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1.  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.
doi:10.1083/jcb.200407021
PMCID: PMC2171577  PMID: 15657392
2.  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.
doi:10.1371/journal.pone.0088833
PMCID: PMC3925164  PMID: 24551172
3.  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.
doi:10.1080/17482960802160370
PMCID: PMC3211070  PMID: 18608100
Amyotrophic lateral sclerosis; insulin-like growth factor-I; treatment
4.  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.
doi:10.1371/journal.pone.0022374
PMCID: PMC3149057  PMID: 21829620
5.  Motor Neuronal Protection by L-Arginine Prolongs Survival of Mutant Sod1 (G93A) Als Mice 
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive paralysis due to motor neuron degeneration. Despite the fact that many different therapeutic strategies have been applied to prevent disease progression, no cure or effective therapy is currently available for ALS. We found that L-arginine protects cultured motor neurons from excitotoxic injury. We also found that L-arginine supplementation both prior to and after the onset of motor neuron degeneration in mtSOD1 (G93A) transgenic ALS mice significantly slowed the progression of neuropathology in lumbar spinal cord, delayed onset of motor dysfunction, and prolonged lifespan. Moreover, L-arginine treatment was associated with preservation of arginase I activity and neuroprotective polyamines in spinal cord motor neurons. Our findings show that L-arginine has potent in vitro and in vivo neuroprotective properties and may be a candidate for therapeutic trials in ALS.
doi:10.1016/j.bbrc.2009.05.015
PMCID: PMC2744197  PMID: 19427829
amyotrophic lateral sclerosis (ALS); L-arginine; motor neuron; polyamines; superoxide dismutase 1 (SOD1)
6.  Intravascular AAV9 preferentially targets neonatal-neurons and adult-astrocytes in CNS 
Nature biotechnology  2008;27(1):59-65.
Delivery of therapeutics to the brain and spinal cord remains a challenge for neurodegenerative diseases, such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). The blood-brain-barrier (BBB) hampers delivery of therapeutics to neurons, glia, and surrounding cell types of the central nervous system (CNS) that may be involved in disease progression. Here, we describe an intravenous injection of adeno-associated-virus-9 (AAV-9) in mouse that efficiently targets brain, dorsal root ganglia and spinal cord motor neurons in neonatal animals and astrocytes in adult mouse brain and spinal cords, offering a new therapeutic delivery approach to deliver genes to widespread regions within the CNS.
doi:10.1038/nbt.1515
PMCID: PMC2895694  PMID: 19098898
7.  Acute glial activation by stab injuries does not lead to overt damage or motor neuron degeneration in the G93A mutant SOD1 rat model of Amyotrophic Lateral Sclerosis 
Experimental neurology  2009;221(2):346-352.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease where motor neurons within the brain and spinal cord are lost, leading to paralysis and death. Recently, a correlation has been reported between head trauma and the incidence of ALS. Furthermore, new invasive neurosurgical studies are being planned which involve inserting needles directly to the spinal cord. We therefore tested whether acute trauma to the spinal cord via a knife wound injury would lead to accelerated disease progression in rodent models of ALS (SOD1G93A rats). A longitudinal stab injury using a small knife was performed within the lumbar spinal cord region of presymptomatic SOD1G93A rats. Host glial activation was detected in the lumbar area surrounding a micro-knife lesion at 2 weeks post-surgery in both wild type and SOD1G93A animals. However, there was no sign of motor neuron loss in the injured spinal cord of any animal and normal motor function was maintained in the ipsilateral limb. These results indicate that motor neurons in pre-symptomatic G93A animals are not affected by an invasive puncture wound injury involving reactive astrocytes. Furthermore, acute trauma alone does not accelerate disease onset or progression in this ALS model which is important for future strategies of gene and cell therapies directly targeting the spinal cord of ALS patients.
doi:10.1016/j.expneurol.2009.12.004
PMCID: PMC2839070  PMID: 20005223
Amyotrophic lateral sclerosis (ALS); SOD1G93A rats; acute trauma; stab injury; astrocyte activation; motor neuron; spinal cord
8.  Adenylyl Cyclase type 3, a marker of primary cilia, is reduced in primary cell culture and in lumbar spinal cord in situ in G93A SOD1 mice 
BMC Neuroscience  2011;12:71.
