Survival Factor For Motor Neurons Research Articles

GDNF to the rescue: GDNF delivery effects on motor neurons and nerves, and muscle re-innervation after peripheral nerve injuries.

Peripheral nerve injuries commonly occur due to trauma, like a traffic accident. Peripheral nerves get severed, causing motor neuron death and potential muscle atrophy. The current golden standard to treat peripheral nerve lesions, especially lesions with large (≥ 3 cm) nerve gaps, is the use of a nerve autograft or reimplantation in cases where nerve root avulsions occur. If not tended early, degeneration of motor neurons and loss of axon regeneration can occur, leading to loss of function. Although surgical procedures exist, patients often do not fully recover, and quality of life deteriorates. Peripheral nerves have limited regeneration, and it is usually mediated by Schwann cells and neurotrophic factors, like glial cell line-derived neurotrophic factor, as seen in Wallerian degeneration. Glial cell line-derived neurotrophic factor is a neurotrophic factor known to promote motor neuron survival and neurite outgrowth. Glial cell line-derived neurotrophic factor is upregulated in different forms of nerve injuries like axotomy, sciatic nerve crush, and compression, thus creating great interest to explore this protein as a potential treatment for peripheral nerve injuries. Exogenous glial cell line-derived neurotrophic factor has shown positive effects in regeneration and functional recovery when applied in experimental models of peripheral nerve injuries. In this review, we discuss the mechanism of repair provided by Schwann cells and upregulation of glial cell line-derived neurotrophic factor, the latest findings on the effects of glial cell line-derived neurotrophic factor in different types of peripheral nerve injuries, delivery systems, and complementary treatments (electrical muscle stimulation and exercise). Understanding and overcoming the challenges of proper timing and glial cell line-derived neurotrophic factor delivery is paramount to creating novel treatments to tend to peripheral nerve injuries to improve patients’ quality of life.

Exercise-dependent regulation of glial cell line-derived neurotrophic factor (GDNF) expression in skeletal muscle and its importance for the neuromuscular system

The focus of this review is to highlight the importance of glial cell line-derived neurotrophic factor (GDNF) for the motor nervous system. GDNF is the most potent survival factor for motor neurons, where it enhances maintenance and survival of both developing and mature motor neurons in vivo and in vitro. GDNF aids in neuromuscular junction formation, maintenance, and plasticity, where skeletal muscle-derived GDNF may be responsible for this phenomenon. Increased levels of physical activity can increase GDNF protein levels in skeletal muscle, where alterations in acetylcholine and acetylcholine receptor activation may be involved in regulation of these changes observed. With inactivity and disuse, GDNF expression shows different patterns of regulation in the central and peripheral nervous systems. Due to its potent effects for motor neurons, GDNF is being extensively studied in neuromuscular diseases.

Ghrelin Protects Spinal Cord Motoneurons Against Chronic Glutamate Excitotoxicity by Inhibiting Microglial Activation

Glutamate excitotoxicity is emerging as a contributor to degeneration of spinal cord motoneurons in amyotrophic lateral sclerosis (ALS). Recently, we have reported that ghrelin protects motoneurons against chronic glutamate excitotoxicity through the activation of extracellular signal-regulated kinase 1/2 and phosphatidylinositol-3-kinase/Akt/glycogen synthase kinase-3β pathways. Previous studies suggest that activated microglia actively participate in the pathogenesis of ALS motoneuron degeneration. However, it is still unknown whether ghrelin exerts its protective effect on motoneurons via inhibition of microglial activation. In this study, we investigate organotypic spinal cord cultures (OSCCs) exposed to threohydroxyaspartate (THA), as a model of excitotoxic motoneuron degeneration, to determine if ghrelin prevents microglial activation. Exposure of OSCCs to THA for 3 weeks produced typical motoneuron death, and treatment of ghrelin significantly attenuated THA-induced motoneuron loss, as previously reported. Ghrelin prevented THA-induced microglial activation in the spinal cord and the expression of pro-inflammatory cytokines tumor necrosis factor-α and interleukin-1β. Our data indicate that ghrelin may act as a survival factor for motoneurons by functioning as a microglia-deactivating factor and suggest that ghrelin may have therapeutic potential for the treatment of ALS and other neurodegenerative disorders where inflammatory responses play a critical role.

