). Alterations of Muller glia function and elevated levels of extracellular glutamate have been described in the pathophysiology of neuronal loss in ophthalmic disorders such as glaucoma. Mechanical stimulation of retinal ganglion axons due to elevation of the intraocular pressure is the major risk for glaucoma and causes reduced GLAST activity, decreased glutamate uptake and excitotoxic damage to the retina. Disruption of glutamate transport in Muller cells following the increase in the intraocular pressure has been shown to be also caused by inner retinal hypoxia (due to compression of blood vessels), increased formation of free radicals in the mitochondria and lipid peroxidation (Mysona et al., 2009). Glial dysfunction linked to ophthalmic diseases has been found to be paralleled by aberrant production and activity of pro-NGF. Under physiological conditions, TrkA is expressed in retinal ganglion cells (RGC), whereas p75NTR is mainly present on glial Muller cells. Alteration of NGF and NGF receptors has been shown to occur in several ophthalmic pathologies, such as glaucoma and retinal injury (Sposato et al., 2008; Colafrancesco et al., 2011). Induction of reactive gliosis in these pathological conditions seems to involve up-regulation of p75 and proNGF which causes RGC death through an indirect mechanism: activation of a p75NTR/sortilin/NRAGE-dependent pathway and production of TNFα by Müller glial cells (Bai et al., 2010; Lebrun-Julien et al., 2010).

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7. Neurotrophin-derived drug candidates for neuroprotection Based on the extensive literature regarding the several functions of neurotrophins in the nervous system, it is generally recognized that decreased neurotrophin availability and signaling play a crucial role in the pathophysiology of many neurological and psychiatric disorders (Weickert et al., 2005; Calissano et al., 2010). Moreover, the functional link between all the components of reactive gliosis (excitotoxicity, decreased neuronal metabolism and antioxidant properties, alteration of neurotrophins metabolism/signaling, and impaired synaptic plasticity), is all indicative of the impact that alterations of this complex neuro-glial network have on disease progression both during brain injury and in neurodegenerative diseases and age-related disorders. Because of their crucial activity in modulating multiple pathways underlying synaptic homeostasis and function in the nervous system, neurotrophins are the most likely drug candidates for neuroprotection in many neurological diseases. For instance, the therapeutic potential of NGF in restoring the cholinergic function in AD has been largely demonstrated in several experimental models (Williams et al., 1986; Koliatsos et al., 1991) and confirmed by small clinical trials (Tuszynski et al., 2005). Experimental and clinical findings are also available about the therapeutic activity of topic application of murine NGF (mNGF) in several ophthalmic pathologies (neurotrophic keratites, dry eye, glaucoma, etc.) (Aloe et al., 2008; Lambiase et al., 2010; Bagnis et al., 2011), as well as on cutaneous ulcers (Aloe et al., 2008; Sun et al., 2010) and peripheral neuropathies (Apfel, 2002). Neuroprotection by other neurotrophins, such as BDNF, in AD and other neurodegenerative conditions has also been largely demonstrated (Monteggia, 2011; Nagahara et al., 2009). However, the development of neurotrophin proteins as drugs holds a number of intrinsic drawbacks, including: i) difficulties to obtain large amount of correctly folded recombinant proteins; ii) poor pharmacokinetic properties due to their in vivo instability (as target of proteinases) and low permeability through the blood–brain barrier, thus raising delivery problems; and iii) pleiotropic actions due to their interaction with receptors and co-receptors (p75 and sortilin) that activate unwanted effects (p75-mediated neuronal death). More recently, however, a valid alternative for neurotrophinbased therapies is the construction of small molecules that can interact and activate specific receptor(s). This strategy has been employed to construct small functional mimetics of NGF, BDNF and NT-3 with activity of agonists or antagonists (Zaccaro et al., 2005; Peleshok and Saragovi, 2006; Massa et al., 2006, 2010; MolinaHolgado et al., 2008). This approach might be even more suitable for both pharmaceutical development and therapeutic properties of the molecules. Advantages also include: i) specific receptor targeting, ii) lower molecular weight, and better pharmacological properties, iii) stability to proteinases, iv) lack of immunogenicity, and v) lower cost of production. For instance, given the role of p75 in mediating proneurotrophin-death signals, NGF peptidomimetics endowed with agonist activity for TrkA or antagonism for p75 might be better neurotherapeutics by targeting more specifically Trk-dependent survival and neuronal function, or blocking the p75-mediated death signaling. Some of these molecules have been found to display some efficacy both in a model of cholinergic dysfunction (Bruno et al., 2004) and in animal models of glaucoma (Shi et al., 2007). More recently, a TrkB antagonist also showed to be useful in reducing anxiety and depression-related behaviors (Cazorla et al., 2011). Our group recently developed a NGF-like peptide, BB14, that behaved as a strong TrkA agonist both in vitro, as demonstrated by its neurotrophic activity on DRG and PC12 cell differentiation through TrkA phosphorylation, and in a rat model of peripheral nerve injury by CCI where BB14 showed to be effective in reducing reactive gliosis and neuropathic behavior (Colangelo et al., 2008). Based on the impact of reactive astrogliosis in most neurodegenerative pathologies, this novel anti-gliosis function displayed by BB14 might render this

