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Despite having disease-specific pathologic features and symptoms, neurodegenerative diseases share common mechanisms, such as excitotoxicity, neuroinflammation, and neurotransmitter dysregulation. Although the common underlying cause of these neurodegenerative processes has yet to be identified, accumulating evidence suggests that branched-chain amino acids (BCAAs) could be involved. BCAAs have been shown to not only influence the central levels of neurotransmitters but also to induce excitotoxicity, hyperexcitability, inflammation, and oxidative stress. Furthermore, emerging evidence indicates that BCAA metabolism may be dysregulated in major neurodegenerative diseases, namely Alzheimer's and Parkinson's diseases and amyotrophic lateral sclerosis. In this review, we identified the neurodegenerative mechanisms of BCAAs and outlined their potential roles in neurodegenerative diseases, suggesting that targeting BCAA metabolism may represent a new approach to identifying new therapeutic targets for multifaceted neurodegenerative diseases.

Stroke is a leading cause of disability and death world-wide and nutrition is a modifiable risk factor for stroke. Metheylenetetrahydrofolate reductase (MTHFR) is an enzyme involved in the metabolism of folic acid, a B-vitamin. In humans, a polymorphism in MTHFR (677C→T) is linked to increased risk of stroke, but the mechanisms remain unknown. The Mthfr+/− mice mimic a phenotype described in humans at bp677. Using this mouse model, the aim of this study was to investigate the impact of MTHFR deficiency on stroke outcome. Male Mthfr+/− and wildtype littermate control mice were aged (~1.5-year-old) and trained on the single pellet reaching task. After which the sensorimotor cortex was then damaged using photothrombosis (PT), a model for ischemic stroke. Post-operatively, animals were tested for skilled motor function, and brain tissue was processed to assess cell death. Mthfr+/− mice were impaired in skilled reaching 2-weeks after stroke but showed some recovery at 5-weeks compared to wild types after PT damage. Within the ischemic brain, there was increased expression of active caspase-3 and reduced levels of phospho-AKT in neurons of Mthfr+/− mice. Recent data suggests that astrocytes may play a significant role after damage, the impact of MTHFR and ischemic investigated the impact of MTHFR-deficiency on astrocyte function. MTHFR-deficient primary astrocytes showed reduced cell viability after exposure to hypoxia compared to controls. Increased immunofluorescence staining of active caspase-3 and hypoxia-inducible factor 1-alpha were also observed. The data suggest that MTHFR deficiency decreases recovery after stroke by reducing neuronal and astrocyte viability.

Pentraxins are a superfamily of evolutionarily conserved proteins that are characterized by their multimeric architecture and their calcium-dependent binding. They can be broadly grouped into two subfamilies: short pentraxins and long pentraxins. Pentraxins regulate many processes in the brain as well as the periphery. Neuronal pentraxin 2 (NP2/NPTX2), also known as neuronal activity-regulated pentraxin (Narp), is an immediate-early gene that has been shown to play a critical role in guiding synaptic plasticity. NP2 has been previously linked to excitatory neurotransmission, based on its ability to aggregate excitatory receptors in the central nervous system. The mechanisms mediating the effects of NP2 on excitatory neurotransmission remain unclear and warrants further investigation. This review article focuses on the biological features of NP2 and discusses the literature supporting a role for NP2 and other pentraxins in glutamatergic signaling. An analysis of evidence around the role of pentraxins in neuropathology is also reviewed.

Peripheral nervous system (PNS) neurons support axon regeneration into adulthood, whereas central nervous system (CNS) neurons lose regenerative ability after development. To better understand this decline whilst aiming to improve regeneration, we focused on phosphoinositide 3‐kinase (PI3K) and its product phosphatidylinositol (3,4,5)‐trisphosphate (PIP3). We demonstrate that adult PNS neurons utilise two catalytic subunits of PI3K for axon regeneration: p110α and p110δ. However, in the CNS, axonal PIP3 decreases with development at the time when axon transport declines and regenerative competence is lost. Overexpressing p110α in CNS neurons had no effect; however, expression of p110δ restored axonal PIP3 and increased regenerative axon transport. p110δ expression enhanced CNS regeneration in both rat and human neurons and in transgenic mice, functioning in the same way as the hyperactivating H1047R mutation of p110α. Furthermore, viral delivery of p110δ promoted robust regeneration after optic nerve injury. These findings establish a deficit of axonal PIP3 as a key reason for intrinsic regeneration failure and demonstrate that native p110δ facilitates axon regeneration by functioning in a hyperactive fashion.

ObjectiveElevated homocysteine concentrations are a risk factor for stroke. A common genetic polymorphism in methylenetetrahydrofolate reductase (MTHFR 677 C➔T) results in elevated levels of homocysteine. MTHFR plays a critical role in the synthesis of S-adenosylmethionine (SAM), a global methyl donor. Our previous work has demonstrated that Mthfr+/− mice, which model the MTHFR polymorphism in humans, are more vulnerable to ischemic damage. The aim of this study was to investigate the cellular mechanisms by which the MTHFR-deficiency changes the brain in the context of ischemic stroke injury.MethodsIn the present study, three-month-old male Mthfr+/− and wild-type littermate mice were subjected to photothrombosis (PT) damage. Four weeks after PT damage, animals were tested on behavioral tasks, in vivo imaging was performed using T2-weighted MRI, and brain tissue was collected.ResultsMthfr+/− animals used their non-impaired forepaw more during to explore the cylinder and had a larger damage volume compared to wild-type littermates. In brain tissue of Mthfr+/− mice methionine adenosyltransferase II alpha (MAT2A) protein levels were decreased within the damage hemisphere and increased levels in hypoxia induced factor 1 alpha (HIF-1α) in non-damage hemisphere. There was an increased antioxidant response in the damage site as indicated by higher levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and superoxide dismutase 2 (SOD2).ConclusionsOur results suggest that Mthfr+/− mice are more vulnerable to PT-induced stroke damage through regulation of the cellular response. The increased antioxidant response we observed may be compensatory to the damage amount.

