Combined intramuscular and intraspinal transplant of bone marrow cells improves neuromuscular function in the SOD1G93A mice.

BACKGROUND: The simultaneous contribution of several etiopathogenic disturbances makes amyotrophic lateral sclerosis (ALS) a fatal and challenging disease. Here, we studied two different cell therapy protocols to protect both central and peripheral nervous system in a murine model of ALS. METHODS: Since ALS begins with a distal axonopathy, in a first assay, we performed injection of bone marrow cells into two hindlimb muscles of transgenic SOD1G93A mice. In a second study, we combined intramuscular and intraspinal injection of bone marrow cells. Fluorescence-activated cell sorting was used to assess the survival of the transplanted cells into the injected tissues. The mice were assessed from 8 to 16 weeks of age by means of locomotion and electrophysiological tests. After follow-up, the spinal cord was processed for analysis of motoneuron survival and glial cell reactivity. RESULTS: We found that, after intramuscular injection, bone marrow cells were able to engraft within the muscle. However, bone marrow cell intramuscular injection failed to promote a general therapeutic effect. In the second approach, we found that bone marrow cells had limited survival in the spinal cord, but this strategy significantly improved motor outcomes. Moreover, we also found that the dual cell therapy tended to preserve spinal motoneurons at late stages of the disease and to reduce microgliosis, although this did not prolong mice survival. CONCLUSION: Overall, our findings suggest that targeting more than one affected area of the motor system at once with bone marrow cell therapy results in a valuable therapeutic intervention for ALS.

Chemotherapeutic agent 5-fluorouracil increases survival of SOD1 mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a lethal motor neuron disease with no cure. Currently there are only two ALS drugs approved by the FDA, both with a limited therapeutic effect. In the search for drug candidates for ALS, we studied the effect of known stem cell mobilizing agents (treatment) and antimetabolite 5-fluorouracil (5-FU) (anti-treatment) in SOD1G93A model of ALS. Surprisingly, we found that anti-cancer drug 5-FU increases lifespan, delays the disease onset and improves motor performance in ALS mice. Although we were not able to demonstrate the mechanistic basis of the beneficial 5-FU action in ALS mice, our findings suggest that 5-FU or similar drugs are possible drug candidates for the treatment of motor neuron diseases through drug repurposing.

Intramuscular transplantation of bone marrow cells prolongs the lifespan of SOD1G93A mice and modulates expression of prognosis biomarkers of the disease.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive muscle weakness, paralysis and death. There is no effective treatment for ALS and stem cell therapy has arisen as a potential therapeutic approach. METHODS: SOD1 mutant mice were used to study the potential neurotrophic effect of bone marrow cells grafted into quadriceps femoris muscle. RESULTS: Bone marrow intramuscular transplants resulted in increased longevity with improved motor function and decreased motoneuron degeneration in the spinal cord. Moreover, the increment of the glial-derived neurotrophic factor and neurotrophin 4 observed in the grafted muscles suggests that this partial neuroprotective effect is mediated by neurotrophic factor release at the neuromuscular junction level. Finally, certain neurodegeneration and muscle disease-specific markers, which are altered in the SOD1G93A mutant mouse and may serve as molecular biomarkers for the early detection of ALS in patients, have been studied with encouraging results. CONCLUSIONS: This work demonstrates that stem cell transplantation in the muscle prolonged the lifespan, increased motoneuron survival and slowed disease progression, which was also assessed by genetic expression analysis.

Comparative study of hematopoietic stem and progenitor cells between sexes in mice under physiological conditions along time.

Hematopoietic stem and progenitor cells (HSPCs) are attractive targets in regenerative medicine, although the differences in their homeostatic maintenance between sexes along time are still under debate. We accurately monitored hematopoietic stem cells (HSCs), common lymphoid progenitors (CLPs), and common myeloid progenitors (CMPs) frequencies by flow cytometry, by performing serial peripheral blood extractions from male and female B6SJL wild-type mice and found no significant differences. Only modest differences were found in the gene expression profile of Slamf1 and Gata2. Our findings suggest that both sexes could be used indistinctly to perform descriptive studies in the murine hematopoietic system, especially for flow cytometry studies in peripheral blood. This would allow diminishing the number of animals needed for the experimental procedures. In addition, the use of serial extractions in the same animals drastically decreases the number of animals needed.