Background
The primary cilium is a solitary organelle important in cellular signaling, that projects from the cell surface of most growth-arrested or post-mitotic cells including neurons in the central nervous system. We hypothesized that primary cilial dysfunction might play a role in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS), and as a first step, report on the prevalence of primary cilial markers on cultured motor neurons from the lumbar spinal cord of embryonic wildtype (WT) and transgenic G93A SOD1 mice, and on motor neurons in situ in the lumbar spinal cord.
Results
At 7 days in culture there is no difference in the proportion of G93A SOD1 and WT motor neurons staining for the cilial marker ACIII. However, at 21 days there is a large relative drop in the proportion of ciliated G93A SOD1 motor neurons. In situ, at 40 days there was a slight relative drop in the proportion of ciliated motor neurons in G93A SOD1 mice. At 98 days of age there was no change in motor neuron ciliation in WT mice, but there was motor neuron loss and a large reduction in the proportion of surviving motor neurons bearing a primary cilium in G93A SOD1 mice.
Conclusions
In primary culture and in situ in G93A SOD1 mice there is a large reduction in the proportion of motor neurons bearing a primary cilium.
doi:10.1186/1471-2202-12-71
PMCID: PMC3199874  PMID: 21767396
9.  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.
doi:10.1038/mt.2008.60
PMCID: PMC2737251  PMID: 18388910
10.  Overexpression of survival motor neuron improves neuromuscular function and motor neuron survival in mutant SOD1 mice 
Neurobiology of Aging  2014;35(4):906-915.
Spinal muscular atrophy results from diminished levels of survival motor neuron (SMN) protein in spinal motor neurons. Low levels of SMN also occur in models of amyotrophic lateral sclerosis (ALS) caused by mutant superoxide dismutase 1 (SOD1) and genetic reduction of SMN levels exacerbates the phenotype of transgenic SOD1G93A mice. Here, we demonstrate that SMN protein is significantly reduced in the spinal cords of patients with sporadic ALS. To test the potential of SMN as a modifier of ALS, we overexpressed SMN in 2 different strains of SOD1G93A mice. Neuronal overexpression of SMN significantly preserved locomotor function, rescued motor neurons, and attenuated astrogliosis in spinal cords of SOD1G93A mice. Despite this, survival was not prolonged, most likely resulting from SMN mislocalization and depletion of gems in motor neurons of symptomatic mice. Our results reveal that SMN upregulation slows locomotor deficit onset and motor neuron loss in this mouse model of ALS. However, disruption of SMN nuclear complexes by high levels of mutant SOD1, even in the presence of SMN overexpression, might limit its survival promoting effects in this specific mouse model. Studies in emerging mouse models of ALS are therefore warranted to further explore the potential of SMN as a modifier of ALS.
doi:10.1016/j.neurobiolaging.2013.09.030
PMCID: PMC3919158  PMID: 24210254
Amyotrophic lateral sclerosis; Spinal muscular atrophy; Superoxide dismutase 1; Survival motor neuron
11.  Human Neural Stem Cell Replacement Therapy for Amyotrophic Lateral Sclerosis by Spinal Transplantation 
PLoS ONE  2012;7(8):e42614.
Background
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.
Conclusions/Significance
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.
doi:10.1371/journal.pone.0042614
PMCID: PMC3423406  PMID: 22916141
12.  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.