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molecule very suitable for therapeutic applications in a wide number of neurological conditions, as well as in ophthalmic pathologies like glaucoma, neurotrophic keratites and dry eye, which so far lack effective therapeutic interventions. 8. Conclusions Molecular dissection of neurodegenerative pathologies strongly indicates that reactive astrogliosis is a complex process that involves a number of changes ranging from alterations in gene expression and morphology to modification of synaptic circuitry. The relevance of neurotrophins in neuronal survival and function, and the strict correlation between glial dysfunction and aberrant NGF metabolisms and signaling, all suggest that the glia-neurotrophin system might be an effective therapeutic target in neurodegenerative diseases. Indeed, the positive effects of recombinant neurotrophins or peptidomimetics in animal models and small clinical trials also provide compelling evidence of the impact that novel biotechnological approaches might have in the development of novel and effective molecules for neuroprotection. It is expected that small neurotrophin molecules endowed with better selectivity and potency, and void of the potential complications of systemic neurotrophin stimulation, might be the next generation therapeutics for neurodegenerative diseases. Acknowledgments This work was supported by grants from Regione Campania (L.R. N.5 Bando 2003 to M.P.), the Italian Minister of Research and University (PRIN2007 to M.P. and to A.M.C.), Regione Campania (Prog. Spec art 12 E.F. 2000 to M.P.), the CNR (Neurobiotecnologie 2003 to M.P.), FIRBITALBIONET to L.A., PRIMM srl, Blueprint Biotech and Associazione LeviMontalcini. References Alberghina L, Colangelo AM. The modular systems biology approach to investigate the control of apoptosis in Alzheimer's disease neurodegeneration. BMC Neurosci 2006;7(Suppl 1):S2. Oct 30. Allegrini P, Fronzoni L, Pirino D. The influence of the astrocyte field on neuronal dynamics and synchronization. J Biol Phys 2009;35:413–23. Aloe L, Tirassa P, Lambiase A. The topical application of nerve growth factor as a pharmacological tool for human corneal and skin ulcers. Pharmacol Res 2008;57: 253–8. Al-Shawi R, Hafner A, Chun S, Raza S, Crutcher K, Thrasivoulou C, et al. ProNGF, sortilin, and age-related neurodegeneration. Ann NY Acad Sci 2007;1119:208–15. Apfel SC. Nerve growth factor for the treatment of diabetic neuropathy: what went wrong, what went right, and what does the future hold? Int Rev Neurobiol 2002;50:393–413. Bagnis A, Papadia M, Scotto R, Traverso CE. Current and emerging medical therapies in the treatment of glaucoma. Expert Opin Emerg Drugs 2011;16:293–307. Bai Y, Dergham P, Nedev H, Xu J, Galan A, Rivera JC, et al. Chronic and acute models of retinal neurodegeneration TrkA activity are neuroprotective whereas p75NTR activity is neurotoxic through a paracrine mechanism. J Biol Chem 2010;285: 39392–400. Bajramovic JJ. Regulation of innate immune responses in the central nervous system. CNS Neurol Disord Drug Targets 2011;10:4–24. Bal-Price A, Brown GC. Inflammatory neurodegeneration mediated by nitric oxide from activated glia-inhibiting neuronal respiration, causing glutamate release and excitotoxicity. J Neurosci 2001;21:6480–91. Beattie MS, Harrington AW, Lee R, Kim JY, Boyce SL, Longo FM, et al. ProNGF induces p75-mediated death of oligodendrocytes following spinal cord injury. Neuron 2002;36:375–86. Benarroch EE. Neuron–astrocyte interactions: partnership for normal function and disease in the central nervous system. Mayo Clin Proc 2005;80:1326–38. Bentley CA, Lee KF. p75 is important for axon growth and Schwann cell migration during development. J Neurosci 2000;20:7706–15. Bolaños JP, Almeida A, Moncada S. Glycolysis: a bioenergetic or a survival pathway? Trends Biochem Sci 2010;35:145–9. Borlongan CV, Koutouzis TK, Sanberg PR. 3-Nitropropionic acid animal model and Huntington's disease. Neurosci Biobehav Rev 1997;21:289–93. Bringmann A, Iandiev I, Pannicke T, Wurm A, Hollborn M, Wiedemann P, et al. Cellular signaling and factors involved in Müller cell gliosis: neuroprotective and detrimental effects. Prog Retin Eye Res 2009;28:423–51. Brouillet E, Jacquard C, Bizat N, Blum D. 3-Nitropropionic acid: a mitochondrial toxin to uncover physiopathological mechanisms underlying striatal degeneration in Huntington's disease. J Neurochem 2005;95:1521–40.