Peripheral nervous system (PNS) neurons support axon regeneration into adulthood, whereas central nervous system (CNS) neurons lose regenerative ability after development. To better understand this decline whilst aiming to improve regeneration, we focused on phosphoinositide 3‐kinase (PI3K) and its product phosphatidylinositol (3,4,5)‐trisphosphate (PIP 3). We demonstrate that adult PNS neurons utilise two catalytic subunits of PI3K for axon regeneration: p110α and p110δ. However, in the CNS, axonal PIP 3 decreases with development at the time when axon transport declines and regenerative competence is lost. Overexpressing p110α in CNS neurons had no effect; however, expression of p110δ restored axonal PIP 3 and increased regenerative axon transport. p110δ expression enhanced CNS regeneration in both rat and human neurons and in transgenic mice, functioning in the same way as the hyperactivating H1047R mutation of p110α. Furthermore, viral delivery of p110δ promoted robust regeneration after optic nerve injury. These findings establish a deficit of axonal PIP 3 as a key reason for intrinsic regeneration failure and demonstrate that native p110δ facilitates axon regeneration by functioning in a hyperactive fashion., CNS axons lose the ability to regenerate with maturity, whilst PNS axons do not. This study shows that PIP3 levels decline in CNS neurons at the time when regenerative ability is lost. CNS overexpression of one isoform of PI3K, p110δ, enhances axonal PIP3, axon transport, and regenerative ability.

Previous evidence suggests that a significant decline in cognitive ability begins during middle-age and continues to deteriorate with increase in age. Recent work has demonstrated the potential rejuvenation impact of growth differentiation factor-11 (GDF-11) in aged mice. We carried out experiments to evaluate the impact of a single dose of recombinant (rGDF-11) on short-term visual and spatial memory in middle-aged male mice. On the novel object recognition task, we observed middle-aged mice treated rGDF-11 showed improved performance on the novel object recognition task. However, middle-aged mice did not show increased expression of phosphorylated-Smad2/3, a downstream effector of GDF-11. We noted however that the expression of the transcription factor, Sox2 was increased within the dentate gyrus. Our data suggest that a single injection of rGDF-11 contributes to improvements in cognitive function of middle-aged animals, which may be critical in the preservation of short-term memory capacity in old age.

Astrocytes have been associated with the failure of axon regeneration in the central nervous system (CNS), as it undergoes reactive gliosis in response to damages to the CNS and functions as a chemical and physical barrier to axon regeneration. However, beneficial roles of astrocytes have been extensively studied in the spinal cord over the years, and a growing body of evidence now suggests that inducing astrocytes to become more growth-supportive can promote axon regeneration after spinal cord injury (SCI). In retina, astrocytes and Müller cells are known to undergo reactive gliosis after damage to retina and/or optic nerve and are hypothesized to be either detrimental or beneficial to survival and axon regeneration of retinal ganglion cells (RGCs). Whether they can be induced to become more growth-supportive after retinal and optic nerve injury has yet to be determined. In this review, we pinpoint the potential molecular pathways involved in the induction of growth-supportive astrocytes in the spinal cord and suggest that stimulating the activation of these pathways in the retina could represent a new therapeutic approach to promoting survival and axon regeneration of RGCs in retinal degenerative diseases.

Peripheral nervous system (PNS) neurons support axon regeneration into adulthood, whereas central nervous system (CNS) neurons lose regenerative ability after development. To better understand this decline whilst aiming to improve regeneration, we focused on phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol(3,4,5)-trisphosphate (PIP3). We found that neuronal PIP3 decreases with maturity in line with regenerative competence, firstly in the cell body and subsequently in the axon. We show that adult PNS neurons utilise two catalytic subunits of PI3K for efficient regeneration: p110 and p110{delta}. Overexpressing p110 in CNS neurons had no effect, however expression of p110{delta} restored axonal PIP3 and enhanced CNS regeneration in rat and human neurons and in transgenic mice, functioning in the same way as the hyperactivating H1047R mutation of p110. Furthermore, viral delivery of p110{delta} promoted robust regeneration after optic nerve injury. These findings demonstrate a deficit of axonal PIP3 as a reason for intrinsic regeneration failure and show that native p110{delta} facilitates axon regeneration by functioning in a hyperactive fashion.