Granulocyte Colony-Stimulating Factor Ameliorates Skeletal Muscle Dysfunction in Amyotrophic Lateral Sclerosis Mice and Improves Proliferation of SOD1-G93A Myoblasts in vitro.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) causes loss of upper and lower motor neurons as well as skeletal muscle (SKM) dysfunction and atrophy. SKM is one of the tissues involved in the development of ALS pathology, and studies in a SOD1-G93A mouse model of ALS have demonstrated alterations in SKM degeneration/regeneration marker expression in vivo and defective mutant myoblast proliferation in vitro. Granulocyte colony-stimulating factor (G-CSF) has been shown to alleviate SOD1-G93A pathology. However, it is unknown whether G-CSF may have a direct effect on SKM or derived myoblasts. OBJECTIVE: To investigate effects of G-CSF and its analog pegfilgrastim (PEGF) on SOD1-G93A- associated SKM markers in vivo and those of G-CSF on myoblast proliferation in vitro. METHODS: The effect of PEGF treatment on hematopoietic stem cell mobilization, survival, and motor function was determined. RNA expression of SKM markers associated with mutant SOD1 expression was quantified in response to PEGF treatment in vivo, and the effect of G-CSF on the proliferation of myoblasts derived from mutant and control muscles was determined in vitro. RESULTS: Positive effects of PEGF on hematopoietic stem cell mobilization, survival, and functional assays in SOD1-G93A animals were confirmed. In vivo PEGF treatment augmented the expression of its receptor Csf3r and alleviated typical markers for mutant SOD1 muscle. Additionally, G-CSF was found to directly increase the proliferation of SOD1-G93A, but not wild-type primary myoblasts in vitro. CONCLUSION: Our results support the beneficial role of the G-CSF analog PEGF in a SOD1-G93A model of ALS. Thus, G-CSF and its analogs may be directly beneficial in diseases where the SKM function is compromised.

Hematopoietic stem and progenitor cells as novel prognostic biomarkers of longevity in a murine model for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a difficult diagnosis and prognosis. In this regard, new and more reliable biomarkers for the disease are needed. We propose peripheral blood, and, more specifically, the hematopoietic stem and progenitor cells (HSPCs) as potential prognostic biomarkers in the SOD1G93A murine model of ALS. We accurately and serially studied three HSPCs-hematopoietic stem cells (HSCs), common lymphoid progenitors (CLPs), and common myeloid progenitors (CMPs)-in both control and SOD1G93A mice along the disease's progression by RT-PCR and flow cytometry analysis. We found interesting differences for every HSPC type in the transgenic mice compared with the control mice at every time point selected, as well as differences along the disease course. The results showed a maintained compensatory increase of HSCs along disease progression. However, the downregulated levels of CLPs and CMPs suggested an exit of these cell populations to the peripheral tissues, probably due to their supporting role to the damaged tissues. In addition, a positive correlation of the percentage of CLPs and CMPs with the longevity was found, as well as a positive correlation of HSCs and CMPs with motor function and weight, thus reinforcing the idea that HSPCs play a relevant role in the longevity of the SOD1G93A mice. On the basis of these results, both CLPs and CMPs could be considered prognostic biomarkers of longevity in this animal model, opening the door to future studies in human patients for their potential clinical use.

Neuroprotective Effect of Non-viral Gene Therapy Treatment Based on Tetanus Toxin C-fragment in a Severe Mouse Model of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is a hereditary childhood disease that causes paralysis and progressive degeneration of skeletal muscles and spinal motor neurons. SMA is associated with reduced levels of full-length Survival of Motor Neuron (SMN) protein, due to mutations in the Survival of Motor Neuron 1 gene. Nowadays there are no effective therapies available to treat patients with SMA, so our aim was to test whether the non-toxic carboxy-terminal fragment of tetanus toxin heavy chain (TTC), which exhibits neurotrophic properties, might have a therapeutic role or benefit in SMA. In this manuscript, we have demonstrated that TTC enhance the SMN expression in motor neurons "in vitro" and evaluated the effect of intramuscular injection of TTC-encoding plasmid in the spinal cord and the skeletal muscle of SMNdelta7 mice. For this purpose, we studied the weight and the survival time, as well as, the survival and cell death pathways and muscular atrophy. Our results showed that TTC treatment reduced the expression of autophagy markers (Becn1, Atg5, Lc3, and p62) and pro-apoptotic genes such as Bax and Casp3 in spinal cord. In skeletal muscle, TTC was able to downregulate the expression of the main marker of autophagy, Lc3, to wild-type levels and the expression of the apoptosis effector protein, Casp3. Regarding the genes related to muscular atrophy (Ankrd1, Calm1, Col19a1, Fbox32, Mt2, Myod1, NogoA, Pax7, Rrad, and Sln), TTC suggest a compensatory effect for muscle damage response, diminished oxidative stress and modulated calcium homeostasis. These preliminary findings suggest the need for further experiments to depth study the effect of TTC in SMA disease.

