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1.  The lack of effect of specific overexpression of IGF-1 in the central nervous system or skeletal muscle on pathophysiology in the G93A SOD-1 mouse model of ALS 
Experimental neurology  2007;207(1):52-63.
The ability of insulin like growth factor 1 (IGF-1) to prevent the pathophysiology associated with amyotrophic lateral sclerosis (ALS) is currently being explored with animal models and in clinical trials with patients. Several studies have reported positive effects of IGF-1 in reducing motor neuron death, delaying the onset of motor performance decline, and increasing life span, in SOD-1 mouse models of ALS and in one clinical trial. However, a second clinical trial produced no positive results raising questions about the therapeutic efficacy of IGF-1. To investigate the effect of specific and sustained IGF-1 expression in skeletal muscle or central nervous system on motor performance, life span, and motor neuron survival, human-IGF-1-transgenic mice were crossed with the G93A SOD-1 mutant model of ALS. No significant differences were found in onset of motor performance decline, life span, or motor neuron survival in the spinal cord, between SOD+/IGF-1+ and SOD+/IGF-1- hybrid mice. IGF-1 concentration levels, measured by radioimmunoassay, were found to be highly increased throughout life in the central nervous system (CNS) and skeletal muscle of IGF-1 transgenic hybrid mice. Additionally, increased CNS weight in SOD+ mice crossbred with CNS IGF-1 transgenic mice demonstrates that IGF-1 overexpression is biologically active even after the disease is fully developed. Taken together, these results raise questions concerning the therapeutic value of IGF-1 and indicate that further studies are needed to examine the relationship between methods of IGF-1 administration and its potential therapeutic value.
PMCID: PMC2043146  PMID: 17597610
Amyotrophic Lateral Sclerosis; Insulin-like growth factor-1; motor neuron; cell death; skeletal muscle; transgenic; G93A SOD-1; motor performance
2.  Two Faces of Chondroitin Sulfate Proteoglycan in Spinal Cord Repair: A Role in Microglia/Macrophage Activation 
PLoS Medicine  2008;5(8):e171.
Chondroitin sulfate proteoglycan (CSPG) is a major component of the glial scar. It is considered to be a major obstacle for central nervous system (CNS) recovery after injury, especially in light of its well-known activity in limiting axonal growth. Therefore, its degradation has become a key therapeutic goal in the field of CNS regeneration. Yet, the abundant de novo synthesis of CSPG in response to CNS injury is puzzling. This apparent dichotomy led us to hypothesize that CSPG plays a beneficial role in the repair process, which might have been previously overlooked because of nonoptimal regulation of its levels. This hypothesis is tested in the present study.
Methods and Findings
We inflicted spinal cord injury in adult mice and examined the effects of CSPG on the recovery process. We used xyloside to inhibit CSPG formation at different time points after the injury and analyzed the phenotype acquired by the microglia/macrophages in the lesion site. To distinguish between the resident microglia and infiltrating monocytes, we used chimeric mice whose bone marrow-derived myeloid cells expressed GFP. We found that CSPG plays a key role during the acute recovery stage after spinal cord injury in mice. Inhibition of CSPG synthesis immediately after injury impaired functional motor recovery and increased tissue loss. Using the chimeric mice we found that the immediate inhibition of CSPG production caused a dramatic effect on the spatial organization of the infiltrating myeloid cells around the lesion site, decreased insulin-like growth factor 1 (IGF-1) production by microglia/macrophages, and increased tumor necrosis factor alpha (TNF-α) levels. In contrast, delayed inhibition, allowing CSPG synthesis during the first 2 d following injury, with subsequent inhibition, improved recovery. Using in vitro studies, we showed that CSPG directly activated microglia/macrophages via the CD44 receptor and modulated neurotrophic factor secretion by these cells.
Our results show that CSPG plays a pivotal role in the repair of injured spinal cord and in the recovery of motor function during the acute phase after the injury; CSPG spatially and temporally controls activity of infiltrating blood-borne monocytes and resident microglia. The distinction made in this study between the beneficial role of CSPG during the acute stage and its deleterious effect at later stages emphasizes the need to retain the endogenous potential of this molecule in repair by controlling its levels at different stages of post-injury repair.
Michal Schwartz and colleagues describe the role of chondroitin sulfate proteoglycan in the repair of injured tissue and in the recovery of motor function during the acute phase after spinal cord injury.
Editors' Summary
Every year, spinal cord injuries paralyze about 10,000 people in the United States. The spinal cord, which contains bundles of nervous system cells called neurons, is the communication superhighway between the brain and the body. Messages from the brain travel down the spinal cord to control movement, breathing, and other bodily functions; messages from the skin and other sensory organs travel up the spinal cord to keep the brain informed about the body. All these messages are transmitted along axons, long extensions on the neurons. The spinal cord is protected by the bones of the spine but if these are displaced or broken, the axons can be compressed or cut, which interrupts the information flow. Damage near the top of the spinal cord paralyzes the arms and legs (tetraplegia); damage lower down paralyzes the legs only (paraplegia). Spinal cord injuries also cause other medical problems, including the loss of bowel and bladder control. Currently there is no effective treatment for spinal cord injuries. Treatment with drugs to reduce inflammation has, at best, only modest effects. Moreover, because damaged axons rarely regrow, most spinal cord injuries are permanent.
Why Was This Study Done?
One barrier to recovery after a spinal cord injury seems to be an inappropriate immune response to the injury. After an injury, microglia (immune system cells that live in the nervous system), and macrophages (blood-borne immune system cells that infiltrate the injury) become activated. Microglia/macrophage activation can be either beneficial (the cells make IGF-1, a protein that stimulates axon growth) or destructive (the cells make TNF-α, a protein that kills neurons), so studies of microglia/macrophage activation might suggest ways to treat spinal cord injuries. Another possible barrier to recovery is “chondroitin sulfate proteoglycan” (CSPG). This is a major component of the scar tissue (the “glial scar”) that forms around spinal cord injuries. CSPG limits axon regrowth, so attempts have been made to improve spinal cord repair by removing CSPG. But if CSPG prevents spinal cord repair, why is so much of it made immediately after an injury? In this study, the researchers investigate this paradox by asking whether CSPG made in the right place and in the right amount might have a beneficial role in spinal cord repair that has been overlooked.