The prevalence of stroke increases with age and the ability to absorb all nutrients from our diets decreases with age. Nutrition is a modifiable risk factor for stroke, which is a leading cause of death and disability in world-wide. Deficiencies in one‑carbon metabolism, including in methyltetrahydrofolate reductase (MTHFR), have been linked to increased risk of stroke. The Mthfr+/− mice mouse model mimic the phenotype of the MTHFR677C➔T polymorphism, such as elevated levels of homocystine. Using this mouse model, the aim of this study was to investigate the impact of dietary supplementation with 5-methylTHF, vitamin B12, and choline after ischemic stroke. Male Mthfr+/− and wildtype littermate control mice were aged (~1.5-year-old) and were placed on control diet (CD) 4-weeks prior to sensorimotor cortex damage using photothrombosis (PT), a model for ischemic stroke. Post-operatively, one group of Mthfr+/− and wildtype littermate mice were placed on 5-methylTHF, vitamin B12, and choline supplemented diet (SD). Four weeks after PT and SD motor function was assessed using the accelerating rotarod, forepaw asymmetry, and ladder beam walking tasks. Total homocysteine and cysteine levels were measured in blood. Brain tissue was processed to assess lesion volume and investigate biochemical and molecular changes. After PT and SD, Mthfr+/− mice were able to stay on the accelerating rotarod longer and used their impaired forepaw to explore more when compared to CD animals. Furthermore, total homocysteine levels in plasma and lesion volume were reduced in Mthfr+/+ and Mthfr+/− SD mice. Within the damage site, there were reduced levels of apoptotic cell death and increased neuroprotective cellular response in the brains of SD treated Mthfr+/− mice. This study reveals a critical role for one‑carbon supplementation, with 5-methylTHF, vitamin B12, and choline, in supporting improvement after ischemic stroke damage.

Paraquat is an herbicide that is commonly used worldwide. Exposure to paraquat results in Parkinson’s disease (PD)-like symptoms including dopaminergic cell loss. Nutrition has also been linked in the pathogenesis of PD, such as reduced levels of folic acid, a B-vitamin, and component of one-carbon metabolism. Within one-carbon metabolism, methylenetetrahydrofolate reductase (MTHFR) catalyzes the irreversible conversion of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. A polymorphism in MTHFR (677 C&→T) has been reported in 5%–15% of North American and European human populations. The MTHFR polymorphism is also prevalent in PD patients. The goal of this study was to investigate the impact of paraquat-induced PD-like pathology in the context of reduced levels of MTHFR. Three-month-old male Mthfr+/− mice, which model the MTHFR polymorphism observed in humans, were administered intraperitoneal injections of paraquat (10 mg/kg) or saline 6 times over 3 weeks. At the end of paraquat treatment, motor and memory function were assessed followed by collection of brain tissue for biochemical analysis. Mthfr+/– mice treated with paraquat showed impaired motor function. There was increased microglial activation within the substantia nigra (SN) of Mthfr+/− mice treated with paraquat. Additionally, all Mthfr+/− mice that were treated with paraquat showed increased oxidative stress within the dorsal striatum, but not the SN. The present results show that paraquat exposure increases PD-like pathology in mice deficient in one-carbon metabolism.

Tissue plasminogen activator (tPA) is a thrombolytic agent commonly used in the treatment of ischemic stroke. While the thrombolytic effects of tPA have been well established, the impact of this blood-brain barrier (BBB) crossing drug on neurons is not known. Given the widespread use of tPA in the clinical setting and the strict therapeutic window established for effective use of the drug, we examined the molecular mechanisms mediating the impact of tPA on postnatal cortical neurons isolated from the mouse brain. Dissociated postnatal primary cortical neurons were treated with tPA and the effects on neuron survival were evaluated. Pharmacological inhibitors of several signaling pathways previously implicated in neuroprotection (mTOR, JAK/STAT, MAPK and PKA-dependent mechanisms) were used to pinpoint the mechanistic effectors of tPA on neuron survival in vitro. We report here that tPA treatment results in a time-dependent neuroprotective effect on postnatal cortical neurons that relies predominantly on Janus kinase (JAK) and mammalian target of rapamycin (mTOR) signaling mechanisms. Taken together, these data suggest that tPA promotes neuroprotection in a temporally-regulated manner and that both JAK and mTOR signaling effectors are critical mediators of this neuroprotective effect. The results suggest the possibility of targeting these defined mechanisms to potentially expand the therapeutic window for tPA.

This protocol outlines the preparation of embryonic mouse retinal explants, which provides an effective technique to analyze neurite outgrowth in central nervous system (CNS) neurons. This validated ex vivo system, which displays limited neuronal death, is highly reproducible and particularly amenable to manipulation. Our previously published studies involving embryonic chick or adult mouse retinal explants were instrumental in the preparation of this protocol; aspects of these previous techniques were combined, adopted and optimized. This protocol thus permits more efficient analysis of neurite growth. Briefly, the retina is dissected from the embryonic mouse eye using precise techniques that take into account the small size of the embryonic eye. The approach applied ensures that the retinal ganglion cell (RGC) layer faces the adhesion substrate on coated cover slips. Neurite growth is clear, well-delineated and readily quantifiable. These retinal explants can therefore be used to examine the neurite growth effects elicited by potential therapeutic agents.

Proteoglycans (PGs) are a diverse family of proteins that consist of one or more glycosaminoglycan (GAG) chains, covalently linked to a core protein. PGs are major components of the extracellular matrix (ECM) and play critical roles in development, normal function and damage-response of the central nervous system (CNS). GAGs are classified based on their disaccharide subunits, into the following major groups: chondroitin sulfate (CS), heparan sulfate (HS), heparin (HEP), dermatan sulfate (DS), keratan sulfate (KS) and hyaluronic acid (HA). All except HA are modified by sulfation, giving GAG chains specific charged structures and binding properties. While significant neuroscience research has focused on the role of one PG family member, chondroitin sulfate proteoglycan (CSPG), there is ample evidence in support of a role for the other PGs in regulating CNS function in normal and pathological conditions. This review discusses the role of all the identified PG family members (CS, HS, HEP, DS, KS and HA) in normal CNS function and in the context of pathology. Understanding the pleiotropic roles of these molecules in the CNS may open the door to novel therapeutic strategies for a number of neurological conditions.

