Neuregulin-1 promotes functional improvement by enhancing collateral sprouting in SOD1G93A ALS mice and after partial muscle denervation

Altres ajuts: Fundació La Marato-TV3(TV3201428-10), AFM-Telethon (Nrg14ALS)Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of motoneurons, which is preceded by loss of neuromuscular connections in a "dying back" process. Neuregulin-1 (Nrg1) is a neurotrophic factor essential for the development and maintenance of neuromuscular junctions, and Nrg1 receptor ErbB4 loss-of-function mutations have been reported as causative for ALS. Our main goal was to investigate the role of Nrg1 type I (Nrg1-I) in SOD1G93A mice muscles. We overexpressed Nrg1-I by means of an adeno-associated viral (AAV) vector, and investigated its effect by means of neurophysiological techniques assessing neuromuscular function, as well as molecular approaches (RT-PCR, western blot, immunohistochemistry, ELISA) to determine the mechanisms underlying Nrg1-I action. AAV-Nrg1-I intramuscular administration promoted motor axon collateral sprouting by acting on terminal Schwann cells, preventing denervation of the injected muscles through Akt and ERK1/2 pathways. We further used a model of muscle partial denervation by transecting the L4 spinal nerve. AAV-Nrg1-I intramuscular injection enhanced muscle reinnervation by collateral sprouting, whereas administration of lapatinib (ErbB receptor inhibitor) completely blocked it. We demonstrated that Nrg1-I plays a crucial role in the collateral reinnervation process, opening a new window for developing novel ALS therapies for functional recovery rather than preservation

Collagen XIX Alpha 1 improves prognosis in amyotrophic lateral sclerosis

The identification of more reliable diagnostic or prognostic biomarkers in age-related neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis (ALS), is urgently needed. The objective in this study was to identify more reliable prognostic biomarkers of ALS mirroring neurodegeneration that could be of help in clinical trials. A total of 268 participants from three cohorts were included in this study. The muscle and blood cohorts were analyzed in two cross-sectional studies, while the serial blood cohort was analyzed in a longitudinal study at 6-monthly intervals. Fifteen target genes and fourteen proteins involved in muscle physiology and differentiation, metabolic processes and neuromuscular junction dismantlement were studied in the three cohorts. In the muscle biopsy cohort, the risk for a higher mortality in an ALS patient that showed high Collagen type XIX, alpha 1 (COL19A1) protein levels and a fast progression of the disease was 70.5% (P < 0.05), while in the blood cohort, this risk was 20% (P < 0.01). In the serial blood cohort, the linear mixed model analysis showed a significant association between increasing COL19A1 gene levels along disease progression and a faster progression during the follow-up period of 24 months (P < 0.05). Additionally, higher COL19A1 levels and a faster progression increased 17.9% the mortality risk (P < 0.01). We provide new evidence that COL19A1 can be considered a prognostic biomarker that could help the selection of homogeneous groups of patients for upcoming clinical trial and may be pointed out as a promising therapeutic target in ALS

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 manufacturer's 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

Efecto de la inhibición dual de GSK-3 y PDE-7 en un modelo murino de esclerosis lateral amiotrófica

La Esclerosis Lateral Amiotrófica (ELA) es una enfermedad neurodegenerativa en la que mueren progresivamente las neuronas motoras de la corteza cerebral y la médula, con la consiguiente atrofia de los músculos inervados por ellas. Si bien últimamente han aparecido distintos modelos animales para el estudio de la ELA, los ratones que sobre-expresan el gen SOD1 humano con la mutación G93A, son los más utilizados. Estos son los animales que empleamos en nuestro estudio, en el cual evaluamos un inhibidor dual de las enzimas GSK-3 y PDE-7 llamado VP1.15. La Glucógeno Sintetasa Kinasa -3 (GSK-3) es una serina-treonina kinasa con múltiples funciones celulares y neurofisiológicas. En el Sistema Nervioso Central, la isoforma más abundante corresponde a GSK-3 β, cuya actividad está regulada negativamente por fosforilación en la Ser9. Su sobre-expresión se asocia con la muerte neuronal, la hiperfosforilación en Tau y la retracción de las neuritas en cultivos de neuronas. Por eso es vista como partícipe de la patofisiología de las enfermedades neurodegenerativas. En particular, su aumento ha sido detectado en la médula de los pacientes con ELAe. La superfamilia de las Fosfodiesterasas (PDEs) comprende enzimas ubicuas con capacidad de metalofosfohidrolasas que hidrolizan AMPc y/o GMPc llevándolos a sus respectivos monofosfatos inactivos. PDE-7 es una PDE específica para AMPc. Su inhibición actúa como neuroprotectora en cultivos celulares y en modelos de infarto experimental, disminuye la activación inflamatoria y la supervivencia de precursores de oligodendrocitos en cultivos primarios de neuronas, reduce los síntomas clínicos en el modelo murino de encefalomielitis autoinmune y la alteración cognitiva en el modelo murino de Enfermedad de Alzheimer (EA). Los mecanismos regulatorios y de señalización celular modulados por GSK-3 y PDE-7, involucran a CREB, β-catenina y los niveles de AMPc, moléculas que serán estudiadas en profundidad en esta investigación..