What Did the Researchers Do and Find?
The researchers bruised a small section of the spinal cord of mice to cause hind limb paralysis, and then monitored the recovery of movement in these animals. They also examined the injured tissue microscopically, looked for microglia and infiltrating macrophages at the injury site, and measured the production of IGF-1 and TNF-α by these cells. Inhibition of CSPG synthesis immediately after injury impaired the functional recovery of the mice and increased tissue loss at the injury site. It also altered the spatial organization of infiltrating macrophages at the injury site, reduced IGF-1 production by these microglia/macrophages, and increased TNF-α levels. In contrast, when CSPG synthesis was not inhibited until two days after the injury, the mice recovered well from spinal cord injury. Furthermore, the interaction of CSPG with a cell-surface protein called CD44 activated microglia/macrophages growing in dishes and increased their production of IGF-1 but not of molecules that kill neurons.
What Do These Findings Mean?
These findings suggest that, immediately after a spinal cord injury, CSPG is needed for the repair of injured neurons and the recovery of movement, but that later on the presence of CSPG hinders repair. The findings also indicate that CSPG has these effects, at least in part, because it regulates the activity and localization of microglia and macrophages at the injury site and thus modulates local immune responses to the damage. Results obtained from experiments done in animals do not always accurately reflect the situation in people, so these findings need to be confirmed in patients with spinal cord injuries. However, they suggest that the effect of CSPG on spinal cord repair is not an inappropriate response to the injury, as is widely believed. Consequently, careful manipulation of CSPG levels might improve outcomes for people with spinal cord injuries.
Additional Information.
Please access these Web sites via the online version of this summary at
The MedlinePlus encyclopedia provides information about spinal cord injuries; MedlinePlus provides an interactive tutorial and a list of links to additional information about spinal cord injuries (in English and Spanish)
The US National Institute of Neurological Disorders and Stroke also provides information about spinal cord injury (in English and Spanish)
Wikipedia has a page on glial scars (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
PMCID: PMC2517615  PMID: 18715114
3.  Exendin-4 Ameliorates Motor Neuron Degeneration in Cellular and Animal Models of Amyotrophic Lateral Sclerosis 
PLoS ONE  2012;7(2):e32008.
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by a progressive loss of lower motor neurons in the spinal cord. The incretin hormone, glucagon-like peptide-1 (GLP-1), facilitates insulin signaling, and the long acting GLP-1 receptor agonist exendin-4 (Ex-4) is currently used as an anti-diabetic drug. GLP-1 receptors are widely expressed in the brain and spinal cord, and our prior studies have shown that Ex-4 is neuroprotective in several neurodegenerative disease rodent models, including stroke, Parkinson's disease and Alzheimer's disease. Here we hypothesized that Ex-4 may provide neuroprotective activity in ALS, and hence characterized Ex-4 actions in both cell culture (NSC-19 neuroblastoma cells) and in vivo (SOD1 G93A mutant mice) models of ALS. Ex-4 proved to be neurotrophic in NSC-19 cells, elevating choline acetyltransferase (ChAT) activity, as well as neuroprotective, protecting cells from hydrogen peroxide-induced oxidative stress and staurosporine-induced apoptosis. Additionally, in both wild-type SOD1 and mutant SOD1 (G37R) stably transfected NSC-19 cell lines, Ex-4 protected against trophic factor withdrawal-induced toxicity. To assess in vivo translation, SOD1 mutant mice were administered vehicle or Ex-4 at 6-weeks of age onwards to end-stage disease via subcutaneous osmotic pump to provide steady-state infusion. ALS mice treated with Ex-4 showed improved glucose tolerance and normalization of behavior, as assessed by running wheel, compared to control ALS mice. Furthermore, Ex-4 treatment attenuated neuronal cell death in the lumbar spinal cord; immunohistochemical analysis demonstrated the rescue of neuronal markers, such as ChAT, associated with motor neurons. Together, our results suggest that GLP-1 receptor agonists warrant further evaluation to assess whether their neuroprotective potential is of therapeutic relevance in ALS.
PMCID: PMC3285661  PMID: 22384126
4.  The Neuroprotective Effects of Intramuscular Insulin-Like Growth Factor-I Treatment in Brain Ischemic Rats 
PLoS ONE  2013;8(5):e64015.
Brain ischemia leads to muscle inactivity-induced atrophy and may exacerbate motor function deficits. Intramuscular insulin-like growth factor I (IGF-I) injection has been shown to alleviate the brain ischemia-induced muscle atrophy and thus improve the motor function. Motor function is normally gauged by the integrity and coordination of the central nervous system and peripheral muscles. Whether brain ischemic regions are adaptively changed by the intramuscular IGF-I injection is not well understood. In this study, the effect of intramuscular IGF-I injection was examined on the central nervous system of brain ischemic rats. Rats were divided into 4 groups: sham control, brain ischemia control, brain ischemia with IGF-I treatment, and brain ischemia with IGF-I plus IGF-I receptor inhibitor treatment. Brain ischemia was induced by right middle cerebral artery occlusion. IGF-I and an IGF-1 receptor inhibitor were injected into the affected calf and anterior tibialis muscles of the treated rats for 4 times. There was an interval of 2 days between each injection. Motor function was examined and measured at the 24 hours and 7 days following a brain ischemia. The affected hind-limb muscles, sciatic nerve, lumbar spinal cord, and motor cortex were collected for examination after euthanizing the rats. IGF-I expression in the central nervous system and affected muscles were significantly decreased after brain ischemia. Intramuscular IGF-I injection increased the IGF-I expression in the affected muscles, sciatic nerve, lumbar spinal cord, and motor cortex. It also increased the p-Akt expression in the affected motor cortex. Furthermore, intramuscular IGF-I injection decreased the neuronal apoptosis and improved the motor function. However, co-administration of the IGF-I receptor inhibitor eliminated these effects. Intramuscular IGF-I injection after brain ischemia attenuated or reversed the decrease of IGF-I in both central and peripheral tissues, and these effects could contribute to neuroprotection and improve motor function.