OBJECTIVES: Stroke is a leading cause of disability and death world-wide. Increased levels of homocysteine are associated with risk for stroke. Metheylenetetrahydrofolate reductase (MTHFR) generates methyl groups to aid in the metabolism of homocysteine. A polymorphism in MTHFR (677C→T) has been identified in 5–15% of North American and European populations. Individuals with the TT genotype have elevated homocysteine concentrations and have increased risk of stroke. Using the MTHFR mouse model, the objective of this study was to investigate the mechanisms through which MTHFR deficiency may increase the risk of stroke. METHODS: We isolated primary neurons and astrocytes cells from MTHFR-deficient mice and exposed cells to hypoxia for 6 hours. Twenty-four hours after damage, we measured cell viability. In vivo, using aged (∼1.5-year-old) male Mthfr(+/−) and wildtype littermate controls, the sensorimotor cortex was damaged using the photothrombosis model to induce ischemic stroke. Post-operatively, animals were tested on skilled motor function, and brain tissue was processed for analysis. Additionally, in adult male mice using MRI we measured lesion volume two and four weeks after damage. RESULTS: Primary neurons and astrocytes from MTHFR-deficient embryos showed reduced cell viability through increased trypan blue staining and MTT release. In vivo, aged Mthfr(+/−) mice were impaired in skilled motor function after damage. Using MRI, adult Mthfr(+/−) brains were more severely damaged compared to wild-type littermates. In vitro and in vivo, we observed increased apoptosis in Mthfr(+/−) animals. Furthermore, methionine adenosyltransferase II alpha (MAT2A) protein levels were increased in the stroke hemisphere of wild-type mice. In the non-PT hemisphere of Mthfr(+/−) mice there was an increase in hypoxia induced factor 1 alpha (HIF-1α) protein and growth differentiation factor 11 (GDF11). CONCLUSIONS: Together, our results suggest that Mthfr(+/−) mice are more vulnerable to ischemic stroke damage. This is through reducing neuronal and astrocyte viability after damage, as well as changes in methylation and cellular response. FUNDING SOURCES: This research was funded by the Natural Sciences and Engineering Research Council, Council of Ontario Universities, and Fonds de la recherché en santé Québec (FRSQ).

Lack of regeneration in the adult central nervous system (CNS) is a major hurdle that limits recovery from neurological ailments. Although accumulating research suggests the possibility of axon regeneration by targeting intrinsic signaling mechanisms, it remains a matter of controversy whether functional recovery can be achieved by manipulating aspects of molecular signaling. Recent studies have shown that granulocyte macrophage colony-stimulating factor (GM-CSF) may be an effective means of targeting repair following CNS injury; how this molecule is able to produce this effect is not known. Indeed, GM-CSF has been shown to promote neuronal survival, potentially through activation of as yet unknown cytokine-dependent signals and potentially through regulation of antiapoptotic mechanisms. It is well established that the loss of intrinsic regenerative ability is highly correlated with development of CNS neurons. We therefore designed experiments, using a well-established in vitro retinal ganglion cell (RGC) culture system, to evaluate the effect of GM-CSF on axon growth and cell survival and define possible mechanisms involved in GM-CSF-mediated effects in vitro. Several developmental stages were evaluated, with particular focus placed on stages at which axon growth is known to be significantly diminished. Our results reveal that GM-CSF not only promotes axon growth in postnatal RGCs but also enhances cell survival through a mammalian target of rapamycin (mTOR)-dependent mechanism. © 2013 Wiley Periodicals, Inc.