doi:10.1016/j.expneurol.2007.05.016
PMCID: PMC2043146  PMID: 17597610
Amyotrophic Lateral Sclerosis; Insulin-like growth factor-1; motor neuron; cell death; skeletal muscle; transgenic; G93A SOD-1; motor performance
13.  More than a bystander: the contributions of intrinsic skeletal muscle defects in motor neuron diseases 
Spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), and spinal-bulbar muscular atrophy (SBMA) are devastating diseases characterized by the degeneration of motor neurons. Although the molecular causes underlying these diseases differ, recent findings have highlighted the contribution of intrinsic skeletal muscle defects in motor neuron diseases. The use of cell culture and animal models has led to the important finding that muscle defects occur prior to and independently of motor neuron degeneration in motor neuron diseases. In SMA for instance, the muscle specific requirements of the SMA disease-causing gene have been demonstrated by a series of genetic rescue experiments in SMA models. Conditional ALS mouse models expressing a muscle specific mutant SOD1 gene develop atrophy and muscle degeneration in the absence of motor neuron pathology. Treating SBMA mice by over-expressing IGF-1 in a skeletal muscle-specific manner attenuates disease severity and improves motor neuron pathology. In the present review, we provide an in depth description of muscle intrinsic defects, and discuss how they impact muscle function in these diseases. Furthermore, we discuss muscle-specific therapeutic strategies used to treat animal models of SMA, ALS, and SBMA. The study of intrinsic skeletal muscle defects is crucial for the understanding of the pathophysiology of these diseases and will open new therapeutic options for the treatment of motor neuron diseases.
doi:10.3389/fphys.2013.00356
PMCID: PMC3866803  PMID: 24391590
mouse models; neuromuscular disease; myofiber degeneration; fusion defect; insulin-like growth factor 1
14.  Intracisternal delivery of AAV9 results in oligodendrocyte and motor neuron transduction in the whole central nervous system of cats 
Gene Therapy  2014;21(5):522-528.
Systemic and intracerebrospinal fluid delivery of adeno-associated virus serotype 9 (AAV9) has been shown to achieve widespread gene delivery to the central nervous system (CNS). However, after systemic injection, the neurotropism of the vector has been reported to vary according to age at injection, with greater neuronal transduction in newborns and preferential glial cell tropism in adults. This difference has not yet been reported after cerebrospinal fluid (CSF) delivery. The present study analyzed both neuronal and glial cell transduction in the CNS of cats according to age of AAV9 CSF injection. In both newborns and young cats, administration of AAV9-GFP in the cisterna magna resulted in high levels of motor neurons (MNs) transduction from the cervical (84±5%) to the lumbar (99±1%) spinal cord, demonstrating that the remarkable tropism of AAV9 for MNs is not affected by age at CSF delivery. Surprisingly, numerous oligodendrocytes were also transduced in the brain and in the spinal cord white matter of young cats, but not of neonates, indicating that (i) age of CSF delivery influences the tropism of AAV9 for glial cells and (ii) AAV9 intracisternal delivery could be relevant for both the treatment of MN and demyelinating disorders.
doi:10.1038/gt.2014.16
PMCID: PMC4015314  PMID: 24572783
15.  Virus-Delivered Small RNA Silencing Sustains Strength in Amyotrophic Lateral Sclerosis 
Annals of neurology  2005;57(5):773-776.
Mutations in superoxide dismutase cause a subset of familial amyotrophic lateral sclerosis and provoke progressive paralysis when expressed in mice. After retrograde transport to the spinal cord following injection into muscles, an adeno-associated virus carrying a gene that encodes a small interfering RNA was shown to target superoxide dismutase messenger RNA for degradation. The corresponding decrease in mutant superoxide dismutase in spinal motor neurons preserved grip strength. This finding provides proof of principle for the selective reduction of any neuronal protein and supports intramuscular injections of a small interfering RNA–encoding virus as a viable therapy for this type of familial amyotrophic lateral sclerosis.
doi:10.1002/ana.20453
PMCID: PMC1351126  PMID: 15852369
16.  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.
doi:10.1371/journal.pone.0023968
PMCID: PMC3161091  PMID: 21887354
17.  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.
doi:10.1038/mt.2013.239
PMCID: PMC3944333  PMID: 24394188
18.  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.