PMCID: PMC3661564  PMID: 23717526
5.  Abnormal Changes in NKT Cells, the IGF-1 Axis, and Liver Pathology in an Animal Model of ALS 
PLoS ONE  2011;6(8):e22374.
Amyotrophic lateral sclerosis (ALS) is a rapidly progressing fatal neurodegenerative disorder characterized by the selective death of motor neurons (MN) in the spinal cord, and is associated with local neuroinflammation. Circulating CD4+ T cells are required for controlling the local detrimental inflammation in neurodegenerative diseases, and for supporting neuronal survival, including that of MN. T-cell deficiency increases neuronal loss, while boosting T cell levels reduces it. Here, we show that in the mutant superoxide dismutase 1 G93A (mSOD1) mouse model of ALS, the levels of natural killer T (NKT) cells increased dramatically, and T-cell distribution was altered both in lymphoid organs and in the spinal cord relative to wild-type mice. The most significant elevation of NKT cells was observed in the liver, concomitant with organ atrophy. Hepatic expression levels of insulin-like growth factor (IGF)-1 decreased, while the expression of IGF binding protein (IGFBP)-1 was augmented by more than 20-fold in mSOD1 mice relative to wild-type animals. Moreover, hepatic lymphocytes of pre-symptomatic mSOD1 mice were found to secrete significantly higher levels of cytokines when stimulated with an NKT ligand, ex-vivo. Immunomodulation of NKT cells using an analogue of α-galactosyl ceramide (α-GalCer), in a specific regimen, diminished the number of these cells in the periphery, and induced recruitment of T cells into the affected spinal cord, leading to a modest but significant prolongation of life span of mSOD1 mice. These results identify NKT cells as potential players in ALS, and the liver as an additional site of major pathology in this disease, thereby emphasizing that ALS is not only a non-cell autonomous, but a non-tissue autonomous disease, as well. Moreover, the results suggest potential new therapeutic targets such as the liver for immunomodulatory intervention for modifying the disease, in addition to MN-based neuroprotection and systemic treatments aimed at reducing oxidative stress.
PMCID: PMC3149057  PMID: 21829620
6.  Delivery of AAV-IGF-1 to the CNS Extends Survival in ALS Mice Through Modification of Aberrant Glial Cell Activity 
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor system. Recent work in rodent models of ALS has shown that insulin-like growth factor-1 (IGF-1) slows disease progression when delivered at disease onset. However, IGF-1’s mechanism of action along the neuromuscular axis remains unclear. In this study, symptomatic ALS mice received IGF-1 through stereotaxic injection of an IGF-1-expressing viral vector to the deep cerebellar nuclei (DCN), a region of the cerebellum with extensive brain stem and spinal cord connections. We found that delivery of IGF-1 to the central nervous system (CNS) reduced ALS neuropathology, improved muscle strength, and significantly extended life span in ALS mice. To explore the mechanism of action of IGF-1, we used a newly developed in vitro model of ALS. We demonstrate that IGF-1 is potently neuroprotective and attenuates glial cell–mediated release of tumor necrosis factor-α (TNF-α) and nitric oxide (NO). Our results show that delivering IGF-1 to the CNS is sufficient to delay disease progression in a mouse model of familial ALS and demonstrate for the first time that IGF-1 attenuates the pathological activity of non-neuronal cells that contribute to disease progression. Our findings highlight an innovative approach for delivering IGF-1 to the CNS.
PMCID: PMC2737251  PMID: 18388910
7.  Upregulation of the E3 ligase NEDD4-1 by oxidative stress degrades IGF-1 receptor protein in neurodegeneration 
The importance of ubiquitin E3 ligases in neurodegeneration is being increasingly recognized. The crucial role of NEDD4-1 (neural precursor cell-expressed developmentally down-regulated gene 4) in neural development is well-appreciated; however, its role in neurodegeneration remains unexplored. Herein, we report increased NEDD4-1 expression in the degenerated tissues of several major neurodegenerative diseases. Moreover, its expression is upregulated in cultured neurons in response to various neurotoxins, including zinc and hydrogen superoxide, via transcriptional activation likely mediated by the ROS-responsive FOXM1B. Reduced protein levels of the insulin-like growth factor receptor (IGF-1Rβ) were observed as a consequence of upregulated NEDD4-1 via the ubiquitin-proteasome system (UPS). Overexpression of a familial mutant form of SOD-1 (G93A) in neuroblastoma cells resulted in a similar reduction of IGF-1Rβ protein. This inverse correlation between NEDD4-1 and IGF-1Rβ was also observed in the cortex and spinal cords of mutant (G93A) SOD-1 transgenic mice at a pre-symptomatic age, which was similarly induced by in vivo administered zinc in WT C57BL/6 mice. Furthermore, histochemistry reveals markedly increased NEDD4-1 immunoreactivity in the degenerating/degenerated motor neurons in the lumbar anterior horn of the spinal cord, suggesting a direct causative role for NEDD4-1 in neurodegeneration. Indeed, downregulation of NEDD4-1 by shRNA or overexpression of a catalytically inactive form rescued neurons from zinc-induced cell death. Similarly, neurons with a NEDD4-1 haplotype are more resistant to apoptosis, largely due to expression of higher levels of IGF-1Rβ. Taken together, our work identifies a novel molecular mechanism for ROS-upregulated NEDD4-1 and the subsequently reduced IGF-1Rβ signaling in neurodegeneration.
PMCID: PMC3681290  PMID: 22875931
8.  Upregulation of the E3 ligase NEDD4-1 by Oxidative Stress Degrades IGF-1 Receptor Protein in Neurodegeneration 
The Journal of Neuroscience  2012;32(32):10971-10981.
