Raquel Manzano

MicroRNA-206: A Potential Circulating Biomarker Candidate for Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a lethal motor neuron disease that progressively debilitates neuronal cells that control voluntary muscle activity. Biomarkers are urgently needed to facilitate ALS diagnosis and prognosis, and as indicators of therapeutic response in clinical trials. microRNAs (miRNAs), small posttranscriptional modifiers of gene expression, are frequently altered in disease conditions. Besides their important regulatory role in variety of biological processes, miRNAs can also be released into the circulation by pathologically affected tissues and display remarkable stability in body fluids. In a mouse model of ALS that expresses mutated human superoxide dismutase 1 (SOD1-G93A) skeletal muscle is one of the tissues affected early by mutant SOD1 toxicity. To find biomarkers for ALS, we studied miRNA alterations from skeletal muscle and plasma of SOD1-G93A mice, and subsequently tested the levels of the affected miRNAs in the serum from human ALS patients. Fast-twitch and slow-twitch muscles from symptomatic SOD1-G93A mice (age 90 days) and their control littermates were first studied using miRNA microarrays and then evaluated with quantitative PCR from five age groups from neonatal to the terminal disease stage (10–120 days). Among those miRNA changed in various age/gender/muscle groups (miR-206, -1, -133a, -133b, -145, -21, -24), miR-206 was the only one consistently altered during the course of the disease pathology. In both sexes, mature miR-206 was increased in fast-twitch muscles preferably affected in the SOD1-G93A model, with highest expression towards the most severely affected animals. Importantly, miR-206 was also increased in the circulation of symptomatic animals and in a group of 12 definite ALS patients tested. We conclude that miR-206 is elevated in the circulation of symptomatic SOD1-G93A mice and possibly in human ALS patients. Although larger scale studies on ALS patients are warranted, miR-206 is a promising candidate biomarker for this motor neuron disease.

Fragment C of tetanus toxin, more than a carrier. Novel perspectives in non-viral ALS gene therapy

The non-toxic carboxy-terminal fragment of tetanus toxin heavy chain (TTC) has been implicated in the activation of cascades responsible for trophic actions and neuroprotection by inhibition of apoptosis. Previous in vitro studies have described signalling pathways that underlie the administration of TTC to neurons. We investigated whether these properties were maintained in a mouse model of neurodegenerative disease. Naked DNA encoding for TTC was injected intramuscularly and neuromuscular function and clinical behaviour were monitored until endstage in the transgenic SOD1G93A mouse model that expresses a mutant variant of human superoxide dismutase 1 (SOD1). Our results indicate that TTC treatment ameliorated the decline of hindlimb muscle innervation, significantly delayed the onset of symptoms and functional deficits, improved spinal motor neuron survival, and prolonged lifespan. Furthermore, we found that caspase-1 and caspase-3 proapoptotic genes were down-regulated in the spinal cord of treated mice. Western blot analysis showed that the active form of caspase-3 was also down-regulated after TTC treatment and survival signals, such as the significant phosphorylation of serine/threonine protein kinase Akt, were also detected. These results suggest that fragment C of tetanus toxin, TTC, provides a potential therapy for neurodegenerative diseases.

Early presymptomatic cholinergic dysfunction in a murine model of amyotrophic lateral sclerosis

Sporadic and familiar amyotrophic lateral sclerosis (ALS) cases presented lower cholinergic activity than in healthy individuals in their still preserved spinal motoneurons (MNs) suggesting that cholinergic reduction might occur before MN death. To unravel how and when cholinergic function is compromised, we have analyzed the spatiotemporal expression of choline acetyltransferase (ChAT) from early presymptomatic stages of the SOD1G93A ALS mouse model by confocal immunohistochemistry. The analysis showed an early reduction in ChAT content in soma and presynaptic boutons apposed onto MNs (to 76%) as well as in cholinergic interneurons in the lumbar spinal cord of the 30‐day‐old SOD1G93A mice. Cholinergic synaptic stripping occurred simultaneously to the presence of abundant surrounding major histocompatibility complex II (MHC‐II)‐positive microglia and the accumulation of nuclear Tdp‐43 and the appearance of mild oxidative stress within MNs. Besides, there was a loss of neuronal MHC‐I expression, which is necessary for balanced synaptic stripping after axotomy. These events occurred before the selective raise of markers of denervation such as ATF3. By the same time, alterations in postsynaptic cholinergic‐related structures were also revealed with a loss of the presence of sigma‐1 receptor, a Ca2+ buffering chaperone in the postsynaptic cisternae. By 2 months of age, ChAT seemed to accumulate in the soma of MNs, and thus efferences toward Renshaw interneurons were drastically diminished. In conclusion, cholinergic dysfunction in the local circuitry of the spinal cord may be one of the earliest events in ALS etiopathogenesis.