Cholinergic neurons regulate secretion of glial cell line-derived neurotrophic factor by skeletal muscle cells in culture

Glial cell line-derived neurotrophic factor (GDNF) has been identified as a potent survival factor for both central and peripheral neurons. GDNF has been shown to be a potent survival factor for motor neurons during programmed cell death and continuous treatment with GDNF maintains hyperinnervation of skeletal muscle in adulthood. However, little is known about factors regulating normal production of endogenous GDNF in skeletal muscle. This study aimed to examine the role that motor neurons play in regulating GDNF secretion by skeletal muscle. A co-culture of skeletal muscle cells (C2C12) and cholinergic neurons, glioma × neuroblastoma hybrid cells (NG108-15) were used to create nerve-muscle interactions in vitro. Acetylcholine receptors (AChRs) on nerve–myotube co-cultures were blocked with alpha-bungarotoxin (α-BTX). GDNF protein content in cells and in culture medium was analyzed by enzyme-linked immunosorbant assay (ELISA) and western blotting. GDNF localization was examined by immunocytochemistry. The nerve–muscle co-culture study indicated that the addition of motor neurons to skeletal muscle cells reduced the secretion of GDNF by skeletal muscle. The results also showed that blocking AChRs with α-BTX reversed the action of neural cells on GDNF secretion by skeletal muscle. Although ELISA results showed no GDNF in differentiated NG108-15 cells grown alone, immunocytochemical analysis showed that GDNF was localized in NG108-15 cells co-cultured with C2C12 myotubes. These results suggest that motor neurons may be regulating their own supply of GDNF secreted by skeletal muscle and that activation of AChRs may be involved in this process.

Immunohistochemical expression of IGF‐I and GSK in the spinal cord of Kii and Guamanian ALS patients

Insulin-like growth factor-I (IGF-I) is a potent survival factor for motor neurons in animals, and glycogen synthase kinase-3beta (GSK-3beta) is suspected to play roles in apoptosis and tau phosphorylation. Here we report the immunological expression of IGF-I, GSK-3beta, phosphorylated-GSK-3alpha/beta (p-GSK-3alpha/beta) and phosphorylated-tau in the spinal cord and hippocampus of Kii and Guam amyotrophic lateral sclerosis (ALS) patients. Sixteen ALS patients (10 Japanese sporadic, 3 Kii and 3 Guam ALS) and 14 neurological controls (10 Japanese and 4 Guamanian) were examined. The immunoreactivity for each antibody was rated by the percentages of positive neurons to total anterior horn neurons in each patient and was analyzed statistically. Many normal-looking neurons from Japanese sporadic ALS, Kii ALS and Guam ALS patients, as well as from Japanese and Guam controls, were positive for anti-IGF-I antibody. A positive correlation between IR scores for anti-IGF-I antibody and clinical durations of Japanese sporadic ALS patients was found in this study (P < 0.0001). This suggested that IGF-I might have a protective effect against ALS degeneration. In Japanese sporadic ALS patients, abnormal as well as normal-looking neurons showed significant high IR scores for anti-GSK-3beta antibody than those of controls. Anterior horn neurons from Guam and Kii ALS patients characteristically showed weak staining for anti-GSK-3beta antibody but were markedly positive for anti-pGSK-3alpha/beta antibody compared to those from both Japanese controls and Japanese sporadic ALS patients, and showed the co-localization of IGF-I and p-GSK-3alpha/beta. This suggested that the IGF-I signaling pathway in Guam and Kii ALS patients might function to phosphorylate GSK-3beta to protect neurons from ALS degeneration. Neurofibrillary tangles (NFTs) in the hippocampus and spinal cord from Kii and Guam ALS patients showed the co-localization of PHF-tau and p-GSK-3alpha/beta by a confocal laser scanning technique. The predominant expression of p-GSK-3alpha/beta compared to GSK-3beta in spinal motor neurons and the co-localization of p-GSK-3alpha/beta and PHF-tau in NFT-laden neurons in the hippocampus and spinal cord were characteristic findings of Kii and Guam ALS patients.