Traditionally, it has been thought that the mammalian central nervous system (CNS) does not regenerate. Possibly due to the inhibitory extracellular environment post-injury as well as the limited intrinsic characteristics of adult post-mitotic neurons (Smith et al., 2015); Figure 1. Modulation of several molecular mechanisms promotes some degree of cell survival and axon regeneration in the adult CNS, but much remains to be elucidated. Of particular note, activating inflammatory mechanisms has been shown to either promote or limit axon regeneration in vivo (Filbin, 2006). The optic nerve crush injury model has been extensively used as a means of studying axon growth in the damaged CNS and has facilitated significant strides in our understanding of possible mechanisms required to enhance regeneration of the damaged adult CNS. While this model represents an initial step in guiding further research into axon regeneration, it is important to note that neurological conditions, such as stroke, are characterized by significant axonal damage and models that mimic this axon-specific damage in the brain are currently being explored. Figure 1 Schematic outlining the impact of damage and regeneration to axons in the central nervous system. Stroke is modelled in animals in order to gain a better understanding of the mechanisms mediating neurodegeneration, which will guide the development of novel therapeutic approaches. One of the most well characterized models of stroke is the middle cerebral artery occulsion (MCAO) model. However, there are challenges associated with reproducibility of this model in animals. The MCAO model results in significant hemipsheric brain damage, resulting in significant loss of brain tissue in human stroke cases. This significant loss of brain tissue correlates with high mortality rates in humans and in mouse models and the investigation of axonal damage mechanisms is understandably very complex. In addition, the damage induced by MCAO is particularly variable between operated animals. Conversely, the endothelin-1 (ET-1) model of stroke can be used to generate a focal and region-specific stroke in animals. Importantly, ET-1 can indeed target axonal projections, dependent upon location in the brain that the ET-1 is introduced. Using the ET-1 mouse model, we have demonstrated targeted damage of axonal projections in the corpus collasum, with associated subcortical white matter (SWM) damage, culminating in forelimb motor deficits (Theoret et al., 2016). We have also reported that granulocyte-macrophage colony-stimulating factor (GM-CSF) plays a critical role in attenuating ET-1-induced motor deficits through activation of the mTOR signalling pathway (Theoret et al., 2016). However, the ET-1 mouse model of stroke has a number of limitations in terms of its utility toward evaluating axon regeneration. Given the well established use of the optic nerve crush injury model in analysis of axon regeneration, this may be highly amenable to further evaluate the potential regenerative impact of GM-CSF. The optic nerve has been well-characterized in terms of morphology and functionality. All the axons in the optic nerve arise from a single neuronal cell population called the retinal ganglion cells (RGCs) which can easily be experimentally manipulate. Moreover, because of its accessibility and anatomy, the eye is suitable for effective application of potential therapeutics such as growth-promoting factors like GM-CSF. Potential therapeutics can readily diffuse to the retinal ganglion cells without the need to overcome the blood-retinal barrier. The optic nerve crush model involves the use of forceps to crush and damage axonal connections in the optic nerve, this can be accompanied with administration of therapeutic agent(s), such as GM-CSF. Interestingly, and with particular relevance to the potential application of GM-CSF in brain pathologies, GM-CSF readily crosses both the blood-brain and blood-spinal barriers (McLay et al., 1997), making it an attractive therapeutic option for brain injury. GM-CSF is a pro-inflammatory cytokine, composed of four alpha helices that bind to the alpha subunit of the GM-CSF transmembrane receptor. The alpha-subunit of the GM-CSF receptor is specific for binding GM-CSF, whereas the beta-subunit is involved in signal transduction. GM-CSF is part of the haematopoietin receptor superfamily and plays a prominent role in promoting proliferation, differentiation and maturation of hematopoietic precursors cells (Burgess and Metcalf, 1980). GM-CSF also regulates various cellular processes through the activation of intracellular signalling mechanisms such as MAPK, PI3K/Akt and JAK/STAT signalling pathways (Schallenberg et al., 2009; Legacy et al., 2013; Theoret et al., 2016). The binding of GM-CSF to the alpha subunit of the GM-CSF receptor leads to the phosphorylation of the beta subunit of the receptor by JAK2, a tyrosine kinase. GM-CSF has been shown to act as a neuronal growth factor and as a neuroprotective agent both in vitro and in vivo (Schabitz et al., 2008; Legacy et al., 2013; Hanea et al., 2016; Theoret et al., 2016). The intracellular MAPK-ERK1/2 pathway activation has been implicated in GM-CSF's role in protecting RGCs from cell death both in vitro and in vivo (Schallenberg et al., 2009). In addition, GM-CSF has been shown to stimulate neurite growth in retinal explants and cultured RGCs (Legacy et al., 2013; Hanea et al., 2016). Overall, these studies suggest that GM-CSF might be a novel therapeutic agent for neural repair following traumatic injury to the CNS. The binding of GM-CSF to the alpha subunit of the GM-CSF receptor leads to the phosphorylation of the beta subunit of the receptor by JAK2, a tyrosine kinase. Interestingly, the expression of GM-CSF alpha-receptor is up-regulated following ischemic brain injury (Schabitz et al., 2008; Theoret et al., 2016). Furthermore, intravenous injection of GM-CSF in the MCAO rat-model resulted in significant reduction in infarct volume (Schabitz et al., 2008). Further research is needed to better understand the role of GM-CSF on axon regeneration and the intracellular signalling mechanisms that induce its proposed neuroprotective effects. Translation of the therapeutic potential of GM-CSF from the laboratory to the clinic is slowly progressing; for instance, Jim et al. (2012) reported that GM-CSF improves cognitive function in cancer patients. Although the mechanism by which GM-CSF reduces cognitive decline is not well understood; it is hypothesized that GM-CSF treatment may result in increased angiogenesis, neurite outgrowth or neuronal survival in brain regions known to be involved in cognitive function. Stroke in humans is often associated with cognitive dysfunction; this is particularly important because depending on location of a stroke, patients may experience significant cognitive decline. Further research is necessary to fully elucidate the mechanisms involved and the role of GM-CSF in post-stroke humans. These key findings regarding the neuroprotective role of GM-CSF and its ability to enhance neurite outgrowth suggest that GM-CSF might be an ideal therapeutic agent following CNS injury. However, there seems to be many downstream intracellular effectors of GM-CSF in the CNS. There is a critical need to define the intracellular mechanisms that contribute to the effects of GM-CSF following CNS injury and to clarify whether there is cross-talk between these mechanisms in order to fully understand the role of GM-CSF in the CNS. Overall, these studies indicate that GM-CSF may be a novel therapeutic target to promote regeneration in the injured CNS and further research is necessary to clarify the impact of this compound on both axon regeneration and plasticity in the CNS.

