Study of Survival Motor Neuron protein regulation and the role of autophagy in Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a genetic disorder caused by loss of the Survival motor neuron 1 gene (SMN1), lead to reduced SMN protein level and selective dysfunction of MNs. SMN reduction causes neurite degeneration and cell death without classical apoptotic features, but the direct events leading to MN degeneration in SMA are still unknown. Autophagy is being a primary target for the treatment of many neurodegenerative diseases. The objective of the present study is to analyze the role of autophagy in SMA pathology, the mechanisms that regulate SMN protein degradation and the origin of neurodegeneration in spinal cord MNs. To this end, we have reduced the Smn protein by using the lentivirus knockdown method. In Smn-reduced MNs from lentivirus Smn knockdown and SMA type I transgenic mice models, we have observed the increase of autophagy markers and autophagosome accumulation. Treatment with autophagy activators or inhibitors or proteasome inhibitors or calpain knockdown induce changes of Smn protein level in MNs suggesting the role of autophagy and proteasome in the regulation of Smn protein in these cells. Therefore the results contribute to new insight about Smn protein regulation in MNs and the possible role of autophagy in SMA neurodegeneration.L'atròfia muscular espinal (SMA) és un trastorn genètic, causada per la pèrdua o la mutació del gen de la supervivencia de les neurones motores 1 (SMN1), cosa que condueix a una reducció dels nivells de la proteïna SMN i una disfunció selectiva de les MN. S’ha descrit que la reducció d’SMN causa la degeneració de les neurites i la mort cel•lular sense les característiques apoptòtiques clàssiques, però els esdeveniments directes que condueixen a la degeneració de les MN en l’SMA encara són desconeguts. L’autofàgia és una diana principal per al tractament de moltes malalties neurodegeneratives. L'objectiu d’aquest estudi és analitzar el paper de l'autofàgia en la patologia de l’SMA, els mecanismes que regulen la degradació de la proteïna SMN i l'origen de la neurodegeneració en les MN de la medul•la espinal. Amb aquesta finalitat, hem reduït la proteïna SMN utilitzant el mètode de silenciament amb lentivirus. Hem analitzat els canvis en els marcadors d’autofàgia en les MN en cultiu amb l’SMN reduïda amb lentivirus i en cultius de MN de models de ratolins transgènics de SMA de tipus I. Hem observat que la reducció de l’SMN provoca un augment dels marcadors d’autofàgia i l'acumulació d’autofagosomes. A més, el tractament amb activadors de l'autofàgia, inhibidors de l'autofàgia o inhibidors del proteasoma o calpaïna indueix canvis en els nivells de la proteïna SMN en les MN, la qual cosa suggereix un paper de l'autofàgia i el proteasoma en la regulació de la proteïna SMN en aquestes cèl•lules. Conjuntament, aquests resultats contribueixen a una nova visió sobre la regulació de la proteïna SMN en les MN i sobre el possible paper de l'autofàgia en la neurodegeneració en l’ SMA.La atrofia muscular espinal (AME) es un trastorno genético causado por la pérdida de la supervivencia de las neuronas motoras del gen 1 (SMN1) que conduce a la reducción de nivel de proteína SMN y a la disfunción selectiva de los MNs. La reducción de SMN causa la degeneración de axones y la muerte celular sin características apoptóticas clásicas; sin embargo, los motivos directos que conducen a la degeneración del MN en AME aún se desconocen. La autofagia está siendo un objetivo principal para el tratamiento de muchas enfermedades neurodegenerativas. El objetivo del presente estudio es analizar el papel de la autofagia en la patología de la AME, los mecanismos que regulan la degradación de la proteína SMN y el origen de la neurodegeneración de los MNs en la médula espinal. Con este fin, hemos reducido la proteína SMN utilizando un método de reducción lentiviral. En la Smn reducida mediante el método de reducción lentiviral y modelos de ratones transgénicos AME de tipo I, hemos observado el aumento de los marcadores de autofagia y la acumulación de autofagosoma. El tratamiento con activadores o inhibidores de la autofagia o inhibidores del proteasoma o calpaína reducida induce cambios del nivel de la proteína SMN en los MNs que demuestran el papel de la autofagia y del proteasoma en la regulación de la proteína SMN en estas células. Por lo tanto, los resultados contribuyen a una nueva visión sobre la regulación de las proteínas Smn en el MN y el posible papel de la autofagia en la neurodegeneración de AME

Survival motor neuron protein reduction deregulates autophagy in spinal cord motoneurons in vitro