Intraspinal cord delivery of IGF-I mediated by adeno-associated virus 2 is neuroprotective in a rat model of familial ALS

Background Amyotrophic lateral sclerosis (ALS) is a devastating disease that is characterized by the progressive loss of motor neurons. Patients with ALS usually die from respiratory failure due to respiratory muscle paralysis. Consequently, therapies aimed at preserving segmental function of the respiratory motor neurons could extend life for these patients. Insulin-like growth factor-I (IGF-I) is known to be a potent survival factor for motor neurons. In this study we induced high levels of IGF-I expression in the cervical spinal cord of hSOD1 G93A rats with intraspinal cord (ISC) injections of an adeno-associated virus serotype 2 vector (CERE-130). This approach reduced the extent of motor neuron loss in the treated segments of the spinal cord. However, a corresponding preservation of motor function was observed in male, but not female, hSOD1 G93A rats. We conclude that ISC injection of CERE-130 has the potential to protect motor neurons and preserve neuromuscular function in ALS.

Changes in GDNF protein content of skeletal muscle following involuntary exercise are long lived

Glial‐cell line derived neurotrophic factor (GDNF) is a neurotrophic factor produced by skeletal muscle. GDNF has been shown to be potent survival factor for motor neurons. We have previously shown that GDNF content in skeletal muscle is altered following exercise and GDNF levels 24 hours after the last bout of exercise are significantly different than those measured immediately after exercise. We sought to determine how long after exercise do GDNF protein levels remain altered. We removed extensor digitorum longus (EDL) and Soleus (SOL), 24 hr, 72 hr and 1 week following the last bout of involuntary exercise. Muscles were processed for GDNF protein content using enzyme linked immunosorbant assay. Contralateral EDL and SOL were removed at the same time points for immunohistochemical analysis. The results showed that 72 hr following exercise there was a significant increase of GDNF protein content in SOL and a significant decrease in EDL. The results suggest exercise causes a relatively long‐lived effect on GDNF protein content and that expression of GDNF protein in type I skeletal muscle maybe regulated differently that that in type II skeletal muscle.This work was supported by NIH Grant 1R15AG022908‐01A2, MSU‐KCMS, and Western Michigan University.

Exercised induced changes of GDNF in rat skeletal muscle at various times after exercise

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for motor neurons. Over expression of GDNF in skeletal muscle has been shown to cause a hyperinnervation of muscle fibers. We have previously shown that exercise increases GDNF expression in skeletal muscle. In our previous studies GDNF content was measured 24 hours after the last bout of exercise. For the current study we wanted to determine if GDNF content of muscle differed in muscles removed immediately following exercise (0h) compared to those taken 24 hours (24h) later. Following 2 weeks of exercise soleus (SOL) and extensor digitorium longus (EDL) were removed at 0h or 24h and analyzed for GDNF content by enzyme-linked immunosorbant assay. The results showed a significant difference (p≤0.05) in GDNF levels between SOL 24h and control but not in any other time point or tissue. These results may indicate that production of GDNF in skeletal muscle could be regulated in an activity dependent manner and that, GDNF levels are increasing subsequent to the last bout of exercise for a duration of at least 24 hr. The deferential expression of GDNF in skeletal muscle with exercise could have implications in the aging and diseased NMJ's of humans. This work was supported by NIH Grant 1R15AG022908-01A2, MSU-KCMS, and Western Michigan University.

Muscle-derived but not centrally derived transgene GDNF is neuroprotective in G93A-SOD1 mouse model of ALS