270

A.M. Colangelo et al. / Biotechnology Advances 30 (2012) 261–271

Bruno MA, Cuello AC. Activity-dependent release of precursor nerve growth factor, conversion to mature nerve growth factor, and its degradation by a protease cascade. Proc Natl Acad Sci USA 2006;103:6735–40. Bruno MA, Clarke PB, Seltzer A, Quirion R, Burgess K, Cuello AC, et al. Long-lasting rescue of age-associated deficits in cognition and the CNS cholinergic phenotype by a partial agonist peptidomimetic ligand of TrkA. J Neurosci 2004;24:8009–18. Calissano P, Matrone C, Amadoro G. Nerve growth factor as a paradigm of neurotrophins related to Alzheimer's disease. Dev Neurobiol 2010;70:372–83. Casaccia-Bonnefil P, Carter BD, Dobrowsky RT, Chao MV. Death of oligodendrocytes mediated by the interaction of nerve growth factor with its receptor p75. Nature 1996;383:716–9. Cavaliere C, Cirillo G, Bianco MR, Rossi F, De Novellis V, Maione S, et al. Gliosis alters expression and uptake of spinal glial amino acid transporters in a mouse neuropathic pain model. NGB 2007;2:141–53. Cazorla M, Prémont J, Mann A, Girard N, Kellendonk C, Rognan D. Identification of a low-molecular weight TrkB antagonist with anxiolytic and antidepressant activity in mice. J Clin Invest 2011;121:1846–57. Chao MV. Neurotrophins and their receptors: a convergence point for many signalling pathways. Nat Rev Neurosci 2003;4:299–309. Chao MV, Bothwell M. Neurotrophins: to cleave or not to cleave. Neuron 2002;33:9–12. Chao MV, Rajagopal R, Lee FS. Neurotrophin signalling in health and disease. Clin Sci 2006;110:167–73. Chaudhry FA, Lehre KP, van Lookeren Campagne M, Ottersen OP, Danbolt NC, StormMathisen J. Glutamate transporters in glial plasma membranes: highly differentiated localizations revealed by quantitative ultrastructural immunocytochemistry. Neuron 1995;15:711–20. Chen LW, Yung KKL, Chan YS, Shum DKY, Bolam JP. The proNGF–p75NTR–sortilin signalling complex as new target for the therapeutic treatment of Parkinson's disease. CNS Neurol Disord Drug Targets 2008;7:512–23. Chung YC, Ko HW, Bok E, Park ES, Huh SH, Nam JH, et al. The role of neuroinflammation on the pathogenesis of Parkinson's disease. BMB Rep 2010;43:225–32. Cirillo G, Cavaliere C, Bianco MR, De Simone A, Colangelo AM, Sellitti S, et al. Intrathecal NGF administration reduces reactive astrocytosis and changes neurotrophin receptors expression pattern in a rat model of neuropathic pain. Cell Mol Neurobiol 2010a;30:51–62. Cirillo G, Maggio N, Bianco MR, Vollono C, Sellitti S, Papa M. Discriminative behavioral assessment unveils remarkable reactive astrocytosis and early molecular correlates in basal ganglia of 3-nitropropionic acid subchronic treated rats. Neurochem Int 2010b;56:152–60. Cirillo G, Bianco MR, Colangelo AM, Cavaliere C, Daniele de L, Zaccaro L, et al. Reactive astrocytosis-induced perturbation of synaptic homeostasis is restored by nerve growth factor. Neurobiol Dis 2011;41:630–9. Colafrancesco V, Parisi V, Sposato V, Rossi S, Russo MA, Coassin M, et al. Ocular application of nerve growth factor protects degenerating retinal ganglion cells in a rat model of glaucoma. J Glaucoma 2011;20:100–8. Colangelo AM, Johnson PF, Mocchetti I. Beta-adrenergic receptor-induced activation of nerve growth factor gene transcription in rat cerebral cortex involves CCAAT/enhancer-binding protein delta. Proc Natl Acad Sci USA 1998;95: 10920–5. Colangelo AM, Bianco MR, Vitagliano L, Cavaliere C, Cirillo G, De Gioia L, et al. A new nerve growth factor-mimetic peptide active on neuropathic pain in rats. J Neurosci 2008;28:2698–709. Cosgaya JM, Chan JR, Shooter EM. The neurotrophin receptor p75NTR as a positive modulator of myelination. Science 2002;298:1245–8. Cragnolini AB, Friedman WJ. The function of p75NTR in glia. Trends Neurosci 2008;31: 99–104. Cragnolini AB, Huang Y, Gokina P, Friedman WJ. Nerve growth factor attenuates proliferation of astrocytes via the p75 neurotrophin receptor. Glia 2009;57:1386–92. Cuello AC, Bruno MA. The failure in NGF maturation and its increased degradation as the probable cause for the vulnerability of cholinergic neurons in Alzheimer's disease. Neurochem Res 2007;32:1041–5. Danbolt NC. Glutamate uptake. Prog Neurobiol 2001;65:1–105. Davalos D, Grutzendler J, Yang G, Kim JV, Zuo Y, Jung S, et al. ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci 2005;8:752–8. de Melo Reis RA, Ventura AL, Schitine CS, de Mello MC, de Mello FG. Müller glia as an active compartment modulating nervous activity in the vertebrate retina: neurotransmitters and trophic factors. Neurochem Res 2008;33:1466–74. Domeniconi M, Hempstead BL, Chao MV. Pro-NGF secreted by astrocytes promotes motor neuron cell death. Mol Cell Neurosci 2007;34:271–9. Dun Y, Mysona B, Van Ells T, Amarnath L, Ola MS, Ganapathy V, et al. Expression of the cystine–glutamate exchanger (xc-) in retinal ganglion cells and regulation by nitric oxide and oxidative stress. Cell Tissue Res 2006;324:189–202. Fahnestock M, Michalski B, Xu B, Coughlin MD. The precursor pro-nerve growth factor is the predominant form of nerve growth factor in brain and is increased in Alzheimer's disease. Mol Cell Neurosci 2001;18:210–20. Faideau M, Kim J, Cormier K, Gilmore R, Welch M, Auregan G, et al. In vivo expression of polyglutamine-expanded huntingtin by mouse striatal astrocytes impairs glutamate transport: a correlation with Huntington's disease subjects. Hum Mol Genet 2010;19: 3053–67. Fellin T, Carmignoto G. Neurone-to-astrocyte signalling in the brain represents a distinct multifunctional unit. J Physiol 2004;559:3–15. Festoff BW, Ameenuddin S, Arnold PM, Wong A, Santacruz KS, Citron BA. Minocycline neuroprotects, reduces microgliosis, and inhibits caspase protease expression early after spinal cord injury. J Neurochem 2006;97:1314–26. Friedman WJ. Neurotrophins induce death of hippocampal neurons via the p75 receptor. J Neurosci 2000;20:6340–6.

Friedman WJ, Thakur S, Seidman L, Rabson AB. Regulation of nerve growth factor mRNA by interleukin-1 in rat hippocampal astrocytes is mediated by NFkappaB. J Biol Chem 1996;271:31115–20. Galve-Roperh I, Malpartida JM, Haro A, Brachet P, Díaz-Laviada I. Regulation of nerve growth factor secretion and mRNA expression by bacterial lipopolysaccharide in primary cultures of rat astrocytes. J Neurosci Res 1997;49:569–75. Giovannoni R, Maggio N, Bianco MR, Cavaliere C, Cirillo G, Lavitrano M, et al. Reactive astrocytosis and glial glutamate transporter clustering are early changes in a spinocerebellar ataxia type 1 transgenic mouse model. Neuron Glia Biol 2007;3: 335–51. Glass CK, Saijo K, Winner B, Marchetto MC, Gage FH. Mechanisms underlying inflammation in neurodegeneration. Cell 2010;140:918–34. Gordon PH, Moore DH, Miller RG, Florence JM, Verheijde JL, Doorish C, et al. Study group. Efficacy of minocycline in patients with amyotrophic lateral sclerosis: a phase III randomised trial. Lancet Neurol 2007;6:1045–53. Gray BC, Skipp P, O'Connor VM, Perry VH. Increased expression of glial fibrillary acidic protein fragments and mu-calpain activation within the hippocampus of prioninfected mice. Biochem Soc Trans 2006;34:51–4. Griffin WS, Liu L, Li Y, Mrak RE, Barger SW. Interleukin-1 mediates Alzheimer and Lewy body pathologies. J Neuroinflammation 2006;3:5. Hanisch UK, Kettenmann H. Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nat Neurosci 2007;10:1387–94. Harrington AW, Leiner B, Blechschmitt C, Arevalo JC, Lee R, Mörl K, et al. Secreted proNGF is a pathophysiological death-inducing ligand after adult CNS injury. Proc Natl Acad Sci USA 2004;16:6226–30. Hashizume H, DeLeo JA, Colburn RW, Weinstein JN. Spinal glial activation and cytokine expression after lumbar root injury in the rat. Spine 2000;25:1206–17. Haydon PG. Glia: listening and talking to the synapse. Nat Rev Neurosci 2001;2:185–93. Heales SJ, Lam AA, Duncan AJ, Land JM. Neurodegeneration or neuroprotection: the pivotal role of astrocytes. Neurochem Res 2004;29:513–9. Heneka MT, Rodriguez JJ, Verkhratsky A. Neuroglia in neurodegeneration. Brain Res Rev 2010;63:189–211. Herrero-Mendez A, Almeida A, Fernández E, Maestre C, Moncada S, Bolaños JP. The bioenergetic and antioxidant status of neurons is controlled by continuous degradation of a key glycolytic enzyme by APC/C-Cdh1. Nat Cell Biol 2009;11: 747–52. Hickey MA, Kosmalska A, Enayati J, Cohen R, Zeitlin S, Levine MS, et al. Extensive early motor and non-motor behavioral deficits are followed by striatal neuronal loss in knock-in Huntington's disease mice. Neuroscience 2008;157:280–95. Hirase H. A multi-photon window onto neuronalglial-vascular communication. Trends Neurosci 2005;28:217–9. Jansen P, Giehl K, Nyengaard JR, Teng K, Lioubinski O, Sjoegaard SS, et al. Roles for the pro-neurotrophin receptor sortilin in neuronal development, aging and brain injury. Nat Neurosci 2007;10:1449–57. Ji RR, Strichartz G. Cell signaling and the genesis of neuropathic pain. Science STKE 2004:reE14. Keller AF, Gravel M, Kriz J. Treatment with minocycline after disease onset alters astrocyte reactivity and increases microgliosis in SOD1 mutant mice. Exp Neurol 2011;228(1):69–79. Knight RA, Verkhratsky A. Neurodegenerative diseases: failures in brain connectivity? Cell Death Differ 2010;17:1069–70. Kofuji P, Newman EA. Potassium buffering in the central nervous system. Neuroscience 2004;129:1045–56. Koliatsos VE, Clatterbuck RE, Nauta HJW, Knusel B, Burton LE, Hefti FF, et al. Human Nerve Growth Factor prevents degeneration of basal forebrain cholinergic neurons in primates. Ann Neurol 1991;30:831–40. Kuang X, Scofield VL, Yan M, Stoica G, Liu N, Wong PK. Attenuation of oxidative stress, inflammation and apoptosis by minocycline prevents retrovirus-induced neurodegeneration in mice. Brain Res 2009;1286:174–84. Kuczewski N, Porcher C, Gaiarsa JL. Activity-dependent dendritic secretion of brainderived neurotrophic factor modulates synaptic plasticity. Eur J Neurosci 2010;32: 1239–44. Lambiase A, Mantelli F, Bonini S. Nerve growth factor eye drops to treat glaucoma. Drug News Perspect 2010;23:361–7. Landles C, Bates GP. Huntingtin and the molecular pathogenesis of Huntington's disease. Fourth in molecular medicine review series. EMBO Rep 2004;5:958–63. Lebrun-Julien F, Bertrand MJ, De Backer O, Stellwagen D, Morales CR, Di Polo A, et al. ProNGF induces TNFalpha-dependent death of retinal ganglion cells through a p75NTR non-cell-autonomous signaling pathway. Proc Natl Acad Sci USA 2010;107:3817–22. Lee YB, Du S, Rhim H, Lee EB, Markelonis GJ, Oh TH. Rapid increase in immunoreactivity to GFAP in astrocytes in vitro induced by acidic pH is mediated by calcium influx and calpain I. Brain Res 2000;864:220–9. Lee R, Kermani P, Teng KK, Hempstead BL. Regulation of cell survival by secreted proneurotrophins. Science 2001;294:1945–58. Lehre KP, Levy LM, Ottersen OP, Storm-Mathisen J, Danbolt NC. Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations. J Neurosci 1995;15:1835–53. Levi-Montalcini R. Effects of mouse tumor transplantation on the nervous system. Ann NY Acad Sci 1952;55:330–44. Lewerenz J, Klein M, Methner A. Cooperative action of glutamate transporters and cystine/glutamate antiporter system Xc- protects from oxidative glutamate toxicity. J Neurochem 2006;98:916–25. Li Z, Hogan EL, Banik NL. Role of calpain in spinal cord injury: increased calpain immunoreactivity in rat spinal cord after impact trauma. Neurochem Res 1996;21: 441–8.