doi:10.3858/emm.2009.41.7.054
PMCID: PMC2721146  PMID: 19322031
amyotrophic lateral sclerosis; cell differentiation; glial cell line-derived neurotrophic factor; nerve growth factors; stem cell transplantation; stem cells
19.  Differential effects of brain-derived neurotrophic factor and neurotrophin-3 on hindlimb function in paraplegic rats 
We compared the effect of viral administration of brain-derived neurotrophic factor (BDNF) or neurotrophin 3 (NT-3) on locomotor recovery in adult rats with complete thoracic (T10) spinal cord transection injuries, in order to determine the effect of chronic neurotrophin expression on spinal plasticity. At the time of injury, BDNF, NT-3 or green fluorescent protein (GFP) (control) was delivered to the lesion via adeno-associated virus (AAV) constructs. AAV–BDNF was significantly more effective than AAV–NT-3 in eliciting locomotion. In fact, AAV–BDNF-treated rats displayed plantar, weight-supported hindlimb stepping on a stationary platform, that is, without the assistance of a moving treadmill and without step training. Rats receiving AAV–NT-3 or AAV–GFP were incapable of hindlimb stepping during this task, despite provision of balance support. AAV–NT-3 treatment did promote the recovery of treadmill-assisted stepping, but this required continuous perineal stimulation. In addition, AAV–BDNF-treated rats were sensitized to noxious heat, whereas AAV–NT-3-treated and AAV–GFP-treated rats were not. Notably, AAV–BDNF-treated rats also developed hindlimb spasticity, detracting from its potential clinical applicability via the current viral delivery method. Intracellular recording from triceps surae motoneurons revealed that AAV–BDNF significantly reduced motoneuron rheobase, suggesting that AAV–BDNF promoted the recovery of over-ground stepping by enhancing neuronal excitability. Elevated nuclear c-Fos expression in interneurons located in the L2 intermediate zone after AAV–BDNF treatment indicated increased activation of interneurons in the vicinity of the locomotor central pattern generator. AAV–NT-3 treatment reduced motoneuron excitability, with little change in c-Fos expression. These results support the potential for BDNF delivery at the lesion site to reorganize locomotor circuits.
doi:10.1111/j.1460-9568.2011.07950.x
PMCID: PMC3509221  PMID: 22211901
c-Fos; locomotion; neurotrophin; plasticity; rheobase; spinal cord injury
20.  Progressive changes in microglia and macrophages in spinal cord and peripheral nerve in the transgenic rat model of amyotrophic lateral sclerosis 
Background
The role of neuroinflammation in motor neuron death of amyotrophic lateral sclerosis (ALS) is unclear. The human mutant superoxide dismutase-1 (hmSOD1)-expressing murine transgenic model of ALS has provided some insight into changes in microglia activity during disease progression. The purpose of this study was to gain further knowledge by characterizing the immunological changes during disease progression in the spinal cord and peripheral nerve using the more recently developed hmSOD1 rat transgenic model of ALS.
Methods
Using immunohistochemistry, the extent and intensity of tissue CD11b expression in spinal cord, lumbar nerve roots, and sciatic nerve were evaluated in hmSOD1 rats that were pre-clinical, at clinical onset, and near disease end-stage. Changes in CD11b expression were compared to the detection of MHC class II and CD68 microglial activation markers in the ventral horn of the spinal cord, as well as to the changes in astrocytic GFAP expression.
Results
Our study reveals an accumulation of microglia/macrophages both in the spinal cord and peripheral nerve prior to clinical onset based on CD11b tissue expression. The microglia formed focal aggregates in the ventral horn and became more widespread as the disease progressed. Hypertrophic astrocytes were not prominent in the ventral horn until after clinical onset, and the enhancement of GFAP did not have a strong correlation to increased CD11b expression. Detection of MHC class II and CD68 expression was found in the ventral horn only after clinical onset. The macrophages in the ventral nerve root and sciatic nerve of hmSOD1 rats were observed encircling axons.
Conclusions
These findings describe for the first time in the hmSOD1 rat transgenic model of ALS that enhancement of microglia/macrophage activity occurs pre-clinically both in the peripheral nerve and in the spinal cord. CD11b expression is shown to be a superior indicator for early immunological changes compared to other microglia activation markers and astrogliosis. Furthermore, we suggest that the early activity of microglia/macrophages is involved in the early phase of motor neuron degeneration and propose that studies involving immunomodulation in hmSOD1transgenic models need to consider effects on macrophages in peripheral nerves as well as to microglia in the spinal cord.
doi:10.1186/1742-2094-7-8
PMCID: PMC2825214  PMID: 20109233
21.  The advent of AAV9 expands applications for brain and spinal cord gene delivery 
Introduction
Straightforward studies compared adeno-associated virus (AAV) serotypes to determine the most appropriate one for robust expression in the CNS. AAV9 was efficient when directly injected into the brain, but more surprisingly, AAV9 produced global expression in the brain and spinal cord after a peripheral, systemic route of administration to neonatal mice.