Genetic Biomarkers for ALS Disease in Transgenic SOD1G93A Mice

The pathophysiological mechanisms of both familial and sporadic Amyotrophic Lateral Sclerosis (ALS) are unknown, although growing evidence suggests that skeletal muscle tissue is a primary target of ALS toxicity. Skeletal muscle biopsies were performed on transgenic SOD1G93A mice, a mouse model of ALS, to determine genetic biomarkers of disease longevity. Mice were anesthetized with isoflurane, and three biopsy samples were obtained per animal at the three main stages of the disease. Transcriptional expression levels of seventeen genes, Ankrd1, Calm1, Col19a1, Fbxo32, Gsr, Impa1, Mef2c, Mt2, Myf5, Myod1, Myog, Nnt, Nogo A, Pax7, Rrad, Sln and Snx10, were tested in each muscle biopsy sample. Total RNA was extracted using TRIzol Reagent according to the manufacturers protocol, and variations in gene expression were assayed by real-time PCR for all of the samples. The Pearson correlation coefficient was used to determine the linear correlation between transcriptional expression levels throughout disease progression and longevity. Consistent with the results obtained from total skeletal muscle of transgenic SOD1G93A mice and 74-day-old denervated mice, five genes (Mef2c, Gsr, Col19a1, Calm1 and Snx10) could be considered potential genetic biomarkers of longevity in transgenic SOD1G93A mice. These results are important because they may lead to the exploration of previously unexamined tissues in the search for new disease biomarkers and even to the application of these findings in human studies.

Lack of a synergistic effect of a non-viral ALS gene therapy based on BDNF and a TTC fusion molecule.

BackgroundAmyotrophic 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.MethodsRecombinant 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).ResultsTreatment 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.ConclusionsSignificant 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.

Altered Expression of Myogenic Regulatory Factors in the Mouse Model of Amyotrophic Lateral Sclerosis

Background: In the superoxide dismutase 1 (SOD1)-G93A mouse model of amyotrophic lateral sclerosis (ALS), skeletal muscle is a key target of mutant SOD1 toxicity. However, the expression of factors that control the regenerative potential of the muscle is unknown in this model. Objective: To characterize the expression of satellite cell marker Pax7 and myogenic regulatory factors (MRF) in skeletal muscle of SOD1-G93A mice at different stages of the disease. Methods: The expressions of Pax7, Myod1, Myf5 and myogenin (Myog) were determined by quantitative real-time PCR and by Western blotting from the grouped gastrocnemius, quadriceps and soleus muscles of SOD1-G93A mice at presymptomatic, symptomatic and terminal stages of the disease, and from surgically denervated wild-type gastrocnemius muscles. Results:Pax7 mRNA and MYF5 protein were upregulated in presymptomatic mice, coinciding with increased muscle damage marker Rrad and chemokine Ccl5. All MRF transcripts and most proteins (excluding MYOG) were increased, starting from 3 months of age, simultaneously with increased expression of denervation marker Chrna1. However, in the terminal stage, no protein increase was evident for Pax7 or any of the MRF despite the increased mRNA levels. The transcripts for chemokine Ccl2 and chemokine receptor Cxcr4 were increased starting from the onset of symptoms. Conclusions: The characterization of Pax7 and MRF in SOD1-G93A mice reveals a progressive induction of the myogenic program at the RNA level, but a blunted protein level response at late stages of the disease. Altered posttranscriptional and posttranslational mechanisms likely to operate, as well as the potential role of chemokine signaling in mutant SOD1 muscle, are discussed.

Effect of genetic background on onset and disease progression in the SOD1-G93A model of amyotrophic lateral sclerosis

Abstract Knowledge of the potential effect of genetic background in disease models is important. The SOD1-G93A transgenic mouse is the most widely used model in amyotrophic lateral sclerosis (ALS). Since these animals show considerable variability both in the onset and the progression of the disease, this study aimed to characterize the potential differences between the two most widely used strains, C56BL/6 (B6) and B6SJL. A rotarod test was carried out to assess strength and motor coordination, while electrophysiology tests were performed to evaluate the function of upper and lower motor neurons. Survival of the animals and motor neuron loss were also studied. The results did not show any background effect regarding the rotarod test, despite the differences in the pattern of decline in central and peripheral motor conduction. The onset of motor neuron abnormalities was later in B6SJL mice, but progressed more rapidly. Lifespan was longer for B6 than for B6SJL animals. In conclusion, background differences in disease onset and progression are important. The characteristics of the strain should be taken into account in experimental design of therapeutic studies.