Comparative study of behavioural tests in the SOD1G93A mouse model of amyotrophic lateral sclerosis.

In preclinical trials, a sensitive functional test is required to detect changes in the motor behaviour of the SOD1G93A mouse model of amyotrophic lateral sclerosis (ALS). We evaluated changes in body weight and motor impairment in behavioural tests, such as the rotarod, the hanging-wire test and the treadmill, of transgenic and wild type mice. We found differences in detection of the onset of symptoms and progression of the disease between the different tests assessed. Moreover, the data showed significant gender differences in the motor behaviour of this mouse model. The rotarod and the hanging-wire test were more sensitive to detect early motor impairment. Moreover, the results suggested that the rotarod and hanging-wire became the most accurate tests rather than treadmill to characterise the ALS disease phenotype.

Neuroprotective efficiency of tetanus toxin C fragment in model of global cerebral ischemia in Mongolian gerbils.

The tetanus toxin C (TTC) fragment capacity of being transported in a retrograde way through motoneurons and its nontoxic nature opens the door to a new promising therapeutic strategy for neurodegenerative diseases. In this study, the TTC effect was tested for the first time in animal model of global cerebral ischemia induced by 10-min occlusion of both common carotid arteries. The aim was to evaluate the effect of TTC gene therapy treatment on the development and expression of global cerebral ischemia/reperfusion-induced oxidative stress and motor hyperactivity in Mongolian gerbils. Several oxidative stress and motor behavioral parameters were investigated between 2 h and 14 days after reperfusion. Neuroprotective efficiency of TTC was observed in the forebrain cortex, striatum, hippocampus, and cerebellum at the level of each examined oxidative stress parameter (nitric oxide level, superoxide production, superoxide dismutase activity, and index of lipid peroxidation). Additionally, TTC significantly decreased ischemia-induced motor hyperactivity based on tested parameters (locomotion, stereotypy, and rotations). As judged by biochemical as well as behavioral data, treatment with TTC for the first time showed neuroprotective efficiency by reduction of ischemia-induced oxidative stress and motor hyperactivity and can be a promising strategy for ischemia-induced neuronal damage treatment.

Sigma-1R agonist improves motor function and motoneuron survival in ALS mice.

Amyotrophic lateral sclerosis is a neurodegenerative disorder characterized by progressive weakness, muscle atrophy, and paralysis due to the loss of upper and lower motoneurons (MNs). Sigma-1 receptor (sigma-1R) activation promotes neuroprotection after ischemic and traumatic injuries to the central nervous system. We recently reported that sigma-1R agonist (PRE-084) improves the survival of MNs after root avulsion injury in rats. Moreover, a mutation of the sigma-1R leading to frontotemporal lobar degeneration/amyotrophic lateral sclerosis (ALS) was recently described in human patients. In the present study, we analyzed the potential therapeutic effect of the sigma-1R agonist (PRE-084) in the SOD1(G93A) mouse model of ALS. Mice were daily administered with PRE-084 (0.25 mg/kg) from 8 to 16 weeks of age. Functional outcome was assessed by electrophysiological tests and computerized analysis of locomotion. Histological, immunohistochemical analyses and Western blot of the spinal cord were performed. PRE-084 administration from 8 weeks of age improved the function of MNs, which was manifested by maintenance of the amplitude of muscle action potentials and locomotor behavior, and preserved neuromuscular connections and MNs in the spinal cord. Moreover, it extended survival in both female and male mice by more than 15 %. Delayed administration of PRE-084 from 12 weeks of age also significantly improved functional outcome and preservation of the MNs. There was an induction of protein kinase C-specific phosphorylation of the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor in SOD1(G93A) animals, and a reduction of the microglial reactivity compared with untreated mice. PRE-084 exerts a dual therapeutic contribution, modulating NMDA Ca(2+) influx to protect MNs, and the microglial reactivity to ameliorate the MN environment. In conclusion, sigma-1R agonists, such as PRE-084, may be promising candidates for a therapeutical strategy of ALS.