Spinal muscular atrophy (SMA) is a genetic disorder characterized by degeneration of spinal cord motoneurons (MNs), resulting in muscular atrophy and weakness. SMA is caused by mutations in the Survival Motor Neuron 1 (SMN1) gene and decreased SMN protein. SMN is ubiquitously expressed and has a general role in the assembly of small nuclear ribonucleoproteins and pre-mRNA splicing requirements. SMN reduction causes neurite degeneration and cell death without classical apoptotic features, but the direct events leading to SMN degeneration in SMA are still unknown. Autophagy is a conserved lysosomal protein degradation pathway whose precise roles in neurodegenerative diseases remain largely unknown. In particular, it is unclear whether autophagosome accumulation is protective or destructive, but the accumulation of autophagosomes in the neuritic beadings observed in several neurite degeneration models suggests a close relationship between the autophagic process and neurite collapse. In the present work, we describe an increase in the levels of the autophagy markers including autophagosomes, Beclin1 and light chain (LC)3-II proteins in cultured mouse spinal cord MNs from two SMA cellular models, suggesting an upregulation of the autophagy process in Smn (murine survival motor neuron protein)-reduced MNs. Overexpression of Bcl-xL counteracts LC3-II increase, contributing to the hypothesis that the protective role of Bcl-xL observed in some SMA models may be mediated by its role in autophagy inhibition. Our in vitro experimental data indicate an upregulation in the autophagy process and autophagosome accumulation in the pathogenesis of SMA, thus providing a valuable clue in understanding the mechanisms of axonal degeneration and a possible therapeutic target in the treatment of SMA

Regulation of survival motor neuron protein by the nuclear factor-kappa B pathway in mouse spinal cord motoneurons

Survival motor neuron (SMN) protein deficiency causes the genetic neuromuscular disorder spinal muscular atrophy (SMA), characterized by spinal cord motoneuron degeneration. Since SMN protein level is critical to disease onset and severity, analysis of the mechanisms involved in SMN stability is one of the central goals of SMA research. Here, we describe the role of several members of the NF-κB pathway in regulating SMN in motoneurons. NF-κB is one of the main regulators of motoneuron survival and pharmacological inhibition of NF-κB pathway activity also induces mouse survival motor neuron (Smn) protein decrease. Using a lentiviral-based shRNA approach to reduce the expression of several members of NF-κB pathway, we observed that IKK and RelA knockdown caused Smn reduction in mouse-cultured motoneurons whereas IKK or RelB knockdown did not. Moreover, isolated motoneurons obtained from the severe SMA mouse model showed reduced protein levels of several NF-κB members and RelA phosphorylation. We describe the alteration of NF-κB pathway in SMA cells. In the context of recent studies suggesting regulation of altered intracellular pathways as a future pharmacological treatment of SMA, we propose the NF-κB pathway as a candidate in this new therapeutic approach

Autophagy modulators regulate survival motor neuron protein stability in motoneurons

SpinalMuscular Atrophy (SMA), a neurodegenerative disorder primarily affecting motoneurons (MNs), is caused by the loss of the Survival Motor Neuron 1 (SMN1) gene and reduced levels of full-length survival motor neuron (SMN) protein. The exact cellular/molecular mechanisms involved in SMN-induced MN degeneration are under study. Autophagy is a degradation pathway whose precise roles in neurodegeneration remain largely unknown, but abnormal autophagy has a central role in some neurodegenerative diseases, including MN disorders. The analysis of the autophagy response in SMA and its role in the development of the disease could be essential to understand the disease. In the present work, we describe an increase of autophagosomes and LC3-II protein in spinal cord MNs of severe SMA mouse model. A time-course experiment demonstrated increased LC3-II levels fromembryonic to postnatal stage, suggesting a deregulation of the autophagy process as the disease progressed. Using an in vitro model ofMN culture, we analyzed the effect of autophagy modulators on Smn (murine survival motor neuron) protein level. Results suggest that the inhibitors of the autophagy flux cause reduction ofSmn protein, whereas autophagy inducers increase the level of Smn protein inMNs. In order to evaluate other proteolytic systems involved to SMN degradation,we also studied the effect of the inhibition of the calcium-dependent protease, calpain, on Smn protein level. Our results demonstrate that calpain reduction increases Smn and LC3-II level in cultured MNs. Collectively, these results provide new insight into the role of autophagy and its modulation in SMN protein regulation

Murine cytomegalovirus infection exacerbates complex IV deficiency in a model of mitochondrial disease

The influence of environmental insults on the onset and progression of mitochondrial diseases is unknown. To evaluate the effects of infection on mitochondrial disease we used a mouse model of Leigh Syndrome, where a missense mutation in the Taco1 gene results in the loss of the translation activator of cytochrome c oxidase subunit I (TACO1) protein. The mutation leads to an isolated complex IV deficiency that mimics the disease pathology observed in human patients with TACO1 mutations. We infected Taco1 mutant and wild-type mice with a murine cytomegalovirus and show that a common viral infection exacerbates the complex IV deficiency in a tissue-specific manner. We identified changes in neuromuscular morphology and tissue-specific regulation of the mammalian target of rapamycin pathway in response to viral infection. Taken together, we report for the first time that a common stress condition, such as viral infection, can exacerbate mitochondrial dysfunction in a genetic model of mitochondrial disease

Publisher Correction: Dynamic regulation of Z-DNA in the mouse prefrontal cortex by the RNA-editing enzyme Adar1 is required for fear extinction

An amendment to this paper has been published and can be accessed via a link at the top of the paper