The importance of ubiquitin E3 ligases in neurodegeneration is being increasingly recognized. The crucial role of NEDD4-1 in neural development is well appreciated; however, its role in neurodegeneration remains unexplored. Herein, we report increased NEDD4-1 expression in the degenerated tissues of several major neurodegenerative diseases. Moreover, its expression is upregulated in cultured neurons in response to various neurotoxins, including zinc and hydrogen superoxide, via transcriptional activation likely mediated by the reactive oxygen species (ROS)-responsive FOXM1B. Reduced protein levels of the insulin-like growth factor receptor (IGF-1Rβ) were observed as a consequence of upregulated NEDD4-1 via the ubiquitin-proteasome system. Overexpression of a familial mutant form of superoxide dismutase 1 (SOD1) (G93A) in neuroblastoma cells resulted in a similar reduction of IGF-1Rβ protein. This inverse correlation between NEDD4-1 and IGF-1Rβ was also observed in the cortex and spinal cords of mutant (G93A) SOD1 transgenic mice at a presymptomatic age, which was similarly induced by in vivo-administered zinc in wild-type C57BL/6 mice. Furthermore, histochemistry reveals markedly increased NEDD4-1 immunoreactivity in the degenerating/degenerated motor neurons in the lumbar anterior horn of the spinal cord, suggesting a direct causative role for NEDD4-1 in neurodegeneration. Indeed, downregulation of NEDD4-1 by shRNA or overexpression of a catalytically inactive form rescued neurons from zinc-induced cell death. Similarly, neurons with a NEDD4-1 haplotype are more resistant to apoptosis, largely due to expression of higher levels of IGF-1Rβ.Together, our work identifies a novel molecular mechanism for ROS-upregulated NEDD4-1 and the subsequently reduced IGF-1Rβ signaling in neurodegeneration.
PMCID: PMC3681290  PMID: 22875931
9.  Growth factor-expressing human neural progenitor cell grafts protect motor neurons but do not ameliorate motor performance and survival in ALS mice 
Experimental & Molecular Medicine  2009;41(7):487-500.
Neural progenitor cells (NPs) have shown several promising benefits for the treatment of neurological disorders. To evaluate the therapeutic potential of human neural progenitor cells (hNPs) in amyotrophic lateral sclerosis (ALS), we transplanted hNPs or growth factor (GF)-expressing hNPs into the central nervous system (CNS) of mutant Cu/Zn superoxide dismutase (SOD1G93A) transgenic mice. The hNPs were engineered to express brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1), VEGF, neurotrophin-3 (NT-3), or glial cell-derived neurotrophic factor (GDNF), respectively, by adenoviral vector and GDNF by lentiviral vector before transplantation. Donor-derived cells engrafted and migrated into the spinal cord or brain of ALS mice and differentiated into neurons, oligodendrocytes, or glutamate transporter-1 (GLT1)-expressing astrocytes while some cells retained immature markers. Transplantation of GDNF- or IGF-1-expressing hNPs attenuated the loss of motor neurons and induced trophic changes in motor neurons of the spinal cord. However, improvement in motor performance and extension of lifespan were not observed in all hNP transplantation groups compared to vehicle-injected controls. Moreover, the lifespan of GDNF-expressing hNP recipient mice by lentiviral vector was shortened compared to controls, which was largely due to the decreased survival times of female animals. These results imply that although implanted hNPs differentiate into GLT1-expressing astrocytes and secrete GFs, which maintain dying motor neurons, inadequate trophic support could be harmful and there is sexual dimorphism in response to GDNF delivery in ALS mice. Therefore, additional therapeutic approaches may be required for full functional recovery.
PMCID: PMC2721146  PMID: 19322031
amyotrophic lateral sclerosis; cell differentiation; glial cell line-derived neurotrophic factor; nerve growth factors; stem cell transplantation; stem cells
10.  Human Neural Stem Cell Replacement Therapy for Amyotrophic Lateral Sclerosis by Spinal Transplantation 
PLoS ONE  2012;7(8):e42614.
Mutation in the ubiquitously expressed cytoplasmic superoxide dismutase (SOD1) causes an inherited form of Amyotrophic Lateral Sclerosis (ALS). Mutant synthesis in motor neurons drives disease onset and early disease progression. Previous experimental studies have shown that spinal grafting of human fetal spinal neural stem cells (hNSCs) into the lumbar spinal cord of SOD1G93A rats leads to a moderate therapeutical effect as evidenced by local α-motoneuron sparing and extension of lifespan. The aim of the present study was to analyze the degree of therapeutical effect of hNSCs once grafted into the lumbar spinal ventral horn in presymptomatic immunosuppressed SOD1G93A rats and to assess the presence and functional integrity of the descending motor system in symptomatic SOD1G93A animals.
Methods/Principal Findings
Presymptomatic SOD1G93A rats (60–65 days old) received spinal lumbar injections of hNSCs. After cell grafting, disease onset, disease progression and lifespan were analyzed. In separate symptomatic SOD1G93A rats, the presence and functional conductivity of descending motor tracts (corticospinal and rubrospinal) was analyzed by spinal surface recording electrodes after electrical stimulation of the motor cortex. Silver impregnation of lumbar spinal cord sections and descending motor axon counting in plastic spinal cord sections were used to validate morphologically the integrity of descending motor tracts. Grafting of hNSCs into the lumbar spinal cord of SOD1G93A rats protected α-motoneurons in the vicinity of grafted cells, provided transient functional improvement, but offered no protection to α-motoneuron pools distant from grafted lumbar segments. Analysis of motor-evoked potentials recorded from the thoracic spinal cord of symptomatic SOD1G93A rats showed a near complete loss of descending motor tract conduction, corresponding to a significant (50–65%) loss of large caliber descending motor axons.
These data demonstrate that in order to achieve a more clinically-adequate treatment, cell-replacement/gene therapy strategies will likely require both spinal and supraspinal targets.
PMCID: PMC3423406  PMID: 22916141
11.  Microglia and motor neurons during disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis: changes in arginase1 and inducible nitric oxide synthase 
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting the motor system. Although the etiology of the disease is not fully understood, microglial activation and neuroinflammation are thought to play a role in disease progression.
We examined the immunohistochemical expression of two markers of microglial phenotype, the arginine-metabolizing enzymes inducible nitric oxide synthase (iNOS) and arginase1 (Arg1), in the spinal cord of a mouse model carrying an ALS-linked mutant human superoxide dismutase transgene (SOD1G93A) and in non-transgenic wild-type (WT) mice. Immunolabeling for iNOS and Arg1 was evaluated throughout disease progression (6 to 25 weeks), and correlated with body weight, stride pattern, wire hang duration and ubiquitin pathology. For microglia and motor neuron counts at each time point, SOD1G93A and WT animals were compared using an independent samples t-test. A Welch t-test correction was applied if Levene’s test showed that the variance in WT and SOD1G93A measurements was substantially different.