Growing evidence implicates microglia in the loss of dopaminergic neurons in Parkinson's disease (PD). However, factors mediating microglial activation in PD are poorly understood. Proinflammatory cytokines, such as interferon-γ (IFN-γ), orchestrate the actions of microglia. We report here that PD patients express significantly elevated levels of IFN-γ in their blood plasma. After this initial finding, we found thatIFN-γ-deficient mice displayed attenuated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced substantia nigra pars compacta dopaminergic cell loss along with reduced loss of striatal tyrosine hydroxylase and dopamine transporter fiber density. MPTP-induced depletion of striatal dopamine and its metabolite DOPAC (3,4-dihydroxyphenylacetic acid), as well as ΔFosB, a marker of postsynaptic dysfunction, were also attenuated in these knock-out mice. Consistent with the role for IFN-γ in microglial activation, MPTP-induced morphological activation of microglia was abrogated compared with wild-type mice. To examine more mechanistically the role of IFN-γ in microglial activation, we evaluated the interactions between microglia and dopaminergic neurons in anin vitromixed microglia/midbrain neuron rotenone-induced death paradigm. In thisin vitroparadigm, dopaminergic neurons are selectively damaged by rotenone. Exogenous IFN-γ ligand alone and without rotenone resulted in dopaminergic cell loss, but only in the presence of microglia. The addition of an IFN-γ neutralizing antibody attenuated neuronal loss as a result of rotenone treatment. The presence of only wild-type microglia and not those deficient in IFN-γ receptor elicited significant dopaminergic cell loss when exposed to rotenone. Neurons deficient in IFN-γ receptor, however, did not display increased resistance to death. Finally, levels of IFN-γ message increased in microglia in response to rotenone. Together, these data suggest that IFN-γ participates in death of dopaminergic neurons by regulating microglial activity.

Over the last decade, a large number of research articles have been published demonstrating regeneration and/or neuroprotection of retinal ganglion cells following manipulation of specific genetic and molecular targets. Interestingly, of the targets that have been identified to promote repair following visual system damage, many are genes known to be mutated in different types of cancer. This review explores recent literature on the potential for modulating cancer genes as a therapeutic strategy for visual system repair and looks at the potential clinical challenges associated with implementing this type of therapy. We also discuss signalling mechanisms that have been implicated in cancer and consider how similar mechanisms may improve axonal regeneration in the optic nerve.

Clinical stroke usually results from a cerebral ischaemic event, and is frequently a debilitating condition with limited treatment options. A significant proportion of clinical strokes result from specific damage to the subcortical white matter (SWM), but currently there are few animal models available to investigate the pathogenesis and potential therapeutic strategies to promote recovery. Granulocyte macrophage colony-stimulating factor (GM-CSF) is a cytokine that has been previously shown to promote neuroprotective effects after brain damage; however, the mechanisms mediating this effect are not known. Here, it is reported that GM-CSF treatment results in dramatic functional improvement in a white matter model of stroke in mice. SWM stroke was induced in mice by unilateral injections of the vasoconstrictor, endothelin-1 (ET-1). The results reveal that ET-1-induced stroke impairs skilled motor function on the single pellet-reaching task and results in forelimb asymmetry, in adult mice. Treatment with GM-CSF, after stroke, restores motor function and abolishes forelimb asymmetry. The results also indicate that GM-CSF promotes its effects by activating mammalian target of rapamycin signalling mechanisms in the brain following stroke injury. Additionally, a significant increase in GM-CSF receptor expression was found in the ipsilateral hemisphere of the ET-1-injected brain. Taken together, the present study highlights the use of an under-utilized mouse model of stroke (using ET-1) and suggests that GM-CSF treatment can attenuate ET-1-induced functional deficits.

The mechanisms underlying dopamine neuron loss in Parkinson's disease (PD) are not clearly defined. Here, we delineate a pathway by which dopaminergic loss induced by 1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine (MPTP) is controlledin vivo. We reported previously that calpains play a central required role in dopamine loss after MPTP treatment. Here, we provide evidence that the downstream effector pathway of calpains is through cyclin-dependent kinase 5 (cdk5)-mediated modulation of the transcription factor myocyte enhancer factor 2 (MEF2). We show that MPTP-induced conversion of the cdk5 activator p35 to a pathogenic p25 form is dependent on calpain activityin vivo. In addition, p35 deficiency attenuates MPTP-induced dopamine neuron loss and behavioral outcome. Moreover, MEF2 is phosphorylated on Ser444, an inactivating site, after MPTP treatment. This phosphorylation is dependent on both calpain and p35 activity, consistent with the model that calpain-mediated activation of cdk5 results in phosphorylation of MEF2in vivo. Finally, we provide evidence that MEF2 is critical for dopaminergic loss because “cdk5 phosphorylation site mutant” of MEF2D provides neuroprotection in an MPTP mouse model of PD. Together, these data indicate that calpain-p35-p25/cdk5-mediated inactivation of MEF2 plays a critical role in dopaminergic lossin vivo.

Cyclin-dependent kinases (CDKs) are best characterized for their ability to control cell-cycle progression. However, CDK family members are now known to regulate biological processes outside of the cell cycle, including transcription and neuronal development. Multiple CDKs (both cell cycle and non-cell-cycle related) have been associated with neurological conditions, such as Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). This review will explore the emerging evidence supporting a role for CDKs in neuronal death, focusing on mechanisms involved in the dopaminergic neurodegeneration of PD. An improved understanding of CDK involvement in cell death signaling could facilitate the development of innovative strategies to halt or slow down the characteristic death processes that are associated with some neurodegenerative conditions.

Accumulating evidence suggests that apoptotic and inflammatory factors contribute to the demise of dopaminergic neurons. In this respect, Fas, a member of the tumor necrosis factor receptor family with proapoptotic and inflammatory functions, was reported to be elevated within the striatum and substantia nigra pars compacta (SNc) of Parkinson's disease (PD) patients. Accordingly, the present investigation evaluated the function of Fas in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD. Injection of MPTP increased nigral Fas expression, and mice lacking Fas displayed attenuated MPTP-induced SNc dopaminergic loss and microglial activation. In addition, Fas induction was blocked by expression of a dominant-negative c-Jun adenovirus that also protected dopamine neurons from MPTP-induced damage. Together, these data suggest the critical nature of the c-Jun-Fas signaling pathway in MPTP-induced neuronal loss. Although critical for degeneration of the soma, Fas deficiency did not significantly prevent the reduction of dopaminergic terminal fibers within the striatum or normalize the activation of striatal microglia and elevation of the postsynaptic activity marker ΔFosB induced by denervation. Interestingly, Fas-deficient mice displayed a pre-existing reduction in striatal dopamine levels and locomotor behavior when compared with wild-type mice. Despite the reduced terminals, dopamine levels were not further suppressed by MPTP treatment in mutant mice, raising the possibility of a compensatory response in basal ganglia function in Fas-deficient mice.