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for motoneurons (MNs), and is considered a potential agent for the treatment of amyotrophic lateral sclerosis (ALS) and other MN diseases. The effectiveness of GDNF may depend significantly upon its route of delivery to MNs. In this study we tested the neuroprotective effects of target-derived and centrally derived GDNF in the G93A-SOD1 mouse model of ALS using a transgenic approach. We found that overexpression of GDNF in the skeletal muscle ( Myo-GDNF mice) significantly delayed the onset of disease and increased the life span of G93A-SOD1 mice by 17 days. The duration of disease also increased by 8.5 days, indicating that GDNF slowed down the progression of disease. Locomotor performance in Myo-GDNF/ G93A-SOD1 mice was also significantly improved. The behavioral improvement correlated well with anatomical and histological data. We demonstrated that muscle-derived GDNF resulted in increased survival of spinal MNs, and twice as many MNs survived in end-stage double transgenic mice compared to end-stage G93A-SOD1 mice. Muscle-derived GDNF also had profound effects on muscle innervation and axonal degeneration. Significantly higher numbers of completely or partially innervated NMJs and large caliber myelinated axons were found in double transgenic mice. In contrast, we demonstrated that overexpression of GDNF in astrocytes in the CNS ( GFAP-GDNF mice) failed to demonstrate any neuroprotective effects in G93A-SOD1 mice both on behavioral and histological levels. These data indicate that retrograde transport and signaling of GDNF is more physiological and effective for ALS treatment than anterogradely transported GDNF.

Astrocyte-derived transgene GDNF promotes complete and long-term survival of adult facial motoneurons following avulsion and differentially regulates the expression of transcription factors of AP-1 and ATF/CREB families

Glial-cell-line-derived neurotrophic factor (GDNF) is a potent survival factor for motoneurons (MNs). We have previously demonstrated that overexpression of GDNF in astrocytes of GFAP-GDNF mice promotes long-term survival of neonatal MNs after facial nerve axotomy. In the present study, we investigated whether astrocyte-derived GDNF could also have a neuroprotective effect on adult MNs following facial nerve avulsion. We also examined avulsion- and GDNF-induced changes in the expression pattern of several members of the AP-1 and ATF/CREB families of transcription factors, which are involved in the fate determination of neurons following injury. We demonstrated that GDNF promotes complete rescue of avulsed MNs for at least 4 months post-injury. Transgene GDNF significantly upregulates c-Jun expression in naive MNs, further upregulates injury-induced c-Jun expression in facial MNs, and results in its activation in most surviving MNs. No significant changes were found in c-Fos expression. We found that GDNF has an opposing effect on ATF2 and ATF3 expression. It dramatically downregulates increased levels of ATF3 in response to injury, whereas the expression of ATF2, which is normally reduced after injury, is completely preserved in GFAP-GDNF mice. Our data suggest that maintenance of high levels of ATF2 in injured MNs could be crucial in modulating c-Jun function, and c-Jun/ATF2 signaling could be involved in GDNF-mediated survival of mature MNs.

Neural adhesion molecules L1 and CHL1 are survival factors for motoneurons

Many neurotrophic factors with survival activity for motoneurons in vivo were first identified using cultures of purified embryonic motoneurons. The L1 neural cell adhesion molecule has multiple roles in brain development. We showed by in situ hybridization and RT-PCR that L1 mRNA was expressed at significant levels in motoneurons of embryonic and postnatal spinal cord. We therefore cultured purified motoneurons from E14 rat embryos in the absence of trophic factors but with L1-Fc and CHL1-Fc fusion proteins. L1-Fc prevented the death of approximately half of the motoneurons that were saved by BDNF in a dose-dependent manner (EC50 = 10 pM). CHL1-Fc saved the same number of motoneurons as did L1-Fc, whereas P0-Fc had little neurotrophic activity at the same concentrations. Survival induced by L1 and CHL1 was completely inhibited by 20 microM LY294002 and PD98059, indicating that both MEK and PI3K pathways are required for signaling by these molecules. L1 can signal in other cell types through the FGF receptor FGFR1. In cultures of motoneurons, effects of suboptimal concentrations of L1 and suboptimal concentrations of FGF-2 were additive, but the effects of optimal concentrations of FGF-2 (50 ng/ml) were not further increased in the presence of L1-Fc. Thus, in this system, too, FGF and L1 may use similar signaling pathways.