Areas covered
Topics include AAV9 gene delivery from intraparenchymal, intravenous, intrathecal and intrauterine routes of administration, and related preclinical studies and disease models. Systemic AAV9 gene transfer yields remarkably consistent neuronal expression, though only in early development. AAV9 is versatile to study neuropathological proteins: microtubule-associated protein tau and transactive response DNA-binding protein 43 kDa (TDP-43).
Expert opinion
AAV9 will be more widely used based on current data, although other natural serotypes and recombineered vectors may also support or improve upon wide-scale expression. A peripheral-to-central gene delivery that can affect the entire CNS without having to inject the CNS is promising for basic functional experiments, and potentially for gene therapy. Systemic or intra-cerebrospinal fluid routes of AAV9 administration should be considered for spinal muscular atrophy, lysosomal storage diseases and amyotrophic lateral sclerosis, if more neuronal expression can be achieved in adults, or if glial expression can be exploited.
doi:10.1517/14712598.2012.681463
PMCID: PMC3361729  PMID: 22519910
adeno-associated virus; amyotrophic lateral sclerosis; frontotemporal lobar degeneration; gene therapy; gene transfer; lysosomal storage disease; microtubule-associated protein tau; spinal cord; spinal muscular atrophy; TDP-43
22.  Focal Transplantation-based Astrocyte Replacement is Neuroprotective in a Model of Motor Neuron Disease 
Nature neuroscience  2008;11(11):1294-1301.
Cellular abnormalities in amyotrophic lateral sclerosis (ALS) are not limited to motor neurons. Astrocyte dysfunction occurs in human ALS and SOD1G93A animal models. Therefore, the value of focal enrichment of normal astrocytes was investigated using transplantation of lineage-restricted astrocyte precursors, Glial-Restricted Precursors (GRPs). GRPs were transplanted around cervical spinal cord respiratory motor neuron pools, the principal cells responsible for death in this neurodegenerative disease. GRPs survived in diseased tissue, differentiated efficiently into astrocytes, and reduced microgliosis in SOD1G93A rat cervical spinal cord. GRPs extended survival and disease duration, attenuated motor neuron loss, and slowed declines in fore-limb motor and respiratory physiological function. Neuroprotection was mediated in part by the primary astrocyte glutamate transporter, GLT1. These findings demonstrate the feasibility and efficacy of transplantation-based astrocyte replacement, and show that targeted multi-segmental cell delivery to cervical spinal cord is a promising therapeutic strategy for slowing focal motor neuron loss associated with ALS.
doi:10.1038/nn.2210
PMCID: PMC2656686  PMID: 18931666
stem cell; grafting; transplantation; motor neuron; neurodegeneration; replacement; neuroprotection; non-cell autonomous; astroglia; astrocyte; neural precursor cell; progenitor; lineage-restricted precursor; glial precursor; ALS; amyotrophic lateral sclerosis; SOD1
23.  Early gene expression changes in spinal cord from SOD1G93A Amyotrophic Lateral Sclerosis animal model 
Amyotrophic Lateral Sclerosis (ALS) is an adult-onset and fast progression neurodegenerative disease that leads to the loss of motor neurons. Mechanisms of selective motor neuron loss in ALS are unknown. The early events occurring in the spinal cord that may contribute to motor neuron death are not described, neither astrocytes participation in the pre-symptomatic phases of the disease. In order to identify ALS early events, we performed a microarray analysis employing a whole mouse genome platform to evaluate the gene expression pattern of lumbar spinal cords of transgenic SOD1G93A mice and their littermate controls at pre-symptomatic ages of 40 and 80 days. Differentially expressed genes were identified by means of the Bioconductor packages Agi4×44Preprocess and limma. FunNet web based tool was used for analysis of over-represented pathways. Furthermore, immunolabeled astrocytes from 40 and 80 days old mice were submitted to laser microdissection and RNA was extracted for evaluation of a selected gene by qPCR. Statistical analysis has pointed to 492 differentially expressed genes (155 up and 337 down regulated) in 40 days and 1105 (433 up and 672 down) in 80 days old ALS mice. KEGG analysis demonstrated the over-represented pathways tight junction, antigen processing and presentation, oxidative phosphorylation, endocytosis, chemokine signaling pathway, ubiquitin mediated proteolysis and glutamatergic synapse at both pre-symptomatic ages. Ube2i gene expression was evaluated in astrocytes from both transgenic ages, being up regulated in 40 and 80 days astrocytes enriched samples. Our data points to important early molecular events occurring in pre-symptomatic phases of ALS in mouse model. Early SUMOylation process linked to astrocytes might account to non-autonomous cell toxicity in ALS. Further studies on the signaling pathways presented here may provide new insights to better understand the events triggering motor neuron death in this devastating disorder.