Recent evidence indicates that cyclin-dependent kinases (CDKs, cdks) may be inappropriately activated in several neurodegenerative conditions. Here, we report that cdk5 expression and activity are elevated after administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a toxin that damages the nigrostriatal dopaminergic pathway. Supporting the pathogenic significance of the cdk5 alterations are the findings that the general cdk inhibitor, flavopiridol, or expression of dominant-negative cdk5, and to a lesser extent dominant-negative cdk2, attenuates the loss of dopaminergic neurons caused by MPTP. In addition, CDK inhibition strategies attenuate MPTP-induced hypolocomotion and markers of striatal function independent of striatal dopamine. We propose that cdk5 is a key regulator in the degeneration of dopaminergic neurons in Parkinson's disease.

The molecular mechanisms mediating degeneration of midbrain dopamine neurons in Parkinson's disease (PD) are poorly understood. Here, we provide evidence to support a role for the involvement of the calcium-dependent proteases, calpains, in the loss of dopamine neurons in a mouse model of PD. We show that administration ofN-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) evokes an increase in calpain-mediated proteolysis in nigral dopamine neuronsin vivo. Inhibition of calpain proteolysis using either a calpain inhibitor (MDL-28170) or adenovirus-mediated overexpression of the endogenous calpain inhibitor protein, calpastatin, significantly attenuated MPTP-induced loss of nigral dopamine neurons. Commensurate with this neuroprotection, MPTP-induced locomotor deficits were abolished, and markers of striatal postsynaptic activity were normalized in calpain inhibitor-treated mice. However, behavioral improvements in MPTP-treated, calpain inhibited mice did not correlate with restored levels of striatal dopamine. These results suggest that protection against nigral neuron degeneration in PD may be sufficient to facilitate normalized locomotor activity without necessitating striatal reinnervation. Immunohistochemical analyses of postmortem midbrain tissues from human PD cases also displayed evidence of increased calpain-related proteolytic activity that was not evident in age-matched control subjects. Taken together, our findings provide a potentially novel correlation between calpain proteolytic activity in an MPTP model of PD and the etiology of neuronal loss in PD in humans.

Expression of the transcription factor c-Jun is induced in neurons of the central nervous system (CNS) in response to injury. Mechanical transection of the nigrostriatal pathway at the medial forebrain bundle (MFB) results in the delayed retrograde degeneration of the dopamine neurons in the substantia nigra pars compacta (SNc) and induces protracted expression and phosphorylation of c-Jun. However, the role of c-Jun after axotomy of CNS neurons is unclear. Here, we show that adenovirus-mediated expression of a dominant negative form of c-Jun (Ad.c-JunDN) inhibited axotomy-induced dopamine neuron death and attenuated phosphorylation of c-Jun in nigral neurons. Ad.c-JunDN also delayed the degeneration of dopaminergic nigral axons in the striatum after MFB axotomy. Taken together, these findings suggest that activation of c-Jun mediates the loss of dopamine neurons after axotomy injury.

While the requirement of CDKs in cell cycle control is well established, the participation of CDK family members in other important biological processes are now beginning to be uncovered. Paramount in these newly defined roles is the surprising involvement of CDKs in neuronal development and death. These discoveries are somewhat of a paradox considering the terminally differentiated state of neurons. This brief perspective will focus on the role of CDKs in neuronal death and neurodegeneration. In this regard, we will primarily explore two (of potentially many) ways by which CDKs may enable neuronal death. The first involves the effects of ectopic activation of cell cycle related CDKs in a terminal post mitotic environment. The second explores how cdk5, a neuron specific cdk required for normal neuronal function, can be usurped to signal death.

Lack of regeneration in the adult central nervous system (CNS) is a major hurdle that limits recovery from neurological ailments. Although accumulating research suggests the possibility of axon regeneration by targeting intrinsic signaling mechanisms, it remains a matter of controversy whether functional recovery can be achieved by manipulating aspects of molecular signaling. Recent studies have shown that granulocyte macrophage colony-stimulating factor (GM-CSF) may be an effective means of targeting repair following CNS injury; how this molecule is able to produce this effect is not known. Indeed, GM-CSF has been shown to promote neuronal survival, potentially through activation of as yet unknown cytokine-dependent signals and potentially through regulation of antiapoptotic mechanisms. It is well established that the loss of intrinsic regenerative ability is highly correlated with development of CNS neurons. We therefore designed experiments, using a well-established in vitro retinal ganglion cell (RGC) culture system, to evaluate the effect of GM-CSF on axon growth and cell survival and define possible mechanisms involved in GM-CSF-mediated effects in vitro. Several developmental stages were evaluated, with particular focus placed on stages at which axon growth is known to be significantly diminished. Our results reveal that GM-CSF not only promotes axon growth in postnatal RGCs but also enhances cell survival through a mammalian target of rapamycin (mTOR)-dependent mechanism.