Belgian scientists awarded top honors

The Health Prize of the Interbrew-Baillet Latour Fund, worth nearly $200,000, is the most prestigious international scientific prize awarded in Belgium. On May 12, 2005, Desire Collen of the Flanders Interuniversity Institute for Biotechnology and Peter Carmeliet of the University of Leuven will be honored with this award for their pioneering work in genetics and cardiovascular disease. The JCI spoke with the scientists about the prize and the work leading up to it. JCI: How were you selected for this prize? Carmeliet: An international jury of renowned scientists selected the laureates among 21 candidates from 13 different countries. JCI: What aspect of your work directly contributed to your winning this prize? Collen: I started my scientific career studying the molecular mechanisms of blood clot dissolution. These studies ultimately resulted in the isolation and characterization of tissue-type plasminogen activator (tPA), which digests blood clots. This led to the development, with Genentech Inc., of recombinant tPA for the treatment of acute myocardial infarction. Carmeliet: In the early 90s, I teamed up with Desire Collen to study cardiovascular diseases using gene targeting and focused on the molecular basis of angiogenesis in health and disease. These studies elucidated the role of angiogenic factors, in particular of vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), and tested novel strategies to treat cancer, inflammation, and ischemic tissue disease. Recently, our team provided evidence for the therapeutic potential of VEGF for the neurodegenerative disorder amyotrophic lateral sclerosis (ALS). JCI: What are the most direct clinical consequences of your work? Collen: The discovery of tPA and its development as a novel thrombolytic drug has saved the lives of a few percent of the more than 5 million treated patients with myocardial ischemia worldwide. Carmeliet: Mice deficient in a single VEGF allele revealed the key role of VEGF in angiogenesis. Further studies demonstrated that PlGF, a homolog of VEGF, affects angiogenesis in disease but not in health. The implication of these findings is that blocking PlGF might combine efficacy in inhibiting tumor growth with safety of only affecting tumor but not quiescent vessels. Conversely, delivery of PlGF stimulated revascularization of ischemic tissues. Our studies also showed that low levels of VEGF cause motor neuron degeneration, reminiscent of ALS. We have now demonstrated that VEGF prolongs survival in ALS rodent models. Clinical trials are underway. JCI: What is the biggest challenge you face? Collen: To secure a steady flow of funding and talented researchers to compete at the front line of basic research and to efficiently transfer technology for potential medical applications to interested industrial partners. Carmeliet: Understanding the molecular basis of angio-genesis and lymphangiogenesis, using mouse, zebrafish, and frog genetic models. Our focus is evolving from identifying which signals stimulate endothelial cells to migrate and divide, to understanding how blood vessels, lymph vessels, and neurons navigate to their targets. Our discovery that VEGF has neurotrophic effects in animal models of ALS may indicate a new approach to treatment of this and other life-threatening neurodegenerative disorders. A challenge will be to initiate clinical trials, evaluating the therapeutic potential of VEGF for ALS. JCI: In a typical day, how much of your time do you spend in the lab involved in research? Collen: I am primarily involved in the overall coordination of the research programs of approximately 160 investigators and technical staff. Much of my time is devoted to drug development programs spun out of the laboratory into dedicated spin-offs. I primarily focus on drug development and technology transfer issues. Carmeliet: Apart from some teaching and administrative duties, I, together with 4 other senior associates, spend my entire time supervising and coaching a research team of 55 postdocs, PhD students, and technicians. I am primarily involved in the conception of projects, writing grants and papers, and managing and coordinating the mouse, zebra-fish, and frog facilities. JCI: What do you think is the most important discovery you have ever made? Collen: The molecular regulation of physiological fibrinolysis (breaking down blood clots) and its application to fibrin-relative thrombolytic therapy of acute myocardial infarction. Carmeliet: Three discoveries seem most relevant: the critical role of VEGF in vascular development and disorders; the finding that PlGF is involved in angiogenesis in disease but not in health, and that PlGF stimulates revascularization of ischemic tissues, while PlGF blockers inhibit pathological angiogenesis; the finding that VEGF is a key survival factor for motor neurons in ALS and that VEGF prolongs survival of ALS rodents. JCI: What is your ultimate scientific goal? Collen and Carmeliet: Understanding the molecular mechanisms governing cardiovascular and nervous system function, and the development of mechanism-based novel treatments for life-threatening cardiovascular and neurological diseases.