Fragment C of Tetanus Toxin: New Insights into Its Neuronal Signaling Pathway

When Clostridium tetani was discovered and identified as a Gram-positive anaerobic bacterium of the genus Clostridium, the possibility of turning its toxin into a valuable biological carrier to ameliorate neurodegenerative processes was inconceivable. However, the non-toxic carboxy-terminal fragment of the tetanus toxin heavy chain (fragment C) can be retrogradely transported to the central nervous system; therefore, fragment C has been used as a valuable biological carrier of neurotrophic factors to ameliorate neurodegenerative processes. More recently, the neuroprotective properties of fragment C have also been described in vitro and in vivo, involving the activation of Akt kinase and extracellular signal-regulated kinase (ERK) signaling cascades through neurotrophin tyrosine kinase (Trk) receptors. Although the precise mechanism of the molecular internalization of fragment C in neuronal cells remains unknown, fragment C could be internalized and translocated into the neuronal cytosol through a clathrin-mediated pathway dependent on proteins, such as dynamin and AP-2. In this review, the origins, molecular properties and possible signaling pathways of fragment C are reviewed to understand the biochemical characteristics of its intracellular and synaptic transport.

Sex, fiber‐type, and age dependent in vitro proliferation of mouse muscle satellite cells

During postnatal growth and after muscle injury, satellite cells proliferate and differentiate into myotubes to form and repair musculature. Comparison of studies on satellite cell proliferation and differentiation characteristics is confounded by the heterogeneity of the experimental conditions used. To examine the influence of sex, age, and fiber‐type origin on in vitro properties of satellite cells derived from postnatal muscles, fast extensor digitorum longus (EDL) and slow soleus (SOL) muscles were extracted from male and female mice of 1 week to 3 months of age. Myoblast proliferation and myogenic regulatory factor (MRF) expression was measured from cultures of freshly isolated satellite cells. Higher proliferation rate and elevated Myod1 expression was found in male EDL and SOL derived cells compared with females at age of 40, 60, and 120 days, whereas inverse tendency for cell proliferation was apparent in EDL of juvenile (7‐day‐old) pups. Myogenin and Mrf4 transcripts were generally elevated in males of 40 and 60 days of age and in female EDL of juveniles. However, these differentiation markers did not significantly correlate with proliferation rate at all ages. Pax7, whose overexpression can block myogenesis, was up‐regulated especially in 40‐day‐old females where MRF expression was low. These results indicate that gender, postnatal age, and muscle fiber origin affect proliferation and muscle transcription factor expression in vitro. The results also support the view that satellite cells originating from slow and fast muscles are intrinsically different and warrant further studies on the effect of cell origin for therapeutic approaches. J. Cell. Biochem. 112: 2825–2836, 2011.

Identification of novel, therapy-responsive protein biomarkers in a mouse model of Duchenne muscular dystrophy by aptamer-based serum proteomics

There is currently an urgent need for biomarkers that can be used to monitor the efficacy of experimental therapies for Duchenne Muscular Dystrophy (DMD) in clinical trials. Identification of novel protein biomarkers has been limited due to the massive complexity of the serum proteome and the presence of a small number of very highly abundant proteins. Here we have utilised an aptamer-based proteomics approach to profile 1,129 proteins in the serum of wild-type and mdx (dystrophin deficient) mice. The serum levels of 96 proteins were found to be significantly altered (P < 0.001, q < 0.01) in mdx mice. Additionally, systemic treatment with a peptide-antisense oligonucleotide conjugate designed to induce Dmd exon skipping and recover dystrophin protein expression caused many of the differentially abundant serum proteins to be restored towards wild-type levels. Results for five leading candidate protein biomarkers (Pgam1, Tnni3, Camk2b, Cycs and Adamts5) were validated by ELISA in the mouse samples. Furthermore, ADAMTS5 was found to be significantly elevated in human DMD patient serum. This study has identified multiple novel, therapy-responsive protein biomarkers in the serum of the mdx mouse with potential utility in DMD patients.