Neuregulin-1 promotes functional improvement by enhancing collateral sprouting in SOD1G93A ALS mice and after partial muscle denervation

Altres ajuts: Fundació La Marato-TV3(TV3201428-10), AFM-Telethon (Nrg14ALS)Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of motoneurons, which is preceded by loss of neuromuscular connections in a "dying back" process. Neuregulin-1 (Nrg1) is a neurotrophic factor essential for the development and maintenance of neuromuscular junctions, and Nrg1 receptor ErbB4 loss-of-function mutations have been reported as causative for ALS. Our main goal was to investigate the role of Nrg1 type I (Nrg1-I) in SOD1G93A mice muscles. We overexpressed Nrg1-I by means of an adeno-associated viral (AAV) vector, and investigated its effect by means of neurophysiological techniques assessing neuromuscular function, as well as molecular approaches (RT-PCR, western blot, immunohistochemistry, ELISA) to determine the mechanisms underlying Nrg1-I action. AAV-Nrg1-I intramuscular administration promoted motor axon collateral sprouting by acting on terminal Schwann cells, preventing denervation of the injected muscles through Akt and ERK1/2 pathways. We further used a model of muscle partial denervation by transecting the L4 spinal nerve. AAV-Nrg1-I intramuscular injection enhanced muscle reinnervation by collateral sprouting, whereas administration of lapatinib (ErbB receptor inhibitor) completely blocked it. We demonstrated that Nrg1-I plays a crucial role in the collateral reinnervation process, opening a new window for developing novel ALS therapies for functional recovery rather than preservation

Functional role and related molecular pathway of the seventeen genes studied.

<p>Functional role and related molecular pathway of the seventeen genes studied.</p

Transcriptional SOD1^G93A fold change in muscle biopsy samples.

<p>The transcriptional expression levels of SOD1^G93A were measured in all the samples obtained from the muscle biopsies corresponding to early symptomatic (first biopsy), symptomatic (second biopsy) and terminal stages (third biopsy). No statistical differences were found in SOD1^G93A levels along disease progression. SOD1^G93A fold change in the symptomatic and terminal stages was calculated respect to the relative expression found in all the muscle samples extracted at the early symptomatic stage.</p

Transcriptional expression levels of the sixteen genes varied significantly throughout disease progression in transgenic SOD1^G93A mice.

<p>Representative graphs showing the fold change in transcriptional levels of the seventeen genes tested in the skeletal muscle of transgenic SOD1^G93A mice throughout disease progression with respect to the early symptomatic stage. Age-matched wild type mice were used as controls in each stage of the disease: early symptomatic (60 days, grey bar), symptomatic (90 days, blue bar) and terminal (120 days, pale blue bar) stages. The highest transcriptional expression levels were found in <i>Ankrd1</i> and <i>Col19a1</i>, which at the terminal stage were almost 27 and 18 times higher than those observed at the early symptomatic stage, respectively. A significant upregulation of transcriptional levels was found in all of the genes, except for <i>Calm1,</i> despite its irregular profile pattern throughout disease progression.</p

<i>Mef2c</i>, <i>Gsr</i>, <i>Col19a1</i>, <i>Calm1</i> and <i>Snx10 are potential genetic biomarkers of longevity in ALS</i>.

<p>Linear correlation graphs of five potential genetic biomarkers of longevity. The graph shows the linear relation between the fold change in transcriptional levels of these genes throughout disease progression in skeletal muscle biopsies at the early symptomatic stage and the longevity of the animals at the terminal stage. The transcriptional levels of these genes can predict longevity in transgenic SOD1^G93A mice.</p