Glial cell-line derived neurotrophic factor-dependent fusimotor neuron survival during development

Glial cell-line derived neurotrophic factor (GDNF) is a potent survival factor for motor neurons. Previous studies have shown that some motor neurons depend upon GDNF during development but this GDNF-dependent motor neuron subpopulation has not been characterized. We examined GDNF expression patterns in muscle and the impact of altered GDNF expression on the development of subtypes of motor neurons. In GDNF hemizygous mice, motor neuron innervation to muscle spindle stretch receptors (fusimotor neuron innervation) was decreased, whereas in transgenic mice that overexpress GDNF in muscle, fusimotor innervation to muscle spindles was increased. Facial motor neurons, which do not contain fusimotor neurons, were not changed in number when GDNF was over expressed by facial muscles during their development. Taken together, these data indicate that fusimotor neurons depend upon GDNF for survival during development. Since the fraction of cervical and lumbar motor neurons lost in GDNF-deficient mice at birth closely approximates the size of the fusimotor neuron pool, these data suggest that motor neuron loss in GDNF-deficient mice may be primarily of fusimotor neuron origin.

GDNF increases the survival of developing oculomotor neurons through a target-derived mechanism

Glial cell line-derived neurotrophic factor (GDNF) is the most potent motoneuron survival factor. We show here that in the chick oculomotor system, endogenous GDNF is derived largely from extraocular muscle but less from glial cells and not from muscle spindles. Increased levels of GDNF exclusively in the target rescued 30% of oculomotor neurons that would normally die during developmental cell death, a rate of rescue similar to that with systemic GDNF application. Thus, GDNF supports motoneuron survival in a retrograde, target-derived fashion, as opposed to a local paracrine route or an indirect route via sensory afferents. Persephin, another member of the GDNF family, did not increase survival with target delivery, despite its retrograde transport from the target. Unlike GDNF, however, persephin increased neurite outgrowth from oculomotor nuclei in vitro. Thus, one GDNF family member acts as a muscle-derived retrograde survival factor, whereas another one has distinct functions on neurite outgrowth.

Free insulin-like growth factor (IGF)-I and IGF binding proteins 2, 5, and 6 in spinal motor neurons in amyotrophic lateral sclerosis

Insulin-like growth factor-I (IGF-I) is a potent survival factor for motor neurons and is being investigated as possible therapeutic agent for amyotrophic lateral sclerosis. However, very little information is available on the components of the IGF-I system in this disease. Insulin-like growth factor binding proteins (IGFBPs) play an important part in regulating the bioavailability of IGF-I. We investigated the components of the IGF-I system in spinal cord sections of ten patients with amyotrophic lateral sclerosis and in ten controls without neurological disease. IGF-I was studied by western immunoblotting. IGFBPs and IGF-I receptors were investigated by immunohistochemistry and Western immunoblotting. Total IGF-I in ventral horn homogenates did not differ between patients and controls. However, free IGF-I was 53% lower in patients than in controls. Compared with controls, immunoreactivity in the spinal motor neurons of patients with amyotrophic lateral sclerosis was 64% higher for IGFBP2, 46% higher for IGFBP5, and 33% higher for IGFBP6, with upregulation of IGF-I receptors. Immunoreactivity for IGFBPs1, 3, and 4 did not differ between patients and controls. In the ventral horns of patients, free IGF-I is reduced, which could be because of specific increases in IGFBPs 2, 5, and 6 in spinal motor neurons. This abnormality might have an important role in the processes leading to motor neuron death, and should be taken into account when developing treatments aimed to stimulate IGF-I receptors in motor neurons.

Leukemia inhibitory factor by systemic administration rescues spinal motor neurons in the SOD1 G93A murine model of familial amyotrophic lateral sclerosis

Leukemia inhibitory factor (LIF) is a survival factor for motoneurons. In this study we investigated whether intense systemic LIF therapy prevents the loss of lumbar motoneurons in the transgenic SOD1 G93A mouse model of familial amyotrophic lateral sclerosis. Treatment involved daily 25 μg/kg intraperitoneal injection for a period of 6 weeks starting at 70 days of age. Using the unbiased optical dissector technique, significant rescue of motoneurons in the LIF-treated group (3809±455) was found compared to the vehicle group (1085±140).