A.M. Colangelo et al. / Biotechnology Advances 30 (2012) 261–271 Lievens JC, Salin P, Had-Aissouni L, Mahy N, Kerkerian-Le GL. Differential effects of corticostriatal and thalamostriatal deafferentation on expression of the glutamate transporter GLT1 in the rat striatum. J Neurochem 2000;74:909–19. Lobsiger CS, Cleveland DW. Glial cells as intrinsic components of non-cell-autonomous neurodegenerative disease. Nat Neurosci 2007;10:1355–60. Loiola EC, Ventura AL. Release of ATP from avian Müller glia cells in culture. Neurochem Int 2011;58:414–22. Lu B, Pang PT, Woo NH. The yin and yang of neurotrophin action. Nat Rev Neurosci 2005;6:603–14. Machado-Vieira R, Manji HK, Zarate CA. The role of the tripartite glutamatergic synapse in the pathophysiology and therapeutics of mood disorders. Neuroscientist 2009;15:525–39. Magistretti PJ. Neuron–glia metabolic coupling and plasticity. J Exp Biol 2006;209: 2304–11. Maragakis NJ, Rothstein JD. Mechanisms of disease: astrocytes in neurodegenerative disease. Nat Clin Pract Neurol 2006;2:679–89. Masoudi R, Ioannou MS, Coughlin MD, Pagadala P, Neet KE, Clewes O, et al. Biological activity of Nerve Growth Factor precursor is dependent upon relative levels of its receptors. J Biol Chem 2009;284:18424–33. Massa SM, Xie Y, Yang T, Harrington AW, Kim ML, Yoon SO, et al. Small, nonpeptide p75NTR ligands induce survival signaling and inhibit proNGF-induced death. J Neurosci 2006;26:5288–300. Massa SM, Yang T, Xie Y, Shi J, Bilgen M, Joyce JN, et al. Small molecule BDNF mimetics activate TrkB signaling and prevent neuronal degeneration in rodents. J Clin Invest 2010;120:1774–85. McMahon SB, Cafferty WB, Marchand F. Immune and glial cell factors as pain mediators and modulators. Exp Neurol 2005;192:444–62. Mennerick S, Shen W, Xu W, Benz A, Tanaka K, Shimamoto K, et al. Substrate turnover by transporters curtails synaptic glutamate transients. J Neurosci 1999;19: 9242–51. Milton ID, Banner SJ, Ince PG, Piggott NH, Fray AE, Thatcher N, et al. Expression of the glial glutamate transporter EAAT2 in the human CNS: an immunohistochemical study. Brain Res Mol Brain Res 1997;52:17–31. Mocchetti I, Colangelo AM. Transcriptional regulation of NGF in the central nervous system. In: Mocchetti I, editor. Neurobiology of the neurotrophins. F.P. Graham Publishing Co.; 2001. p. 631–54. Molina-Holgado F, Doherty P, Williams G. Tandem repeat peptide strategy for the design of neurotrophic factor mimetics. CNS Neurol Disord Drug Targets 2008;7: 110–9. Monteggia LM. Toward neurotrophin-based therapeutics. Am J Psychiatry 2011;168: 114–6. Mrak RE, Griffin WS. Glia and their cytokines in progression of neurodegeneration. Neurobiol Aging 2005;26:349–54. Musholt K, Cirillo G, Cavaliere C, Bianco MR, Bock J, Helmeke C, et al. Neonatal separation stress reduces glial fibrillary acidic protein and S100beta-immunoreactive astrocytes in the rat medial precentral cortex. Dev Neurobiol 2009;69:203–11. Mysona B, Dun Y, Duplantier J, Ganapathy V, Smith SB. Effects of hyperglycemia and oxidative stress on the glutamate transporters GLAST and system xc- in mouse retinal Müller glial cells. Cell Tissue Res 2009;335:477–88. Nagahara AH, Merrill DA, Coppola G, Tsukada S, Schroeder BE, Shaked GM, et al. Neuroprotective effects of brain-derived neurotrophic factor in rodent and primate models of Alzheimer's disease. Nat Med 2009;15:331–7. Narita M, Miyatake M, Narita M, Shibasaki M, Shindo K, Nakamura A, et al. Direct evidence of astrocytic modulation in the development of rewarding effects induced by drugs of abuse. Neuropsychopharmacology 2006;31:2476–88. Nieweg K, Schaller H, Pfrieger FW. Marked differences in cholesterol synthesis between neurons and glial cells from postnatal rats. J Neurochem 2009;109:125–34. Nimmerjahn A, Kirchhoff F, Helmchen F. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 2005;308:1314–8. Orr HT, Zoghbi HY. Trinucleotide repeat disorders. Annu Rev Neurosci 2007;30: 575–621. Paulsen JS, Magnotta VA, Mikos AE, Paulson HL, Penziner E, Andreasen NC, et al. Brain structure in preclinical Huntington's disease. Biol Psychiatry 2006;59:57–63. Paulson HL, Fischbeck KH. Trinucleotide repeats in neurogenetic disorders. Annu Rev Neurosci 1996;19:79–107. Pehar M, Cassina P, Vargas MR, Castellanos R, Viera L, Beckman JS, et al. Astrocytic production of nerve growth factor in motor neuron apoptosis: implications for amyotrophic lateral sclerosis. J Neurochem 2004;89:464–73. Pekny M, Nilsson M. Astrocyte activation and reactive gliosis. Glia 2005;50:427–34. Peleshok J, Saragovi HU. Functional mimetics of neurotrophins and their receptors. Biochem Soc Trans 2006;34:612–7. Pfrieger FW. Roles of glial cells in synapse development. Cell Mol Life Sci 2009;66: 2037–47. Pfrieger FW, Barres BA. New views on synapse–glia interactions. Curr Opin Neurobiol 1996;6:615–21. Raghavendra V, Rutkowski MD, De Leo JA. The role of spinal neuroimmune activation in morphine tolerance/hyperalgesia in neuropathic and sham-operated rats. J Neurosci 2002;22:9980–9. Ransohoff RM, Perry VH. Microglial physiology: unique stimuli, specialized responses. Annu Rev Immunol 2009;27:119–45. Reichardt LF. Neurotrophin-regulated signalling pathways. Philos Trans R Soc Lond B Biol Sci 2006;361:1545–64. Rogers J, Kirby LC, Hempelman SR, Berry DL, McGeer PL, Kaszniak AW, et al. Clinical trial of indomethacin in Alzheimer's disease. Neurology 1993;43:1609–11. Rossi D, Volterra A. Astrocytic dysfunction: insights on the role in neurodegeneration. Brain Res Bull 2009;80:224–32.

271

Rothstein JD, Martin L, Levey AI, Dykes-Hoberg M, Jin L, Wu D, et al. Localization of neuronal and glial glutamate transporters. Neuron 1994;13:713–25. Rothstein JD, Dykes-Hoberg M, Pardo CA, Bristol LA, Jin L, Kuncl RW, et al. Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron 1996;16:675–86. Saijo K, Winner B, Carson TC, Collier JG, Boyer L, Rosenfeld MG, et al. A Nurr1/CoREST pathway in microglia and astrocytes protects dopaminergic neurons from inflammation-induced death. Cell 2009;137:47–59. Schols L, Bauer P, Schmidt T, Schulte T, Riess O. Autosomal dominant cerebellar ataxias: clinical features, genetics, and pathogenesis. Lancet Neurol 2004;3:291–304. Serbest G, Burkhardt MF, Siman R, Raghupathi R, Saatman KE. Temporal profiles of cytoskeletal protein loss following traumatic axonal injury in mice. Neurochem Res 2007;32:2006–14. Servadio A, Koshy B, Armstrong D, Antalffy B, Orr HT, Zoghbi HY. Expression analysis of the ataxin-1 protein in tissues from normal and spinocerebellar ataxia type 1 individuals. Nat Genet 1995;10:94–8. Sheldon AL, Robinson MB. The role of glutamate transporters in neurodegenerative diseases and potential opportunities for intervention. Neurochem Int 2007;51: 333–55. Shi Z, Birman E, Saragovi HU. Neurotrophic rationale in glaucoma: a TrkA agonist, but not NGF or a p75 antagonist, protects retinal ganglion cells in vivo. Dev Neurobiol 2007;67:884–94. Simard M, Nedergaard M. The neurobiology of glia in the context of water and ion homeostasis. Neuroscience 2004;129:877–96. Skaper SD. The biology of neurotrophins, signalling pathways, and functional peptide mimetics of neurotrophins and their receptors. CNS Neurol Disord Drug Targets 2008;7:46–62. Sposato V, Bucci MG, Coassin M, Russo MA, Lambiase A, Aloe L. Reduced NGF level and TrkA protein and TrkA gene expression in the optic nerve of