Disease onset, measured as the earliest change in functional parameters compared to non-transgenic WT mice, occurred at 14 weeks of age in SOD1G93A mice. The ventral horn of the SOD1G93A spinal cord contained more microglia than WT from 14 weeks onwards. In SOD1G93A mice, Arg1-positive and iNOS-positive microglia increased 18-fold and 7-fold, respectively, between 10 and 25 weeks of age (endpoint) in the lumbar spinal cord, while no increase was observed in WT mice. An increasing trend of Arg1- and iNOS-expressing microglia was observed in the cervical spinal cords of SOD1G93A mice. Additionally, Arg1-negative motor neurons appeared to selectively decline in the spinal cord of SOD1G93A mice, suggesting that Arg1 may have a neuroprotective function.
This study suggests that the increase in spinal cord microglia occurs around and after disease onset and is preceded by cellular pathology. The results show that Arg1 and iNOS, thought to have opposing inflammatory properties, are upregulated in microglia during disease progression and that Arg1 in motor neurons may confer protection from disease processes. Further understanding of the neuroinflammatory response, and the Arg1/iNOS balance in motor neurons, may provide suitable therapeutic targets for ALS.
PMCID: PMC3994340  PMID: 24655927
Amyotrophic lateral sclerosis; Microglia; Inducible nitric oxide synthase; Arginase1; Motor neurons; Lumbar spinal cord; Cervical spinal cord; Neuroinflammation
12.  A mutation in the dynein heavy chain gene compensates for energy deficit of mutant SOD1 mice and increases potentially neuroprotective IGF-1 
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive loss of motor neurons. ALS patients, as well as animal models such as mice overexpressing mutant SOD1s, are characterized by increased energy expenditure. In mice, this hypermetabolism leads to energy deficit and precipitates motor neuron degeneration. Recent studies have shown that mutations in the gene encoding the dynein heavy chain protein are able to extend lifespan of mutant SOD1 mice. It remains unknown whether the protection offered by these dynein mutations relies on a compensation of energy metabolism defects.
SOD1(G93A) mice were crossbred with mice harboring the dynein mutant Cramping allele (Cra/+ mice). Dynein mutation increased adipose stores in compound transgenic mice through increasing carbohydrate oxidation and sparing lipids. Metabolic changes that occurred in double transgenic mice were accompanied by the normalization of the expression of key mRNAs in the white adipose tissue and liver. Furthermore, Dynein Cra mutation rescued decreased post-prandial plasma triglycerides and decreased non esterified fatty acids upon fasting. In SOD1(G93A) mice, the dynein Cra mutation led to increased expression of IGF-1 in the liver, increased systemic IGF-1 and, most importantly, to increased spinal IGF-1 levels that are potentially neuroprotective.
These findings suggest that the protection against SOD1(G93A) offered by the Cramping mutation in the dynein gene is, at least partially, mediated by a reversal in energy deficit and increased IGF-1 availability to motor neurons.
PMCID: PMC3111394  PMID: 21521523
13.  Muscle expression of a local Igf-1 isoform protects motor neurons in an ALS mouse model 
The Journal of Cell Biology  2005;168(2):193-199.
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by a selective degeneration of motor neurons, atrophy, and paralysis of skeletal muscle. Although a significant proportion of familial ALS results from a toxic gain of function associated with dominant SOD1 mutations, the etiology of the disease and its specific cellular origins have remained difficult to define. Here, we show that muscle-restricted expression of a localized insulin-like growth factor (Igf) -1 isoform maintained muscle integrity and enhanced satellite cell activity in SOD1G93A transgenic mice, inducing calcineurin-mediated regenerative pathways. Muscle-specific expression of local Igf-1 (mIgf-1) isoform also stabilized neuromuscular junctions, reduced inflammation in the spinal cord, and enhanced motor neuronal survival in SOD1G93A mice, delaying the onset and progression of the disease. These studies establish skeletal muscle as a primary target for the dominant action of inherited SOD1 mutation and suggest that muscle fibers provide appropriate factors, such as mIgf-1, for neuron survival.
PMCID: PMC2171577  PMID: 15657392
14.  Transcriptional profile of primary astrocytes expressing ALS-linked mutant SOD1 
Journal of neuroscience research  2008;86(16):3515-3525.
Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons. Mutations in the Cu/Zn superoxide dismutase (SOD1) are found in about 20% of patients with familial ALS. Mutant SOD1 causes motor neuron death through an acquired toxic property. Although the molecular mechanism underlying this toxic gain-of-function remains unknown, evidence support the role of mutant SOD1 expression in non-neuronal cells in shaping motor neuron degeneration. We have previously found that in contrast to non-transgenic cells, SOD1G93A-expressing astrocytes induced apoptosis of co-cultured motor neurons. This prompted us to investigate whether the effect on motor neuron survival was related to a change in the gene expression profile. Through high-density oligonucleotide microarrays, we found changes in the expression of genes involved in transcription, signaling, cell proliferation, extracellular matrix synthesis, response to stress and steroid and lipid metabolism. The most up-regulated gene was decorin (Dcn), a small multifunctional extracellular proteoglycan. Down-regulated genes included the insulin-like growth factor-1 receptor (Igf-1r) and the RNA binding protein ROD1. Rod1 was also found downregulated in purified motor neurons expressing SOD1G93A. Changes in the expression of Dcn, Igf-1r and Rod1 were found in the spinal cord of asymptomatic animals, suggesting these changes occur before overt neuronal degeneration and potentially influence astrocyte-motor neuron interaction in the course of the disease. The astrocyte-specific gene expression profile might contribute to the identification of possible candidates for cell type-specific therapies in ALS.
PMCID: PMC4048747  PMID: 18683239
motor neurons; decorin; IGF-1R; ROD1; microarray
15.  Bone Marrow Transplantation in Hindlimb Muscles of Motoneuron Degenerative Mice Reduces Neuronal Death and Improves Motor Function 
Stem Cells and Development  2013;22(11):1633-1644.