doi:10.3389/fncel.2013.00216
PMCID: PMC3831149  PMID: 24302897
ALS; SOD1G93A; pre-symptomatic; spinal cord; microarray; laser microdissection; astrocytes
24.  Motor Neuron Degeneration Promotes Neural Progenitor Cell Proliferation, Migration and Neurogenesis in the Spinal Cords of ALS Mice 
Stem cells (Dayton, Ohio)  2005;24(1):34-43.
The organization, distribution, and function of neural progenitor cells (NPCs) in the adult spinal cord during motor neuron degeneration in amyotrophic lateral sclerosis (ALS) remain largely unknown. Using nestin promoter controlled LacZ reporter transgenic mice and mutant G93A-SOD1 transgenic mice mimicking ALS, we showed that there was an increase of NPC proliferation, migration, and neurogenesis in the lumbar region of adult spinal cord in response to motor neuron degeneration. The proliferation of NPCs detected by BrdU incorporation and LacZ staining was restricted to the ependymal zone surrounding central canal (EZ). Once the NPCs moved out from the EZ, they lost the proliferative capability, but maintained migratory function vigorously. During ALS-like disease onset and progression, NPCs in the EZ migrated initially toward the dorsal horn direction, and then to the ventral horn regions, where motor neurons have degenerated. More significantly, there was an increased de novo neurogenesis from NPCs during ALS-like disease onset and progression. The enhanced proliferation, migration, and neurogenesis of (from) NPCs in the adult spinal cord of ALS-like mice may play an important role in attempting to repair the degenerated motor neurons and restore the dysfunctional circuitry which resulted from the pathogenesis of mutant SOD1 in ALS.
doi:10.1634/stemcells.2005-0076
PMCID: PMC1828038  PMID: 16099995
Neural progenitor cells; radial glia; glial progenitor cells; motor neurons; nestin; mutant SOD1; ALS
25.  Differential expression of c-Ret in motor neurons versus non-neuronal cells is linked to the pathogenesis of ALS 
Summary
Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by selective degeneration of motor neurons throughout the central nervous systems. Non-cell autonomous damage induced by glial cells is linked to the selective susceptibility of motor neurons in ALS but the mechanisms underlying this phenomenon are not known. We found the expression of non-phosphorylated and phosphorylated forms (tyrosine residue 905, 1016, and 1062) of c-Ret, a member of the glial cell line-derived neurotrophic factor (GDNF) receptor, are altered in motor neurons of the lumbar spinal cord in ALS transgenic (G93A) mice and ALS (G93A) cell line models. Phosphorylated forms of c-Ret were colocalized with neurofilament aggregates in motor neurons of ALS mice. Consistent with the in vivo data, levels of non-phosphorylated and phosphorylated c-Ret (Tyr 905, 1016, and 1062) were decreased by oxidative stress in motor neuronal cells (NSC-34). Non-phosphorylated and phosphorylated forms of c-Ret immunoreactivity were markedly elevated in active microglia of ALS mice. Our findings suggest that constitutive oxidative stress modulates c-Ret function, thereby reducing GDNF signaling in motor neurons. Furthermore, the induction of c-Ret expression in microglia may contribute to non-cell autonomous cell death of motor neurons by depriving available GDNF in ALS.
doi:10.1038/labinvest.2010.203
PMCID: PMC3085919  PMID: 21283077
Amyotrophic lateral sclerosis; c-Ret; glial cell line-derived neurotrophic factor; motor neuron; microglia; astrocyte

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