The failure of axons to regenerate is a major obstacle for functional recovery after central nervous system (CNS) injury. Removing extracellular inhibitory molecules results in limited axon regeneration in vivo. To test for the role of intrinsic impediments to axon regrowth, we analyzed cell growth control genes using a virus-assisted in vivo conditional knockout approach. Deletion of PTEN (phosphatase and tensin homolog), a negative regulator of the mammalian target of rapamycin (mTOR) pathway, in adult retinal ganglion cells (RGCs) promotes robust axon regeneration after optic nerve injury. In wild-type adult mice, the mTOR activity was suppressed and new protein synthesis was impaired in axotomized RGCs, which may contribute to the regeneration failure. Reactivating this pathway by conditional knockout of tuberous sclerosis complex 1, another negative regulator of the mTOR pathway, also leads to axon regeneration. Thus, our results suggest the manipulation of intrinsic growth control pathways as a therapeutic approach to promote axon regeneration after CNS injury.

Mechanical transection of the nigrostriatal dopamine pathway at the medial forebrain bundle (MFB) results in the delayed degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). We have previously demonstrated that c-Jun activation is an obligate component of neuronal death in this model. Here we identified the small GTPase, cdc42, and mixed lineage kinases (MLKs) as upstream factors regulating neuronal loss and activation of c-Jun following MFB axotomy. Adenovirus-mediated expression of a dominant-negative form of cdc42 in nigral neurons blocked MFB axotomy-induced activation (phosphorylation) of MAP kinase kinase 4 (MKK4) and c-Jun, resulting in attenuation of SNpc neuronal death. Pharmacological inhibition of MLKs, MKK4-activating kinases, significantly reduced the phosphorylation of c-Jun and abrogated dopaminergic neuronal degeneration following MFB axotomy. Taken together, these findings suggest that death of nigral dopaminergic neurons following axotomy can be attenuated by targeting cell signaling events upstream of c-Jun N-terminal mitogen-activated protein kinase/c-Jun.

Mutations of the DJ-1 (PARK7) gene are linked to familial Parkinson's disease. We used gene targeting to generate DJ-1-deficient mice that were viable, fertile, and showed no gross anatomical or neuronal abnormalities. Dopaminergic neuron numbers in the substantia nigra and fiber densities and dopamine levels in the striatum were normal. However, DJ-1–/– mice showed hypolocomotion when subjected to amphetamine challenge and increased striatal denervation and dopaminergic neuron loss induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine. DJ-1–/–embryonic cortical neurons showed increased sensitivity to oxidative, but not nonoxidative, insults. Restoration of DJ-1 expression to DJ-1–/– mice or cells via adenoviral vector delivery mitigated all phenotypes. WT mice that received adenoviral delivery of DJ-1 resisted 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine-induced striatal damage, and neurons overexpressing DJ-1 were protected from oxidative stress in vitro . Thus, DJ-1 protects against neuronal oxidative stress, and loss of DJ-1 may lead to Parkinson's disease by conferring hypersensitivity to dopaminergic insults.

Loss-of-function mutations in the parkin gene, which encodes an E3 ubiquitin ligase, are the major cause of early-onset Parkinson's disease (PD). Decreases in parkin activity may also contribute to neurodegeneration in sporadic forms of PD. Here, we show that bcl-2-associated athanogene 5 (BAG5), a BAG family member, directly interacts with parkin and the chaperone Hsp70. Within this complex, BAG5 inhibits both parkin E3 ubiquitin ligase activity and Hsp70-mediated refolding of misfolded proteins. BAG5 enhances parkin sequestration within protein aggregates and mitigates parkin-dependent preservation of proteasome function. Finally, BAG5 enhances dopamine neuron death in an in vivo model of PD, whereas a mutant that inhibits BAG5 activity attenuates dopaminergic neurodegeneration. This contrasts with the antideath functions ascribed to BAG family members and suggests a potential role for BAG5 in promoting neurodegeneration in sporadic PD through its functional interactions with parkin and Hsp70.

X-linked IAP protein is a potent inhibitor of cell death. Here, we describe a novel transgenic mouse in which the human XIAP gene is expressed under the control of the neuron-specific enolase promoter (NSE-xiap). We demonstrate that nigrostriatal dopamine neurons of NSE-xiap mice were resistant to the damaging effects of the dopaminergic neurotoxin MPTP. MPTP-induced reduction of striatal dopamine metabolism was also attenuated in NSE-xiap mice. Furthermore, NSE-xiap mice treated with MPTP did not exhibit deficits in exploratory behaviour in an open-field test. Taken together, these findings suggest that strategies to enhance neuronal expression of XIAP may provide therapeutic benefit for the treatment of neurodegeneration in Parkinson’s disease.

SummaryAxon regeneration failure accounts for permanent functional deficits following CNS injury in adult mammals. However, the underlying mechanisms remain elusive. In analyzing axon regeneration in different mutant mouse lines, we discovered that deletion of suppressor of cytokine signaling 3 (SOCS3) in adult retinal ganglion cells (RGCs) promotes robust regeneration of injured optic nerve axons. This regeneration-promoting effect is efficiently blocked in SOCS3-gp130 double-knockout mice, suggesting that SOCS3 deletion promotes axon regeneration via a gp130-dependent pathway. Consistently, a transient upregulation of ciliary neurotrophic factor (CNTF) was observed within the retina following optic nerve injury. Intravitreal application of CNTF further enhances axon regeneration from SOCS3-deleted RGCs. Together, our results suggest that compromised responsiveness to injury-induced growth factors in mature neurons contributes significantly to regeneration failure. Thus, developing strategies to modulate negative signaling regulators may be an efficient strategy of promoting axon regeneration after CNS injury.