Bone marrow has proved to be an adequate source of stem cells for the treatment of numerous disorders, including neurodegenerative diseases. Bone marrow can be easily and relatively painlessly extracted from a patient or allogenic donor and then transplanted into the degenerative area. Here, the grafted cells will activate a number of mechanisms in order to protect, repair, and/or regenerate the damaged tissue. These properties make the bone marrow a feasible source for cell therapy. In this work, we transplanted bone marrow cells into a mouse model of motoneuron degeneration, with the particularity of placing the cells in the hindlimb muscles rather than in the spinal cord where neuronal degeneration occurs. To this end, we analyze the possibility for the transplanted cells to increase the survival rate of the spinal cord motoneurons by axonal-guided retrograde neurotrophism. As a result, the mice significantly improved their motor functions. This coincided with an increased number of motoneurons innervating the treated muscle compared with the neurons innervating the non-treated contralateral symmetric muscle. In addition, we detected an increase in glial-derived neurotrophic factor in the spinal cord, a neurotrophic factor known to be involved in the rescue of degenerating motoneurons, exerting a neuroprotective effect. Thus, we have proved that bone marrow injected into the muscles is capable of rescuing these motoneurons from death, which may be a possible therapeutic approach for spinal cord motoneuron degenerative diseases, such as amyotrophic lateral sclerosis.
PMCID: PMC3657290  PMID: 23282201
16.  Adeno-associated Virus–mediated Delivery of a Recombinant Single-chain Antibody Against Misfolded Superoxide Dismutase for Treatment of Amyotrophic Lateral Sclerosis 
Molecular Therapy  2014;22(3):498-510.
There is emerging evidence that the misfolding of superoxide dismutase 1 (SOD1) may represent a common pathogenic event in both familial and sporadic amyotrophic lateral sclerosis (ALS). To reduce the burden of misfolded SOD1 species in the nervous system, we have tested a novel therapeutic approach based on adeno-associated virus (AAV)–mediated tonic expression of a DNA construct encoding a secretable single-chain fragment variable (scFv) antibody composed of the variable heavy and light chain regions of a monoclonal antibody (D3H5) binding specifically to misfolded SOD1. A single intrathecal injection of the AAV encoding the single-chain antibody in SOD1G93A mice at 45 days of age resulted in sustained expression of single-chain antibodies in the spinal cord, and it delayed disease onset and extension of life span by up to 28%, in direct correlation with scFv titers in the spinal cord. The treatment caused attenuation of neuronal stress signals and reduction in levels of misfolded SOD1 in the spinal cord of SOD1G93A mice. From these results, we propose that an immunotherapy based on intrathecal inoculation of AAV encoding a secretable scFv against misfolded SOD1 should be considered as potential treatment for ALS, especially for individuals carrying SOD1 mutations.
PMCID: PMC3944333  PMID: 24394188
17.  Overexpression of the Astrocyte Glutamate Transporter GLT1 Exacerbates Phrenic Motor Neuron Degeneration, Diaphragm Compromise, and Forelimb Motor Dysfunction following Cervical Contusion Spinal Cord Injury 
The Journal of Neuroscience  2014;34(22):7622-7638.
A major portion of spinal cord injury (SCI) cases affect midcervical levels, the location of the phrenic motor neuron (PhMN) pool that innervates the diaphragm. While initial trauma is uncontrollable, a valuable opportunity exists in the hours to days following SCI for preventing PhMN loss and consequent respiratory dysfunction that occurs during secondary degeneration. One of the primary causes of secondary injury is excitotoxic cell death due to dysregulation of extracellular glutamate homeostasis. GLT1, mainly expressed by astrocytes, is responsible for the vast majority of functional uptake of extracellular glutamate in the CNS, particularly in spinal cord. We found that, in bacterial artificial chromosome-GLT1-enhanced green fluorescent protein reporter mice following unilateral midcervical (C4) contusion SCI, numbers of GLT1-expressing astrocytes in ventral horn and total intraspinal GLT1 protein expression were reduced soon after injury and the decrease persisted for ≥6 weeks. We used intraspinal delivery of adeno-associated virus type 8 (AAV8)-Gfa2 vector to rat cervical spinal cord ventral horn for targeting focal astrocyte GLT1 overexpression in areas of PhMN loss. Intraspinal delivery of AAV8-Gfa2-GLT1 resulted in transduction primarily of GFAP+ astrocytes that persisted for ≥6 weeks postinjury, as well as increased intraspinal GLT1 protein expression. Surprisingly, we found that astrocyte-targeted GLT1 overexpression increased lesion size, PhMN loss, phrenic nerve axonal degeneration, and diaphragm neuromuscular junction denervation, and resulted in reduced functional diaphragm innervation as assessed by phrenic nerve-diaphragm compound muscle action potential recordings. These results demonstrate that GLT1 overexpression via intraspinal AAV-Gfa2-GLT1 delivery exacerbates neuronal damage and increases respiratory impairment following cervical SCI.
PMCID: PMC4035523  PMID: 24872566
adeno-associated virus; gene therapy; glutamate transporter; phrenic motor neuron; respiratory; spinal cord injury
18.  Insulin-like growth factor-I for the treatment of amyotrophic lateral sclerosis 
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects both upper and lower motor neurons (MN) resulting in weakness, paralysis and subsequent death. Insulin-like growth factor-I (IGF-I) is a potent neurotrophic factor that has neuroprotective properties in the central and peripheral nervous systems. Due to the efficacy of IGF-I in the treatment of other diseases and its ability to promote neuronal survival, IGF-I is being extensively studied in ALS therapeutic trials. This review covers in vitro and in vivo studies examining the efficacy of IGF-I in ALS model systems and also addresses the mechanisms by which IGF-I asserts its effects in these models, the status of the IGF-I system in ALS patients, results of clinical trials, and the need for the development of better delivery mechanisms to maximize IGF-I efficacy. The knowledge obtained from these studies suggests that IGF-I has the potential to be a safe and efficacious therapy for ALS.