rats with experimentally induced glaucoma. Neurosci Lett 2008;446:20–4. Sun W, Lin H, Chen B, Zhao W, Zhao Y, Xiao Z, et al. Collagen scaffolds loaded with collagen-binding NGF-beta accelerate ulcer healing. J Biomed Mater Res A 2010;92: 887–95. Suzuki A, Stern SA, Bozdagi O, Huntley GW, Walker RH, Magistretti PJ, et al. Astrocyte-neuron lactate transport is required for long-term memory formation. Cell 2011;144:810–23. Takano T, Tian GF, Peng W, Lou N, Libionka W, Han X, et al. Astrocyte-mediated control of cerebral blood flow. Nat Neurosci 2006;9:260–7. Tansey MG, Goldberg MS. Neuroinflammation in Parkinson's disease: its role in neuronal death and implications for therapeutic intervention. Neurobiol Dis 2010;37:510–8. Teng HK, Teng KK, Lee R, Wright S, Tevar S, Almeida RD, et al. ProBDNF induces neuronal apoptosis via activation of a receptor complex of p75NTR and sortilin. J Neurosci 2005;25:5455–63. Trotti D, Aoki M, Pasinelli P, Berger UV, Danbolt NC, Brown Jr RH, et al. Amyotrophic lateral sclerosis-linked glutamate transporter mutant has impaired glutamate clearance capacity. J Biol Chem 2001;276:576–82. Tuszynski MH, Thal L, Pay M, Salmon DP, U HS, Bakay R, et al. A phase 1 clinical trial of nerve growth factor gene therapy for Alzheimer disease. Nat Med 2005;11:551–5. Unger JW. Glial reaction in aging and Alzheimer's disease. Microsc Res Tech 1998;43: 24–8. van Noort JM, van den Elsen PJ, van Horssen J, Geurts JJ, van der Valk P, Amor S. Preactive multiple sclerosis lesions offer novel clues for neuroprotective therapeutic strategies. CNS Neurol Disord Drug Targets 2011;10:68–81. Verkhratsky A, Olabarria M, Noristani HN, Yeh CY, Rodriguez JJ. Astrocytes in Alzheimer's disease. Neurotherapeutics 2010;7:399–412. Vilar M, Murillo-Carretero M, Mira H, Magnusson K, Besset V, Ibáñez CF. Bex1, a novel interactor of the p75 neurotrophin receptor, links neurotrophin signaling to the cell cycle. EMBO J 2006;25:1219–30. Watkins LR, Maier SF. Glia: a novel drug discovery target for clinical pain. Nat Rev Drug Discov 2003;2:973–85. Watkins LR, Milligan ED, Maier SF. Glial activation: a driving force for pathological pain. Trends Neurosci 2001;24:450–5. Weickert CS, Ligons DL, Romanczyk T, Ungaro G, Hyde TM, Herman MM, et al. Reductions in neurotrophin receptor mRNAs in the prefrontal cortex of patients with schizophrenia. Mol Psychiatry 2005;10:637–50. Wieseler-Frank J, Maier SF, Watkins LR. Glial activation and pathological pain. Neurochem Int 2004;45:389–95. Williams LR, Varon S, Peterson GM, Wictorin K, Fisher W, Bjorklund A, et al. Continuous infusion of nerve growth factor prevents basal forebrain neuronal death after fimbria fornix transection. Proc Natl Acad Sci USA 1986;83:9231–5. Wong ST, Henley JR, Kanning KC, Huang KH, Bothwell M, Poo MM. A p75(NTR) and Nogo receptor complex mediates repulsive signaling by myelin-associated glycoprotein. Nat Neurosci 2002;5:1302–8. Yano H, Torkin R, Martin LA, Chao MV, Teng KK. Proneurotrophin-3 is a neuronal apoptotic ligand: evidence for retrograde-directed cell killing. J Neurosci 2009;29: 14790–802. Ye ZC, Wyeth MS, Baltan-Tekkok S, Ransom BR. Functional hemichannels in astrocytes: a novel mechanism of glutamate release. J Neurosci 2003;23:3588–96. Zaccaro MC, Lee HB, Pattarawarapan M, Xia Z, Caron A, L'Heureux PJ, et al. Selective small molecule peptidomimetic ligands of TrkC and TrkA receptors afford discrete or complete neurotrophic activities. Chem Biol 2005;12:1015–28. Zhuang ZY, Gerner P, Woolf CJ, Ji RR. ERK is sequentially activated in neurons, microglia, and astrocytes by spinal nerve ligation and contributes to mechanical allodynia in this neuropathic pain model. Pain 2005;114:149–59.