Blood and Nerve

Vascular endothelial growth factor (VEGF) plays a key role in controlling the growth and permeability of blood vessels. When oxygen levels fall (hypoxia), VEGF is rapidly up-regulated, thereby ensuring that tissues are perfused adequately, through a promoter sequence in the VEGF gene called the hypoxia response element (HRE). Oosthuyse et al. generated mice that were lacking the HRE and therefore produced inadequate amounts of VEGF under hypoxic conditions. Surprisingly, the mice developed an adult-onset neurodegenerative disease with many of the characteristics of amyotrophic lateral sclerosis (Lou Gehrig's disease). The motor neuron degeneration in the mice appeared to be caused in part by a reduction in neural vascular perfusion, but cell culture studies supported the tantalizing possibility that VEGF served as a survival factor for motor neurons.

Reg-2 is a motoneuron neurotrophic factor and a signalling intermediate in the CNTF survival pathway.

Cytokines that are related to ciliary neurotrophic factor (CNTF) are physiologically important survival factors for motoneurons, but the mechanisms by which they prevent neuronal cell death remain unknown. Reg-2/PAP I (pancreatitis-associated protein I), referred to here as Reg-2, is a secreted protein whose expression in motoneurons during development is dependent on cytokines. Here we show that CNTF-related cytokines induce Reg-2 expression in cultured motoneurons. Purified Reg-2 can itself act as an autocrine/paracrine neurotrophic factor for a subpopulation of motoneurons, by stimulating a survival pathway involving phosphatidylinositol-3-kinase, Akt kinase and NF-kappaB. Blocking Reg-2 expression in motoneurons using Reg-2 antisense adenovirus specifically abrogates the survival effect of CNTF on cultured motoneurons, indicating that Reg-2 expression is a necessary step in the CNTF survival pathway. Reg-2 shows a unique pattern of expression in late embryonic spinal cord: it is progressively upregulated in individual motoneurons on a cell-by-cell basis, indicating that only a fraction of motoneurons in a given motor pool may be exposed to cytokines. Thus, Reg-2 is a neurotrophic factor for motoneurons, and is itself an obligatory intermediate in the survival signalling pathway of CNTF-related cytokines.

Gene transfer for neuroprotection in animal models of Parkinson's disease and amyotrophic lateral sclerosis.

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for motoneurons (MN) and dopaminergic (DA) neurons, neurons which selectively die in amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). GDNF gene delivery has been studied in rodent models of ALS and PD. In a mouse model of ALS, implantation of myoblasts retrovirally transduced with GDNF into hindlimb muscles at 6 weeks of age, i.e. prior to the onset of disease symptoms, increased the number of large MNs that maintained projections to treated muscles at 18 weeks of age. GDNF-treated mice also performed better on tests of motor function and had a delayed onset of disease. In a progressive degeneration rat model of PD, effects of in vivo GDNF gene therapy using an adenoviral vector (AdGDNF) were studied in young and aged rats. AdGDNF protected DA neurons against the neurotoxin, 6-hydroxydopamine (6-OHDA), and was effective whether injected either before or after 6-OHDA damage had commenced. However, if AdGDNF was injected prior to 6-OHDA, it was most effective in protecting against DA-dependent changes in the brain when injected near the terminals of the DA neurons. In contrast, if 6-OHDA damage had already commenced, AdGDNF was most effective if injected near the DA soma. These studies suggest that GDNF gene delivery into specific sites in the CNS or into muscle where MNs have access to secreted GDNF may slow the progression of PD and ALS, respectively. Neurotrophic factor gene therapy offers novel interventions not only for PD and ALS, but also other neurodegenerative diseases and injuries to the nervous system.

FGF9: a motoneuron survival factor expressed by medial thoracic and sacral motoneurons.

In the nervous system, fibroblast growth factor-9 (FGF9) is produced mainly by neurons. By whole-mount in situ hybridization, on embryonic rat spinal cord, we observed Fgf9 expression in a subpopulation of motoneurons located in the thoracic and sacral regions of the median motor column that innervate the axial muscles. Furthermore, FGF9 prevented death of purified rat and chicken motoneurons in culture in the same concentration range as FGF2. The targets of FGF9 are more restricted than that of the other FGFs, however, because conversely to FGF1 or FGF2, FGF9 had only weak or inexistent survival effects on chicken ciliary neurons or rat DRG. FGF9 may therefore play a role as an autocrine/paracrine survival factor for motoneurons.