PMCID: PMC3211070  PMID: 18608100
Amyotrophic lateral sclerosis; insulin-like growth factor-I; treatment
19.  Apelin Deficiency Accelerates the Progression of Amyotrophic Lateral Sclerosis 
PLoS ONE  2011;6(8):e23968.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective loss of motor neurons. Recent studies have implicated that chronic hypoxia and insufficient vascular endothelial growth factor (VEGF)-dependent neuroprotection may lead to the degeneration of motor neurons in ALS. Expression of apelin, an endogenous ligand for the G protein-coupled receptor APJ, is regulated by hypoxia. In addition, recent reports suggest that apelin protects neurons against glutamate-induced excitotoxicity. Here, we examined whether apelin is an endogenous neuroprotective factor using SOD1G93A mouse model of ALS. In mouse CNS tissues, the highest expressions of both apelin and APJ mRNAs were detected in spinal cord. APJ immunoreactivity was observed in neuronal cell bodies located in gray matter of spinal cord. Although apelin mRNA expression in the spinal cord of wild-type mice was not changed from 4 to 18 weeks age, that of SOD1G93A mice was reduced along with the paralytic phenotype. In addition, double mutant apelin-deficient and SOD1G93A displayed the disease phenotypes earlier than SOD1G93A littermates. Immunohistochemical observation revealed that the number of motor neurons was decreased and microglia were activated in the spinal cord of the double mutant mice, indicating that apelin deficiency pathologically accelerated the progression of ALS. Furthermore, we showed that apelin enhanced the protective effect of VEGF on H2O2-induced neuronal death in primary neurons. These results suggest that apelin/APJ system in the spinal cord has a neuroprotective effect against the pathogenesis of ALS.
PMCID: PMC3161091  PMID: 21887354
20.  Monoaminergic control of spinal locomotor networks in SOD1G93A newborn mice 
Mutations in the gene that encodes Cu/Zn-superoxide dismutase (SOD1) are the cause of approximately 20% of familial forms of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. While ALS symptoms appear in adulthood, spinal motoneurons exhibit functional alterations as early as the embryonic and postnatal stages in the murine model of ALS, the SOD1 mice. Monoaminergic – i.e., dopaminergic (DA), serotoninergic (5-HT), and noradrenergic (NA) – pathways powerfully control spinal networks and contribute significantly to their embryonic and postnatal maturation. Alterations in monoaminergic neuromodulation during development could therefore lead to impairments in the motoneuronal physiology. In this study, we sought to determine whether the monoaminergic spinal systems are modified in the early stages of development in SOD1 mice. Using a post-mortem analysis by high performance liquid chromatography (HPLC), monoaminergic neuromodulators and their metabolites were quantified in the lumbar spinal cord of SOD1 and wild-type (WT) mice aged one postnatal day (P1) and P10. This analysis underscores an increased content of DA in the SOD1 lumbar spinal cord compared to that of WT mice but failed to reveal any modification of the other monoaminergic contents. In a next step, we compared the efficiency of the monoaminergic compounds in triggering and modulating fictive locomotion in WT and SOD1 mice. This study was performed in P1–P3 SOD1 mice and age-matched control littermates using extracellular recordings from the lumbar ventral roots in the in vitro isolated spinal cord preparation. This analysis revealed that the spinal networks of SOD1G93A mice could generate normal locomotor activity in the presence of NMA-5-HT. Interestingly, we also observed that SOD1 spinal networks have an increased sensitivity to NA compared to WT spinal circuits but exhibited similar DA responses.
PMCID: PMC4081764  PMID: 25071458
serotonin; dopamine; noradrenaline; spinal cord; ALS; fictive locomotion; HPLC; extracellular recordings
21.  Overexpression of BDNF Increases Excitability of the Lumbar Spinal Network and Leads to Robust Early Locomotor Recovery in Completely Spinalized Rats 
PLoS ONE  2014;9(2):e88833.
Strategies to induce recovery from lesions of the spinal cord have not fully resulted in clinical applications. This is a consequence of a number of impediments that axons encounter when trying to regrow beyond the lesion site, and that intraspinal rearrangements are subjected to. In the present study we evaluated (1) the possibility to improve locomotor recovery after complete transection of the spinal cord by means of an adeno-associated (AAV) viral vector expressing the neurotrophin brain-derived neurotrophic factor (BDNF) in lumbar spinal neurons caudal to the lesion site and (2) how the spinal cord transection and BDNF treatment affected neurotransmission in the segments caudal to the lesion site. BDNF overexpression resulted in clear increases in expression levels of molecules involved in glutamatergic (VGluT2) and GABAergic (GABA, GAD65, GAD67) neurotransmission in parallel with a reduction of the potassium-chloride co-transporter (KCC2) which contributes to an inhibitory neurotransmission. BDNF treated animals showed significant improvements in assisted locomotor performance, and performed locomotor movements with body weight support and plantar foot placement on a moving treadmill. These positive effects of BDNF local overexpression were detectable as early as two weeks after spinal cord transection and viral vector application and lasted for at least 7 weeks. Gradually increasing frequencies of clonic movements at the end of the experiment attenuated the quality of treadmill walking. These data indicate that BDNF has the potential to enhance the functionality of isolated lumbar circuits, but also that BDNF levels have to be tightly controlled to prevent hyperexcitability.
PMCID: PMC3925164  PMID: 24551172
22.  Ablation of Proliferating Cells in the CNS Exacerbates Motor Neuron Disease Caused by Mutant Superoxide Dismutase 
PLoS ONE  2012;7(4):e34932.
Proliferation of glia and immune cells is a common pathological feature of many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Here, to investigate the role of proliferating cells in motor neuron disease, SOD1G93A transgenic mice were treated intracerebroventicularly (ICV) with the anti-mitotic drug cytosine arabinoside (Ara-C). ICV delivery of Ara-C accelerated disease progression in SOD1G93A mouse model of ALS. Ara-C treatment caused substantial decreases in the number of microglia, NG2+ progenitors, Olig2+ cells and CD3+ T cells in the lumbar spinal cord of symptomatic SOD1G93A transgenic mice. Exacerbation of disease was also associated with significant alterations in the expression inflammatory molecules IL-1β, IL-6, TGF-β and the growth factor IGF-1.
PMCID: PMC3327706  PMID: 22523565
23.  GDNF Secreting Human Neural Progenitor Cells Protect Dying Motor Neurons, but Not Their Projection to Muscle, in a Rat Model of Familial ALS 
PLoS ONE  2007;2(8):e689.
Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease characterized by rapid loss of muscle control and eventual paralysis due to the death of large motor neurons in the brain and spinal cord. Growth factors such as glial cell line derived neurotrophic factor (GDNF) are known to protect motor neurons from damage in a range of models. However, penetrance through the blood brain barrier and delivery to the spinal cord remains a serious challenge. Although there may be a primary dysfunction in the motor neuron itself, there is also increasing evidence that excitotoxicity due to glial dysfunction plays a crucial role in disease progression. Clearly it would be of great interest if wild type glial cells could ameliorate motor neuron loss in these models, perhaps in combination with the release of growth factors such as GDNF.
Methodology/Principal Findings
Human neural progenitor cells can be expanded in culture for long periods and survive transplantation into the adult rodent central nervous system, in some cases making large numbers of GFAP positive astrocytes. They can also be genetically modified to release GDNF (hNPCGDNF) and thus act as long-term ‘mini pumps’ in specific regions of the rodent and primate brain. In the current study we genetically modified human neural stem cells to release GDNF and transplanted them into the spinal cord of rats over-expressing mutant SOD1 (SOD1G93A). Following unilateral transplantation into the spinal cord of SOD1G93A rats there was robust cellular migration into degenerating areas, efficient delivery of GDNF and remarkable preservation of motor neurons at early and end stages of the disease within chimeric regions. The progenitors retained immature markers, and those not secreting GDNF had no effect on motor neuron survival. Interestingly, this robust motor neuron survival was not accompanied by continued innervation of muscle end plates and thus resulted in no improvement in ipsilateral limb use.
The potential to maintain dying motor neurons by delivering GDNF using neural progenitor cells represents a novel and powerful treatment strategy for ALS. While this approach represents a unique way to prevent motor neuron loss, our data also suggest that additional strategies may also be required for maintenance of neuromuscular connections and full functional recovery. However, simply maintaining motor neurons in patients would be the first step of a therapeutic advance for this devastating and incurable disease, while future strategies focus on the maintenance of the neuromuscular junction.
PMCID: PMC1925150  PMID: 17668067
24.  Death receptor 6 (DR6) antagonist antibody is neuroprotective in the mouse SOD1G93A model of amyotrophic lateral sclerosis 
Cell Death & Disease  2013;4(10):e841-.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the death of motor neurons, axon degeneration, and denervation of neuromuscular junctions (NMJ). Here we show that death receptor 6 (DR6) levels are elevated in spinal cords from post-mortem samples of human ALS and from SOD1G93A transgenic mice, and DR6 promotes motor neuron death through activation of the caspase 3 signaling pathway. Blocking DR6 with antagonist antibody 5D10 promotes motor neuron survival in vitro via activation of Akt phosphorylation and inhibition of the caspase 3 signaling pathway, after growth factor withdrawal, sodium arsenite treatment or co-culture with SOD1G93A astrocytes. Treatment of SOD1G93A mice at an asymptomatic stage starting on the age of 42 days with 5D10 protects NMJ from denervation, decreases gliosis, increases survival of motor neurons and CC1+ oligodendrocytes in spinal cord, decreases phosphorylated neurofilament heavy chain (pNfH) levels in serum, and promotes motor functional improvement assessed by increased grip strength. The combined data provide clear evidence for neuroprotective effects of 5D10. Blocking DR6 function represents a new approach for the treatment of neurodegenerative disorders involving motor neuron death and axon degeneration, such as ALS.
PMCID: PMC3824687  PMID: 24113175
ALS; DR6; motor neuron; neurofilament; neurodegeneration; NMJ
25.  Lack of a synergistic effect of a non-viral ALS gene therapy based on BDNF and a TTC fusion molecule 
Amyotrophic lateral sclerosis (ALS) is one of the most devastating neurodegenerative diseases. Neurotrophic factors have been widely tested to counteract neurodegenerative conditions, despite their unspecific neuronal access. The non-toxic C-terminal fragment of the tetanus toxin (TTC) heavy chain has been studied not only as a carrier molecule to the CNS but also as a neuroprotective agent. Because the neurotrophic effects of BDNF have been demonstrated in vitro and in vivo, the question addressed in this work is whether a fusion molecule of BDNF-TTC may have a synergistic effect and enhance the neuroprotective properties of TTC alone in a mouse model of ALS.
Recombinant plasmid constructs (pCMV-TTC and pCMV-BDNF-TTC) were injected into the quadriceps femoris and triceps brachialis muscles of SOD1G93A transgenic mice at 8 weeks of age. The hanging wire and rotarod tests were performed to assess motor coordination, strength and balance. Electrophysiological tests, morphological assays of spinal cord sections of L2 and L4 segments, and gene and protein expression analyses were performed. The Kaplan-Meier survival analysis test was used for comparisons of survival. Multiple comparisons of data were analyzed using a one-way analysis of variance (ANOVA).
Treatment with the fusion-molecule BDNF-TTC and with TTC alone significantly delayed the onset of symptoms and functional deficits of SOD1G93A mice. Muscle innervation was partially preserved with these treatments, and the number of surviving motoneurons in L2 spinal cord segment was increased particularly by the fusion protein induction. Inhibition of pro-apoptotic protein targets (caspase-3 and Bax) and significant phosphorylation of Akt and ERK were also found in the spinal cord of treated mice.
Significant improvements in behavioral and electrophysiological results, motoneuron survival and anti-apoptotic/survival-activated pathways were observed with BDNF-TTC treatment. However, no synergistic effect was found for this fusion molecule. Although BDNF in the fusion molecule is capable of activating autocrine and neuroprotective pathways, TTC treatment alone yielded similar neuroprotection. Therefore, an accurate study of the neuroprotective effects of TTC fusion molecules should be performed to obtain a better understanding of its effects.
PMCID: PMC3072305  PMID: 21418619

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