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By Jamshed Arslan Pharm.D.
Neuromuscular disorders affect the peripheral nervous system and muscles. Spinal muscular atrophy (SMA) is one such incurable disease in which muscles fail to receive signals from the spinal motor neurons (MNs), and consequently, weaken due to inactivity. MN degeneration and muscle atrophy lead to the premature death of the victims. Like most of the neuromuscular disorders, SMA is genetic, and its genetic causes are known: the inactivation of survival motor neuron 1 (SMN1) gene, which reduces the amount of full-length SMN1 protein; or mutated SMN2 gene, which generates an SMNdelta7 protein with reduced function. What is unknown, however, is the role of autophagic degradation in the process of MN loss. A recent study, using a mouse model of SMA from European researchers, has shown that increased autophagy is involved in MN death to such an extent that inhibiting autophagy extends lifespan.
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Immunoblots of the lumbar spinal cord (LSC) of SMA pups showed increased expression of autophagic markers (e.g., Beclin 1, LC3-II). The increase in autophagosomes (indicated by increased LC3-positive puncta) was not accompanied by changes in proteins that are selectively degraded by autophagy (p62/SQSTM1), suggesting that autophagic degradation activity remained unaffected. Electron microscopy and immunofluorescence confirmed an increase in the autophagic features (autophagosomes and autolysosomes) with cellular abnormalities (such as endoplasmic reticulum swelling and cytoplasmic shrinkage) in the dying lower-MNs of 10-day old SMA pups, relative to the normal/control pups.
"In our paper, we demonstrated the involvement of autophagy in SMA, suggesting for the first time that inhibition of this process by ICV administration of 3-MA may significantly influence disease. Based on our results, we suggested that inhibition of autophagy represents a novel and intriguing therapeutic target for clinical trials in patients diagnosed with SMA. In addition, regulation of autophagy could be considered a valid target, possibly in combination with emerging and promising gene therapy approaches (e.g. antisense oligonucleotides, ASOs) aimed at restoring full-length SMN1 protein expression."
- Antonio Piras, PhD
Senior Research Scientist
Innovative Medicine & Early Development - Respiratory, Inflammation & Autoimmunity - AstraZeneca, Mölndal
After confirming autophagy’s involvement in MN degeneration, the researchers inhibited autophagy by administering 3-methyladenine (3-MA), a phosphoinositide 3-kinase (PI3K) inhibitor, directly into the brain of 3-day old SMA pups. At day 9, Nissl staining showed significantly higher number of MNs in mice treated with 3-MA, as compared to those treated with saline control or autophagy-inducer rapamycin. Likewise, 3-MA increased lifespan of SMA mice from a typical 13 days to more than 15 days, whereas rapamycin (mTOR) showed an opposite trend. Moreover, western blotting of the protein extract from LSC of SMA pups and TUNEL-staining revealed that 3-MA reduced apoptotic activation, evidenced by the reduction of cleaved caspase-3 and increased Bcl2, suggesting that autophagy is intertwined with apoptosis.
To investigate how inhibiting autophagy may affect disease progression, motor coordination of SMA pups was analyzed: posture was evaluated after hanging animals by their tails or hindlimbs; and the time required for all four paws to contact a flat surface after a pup is inverted, was recorded. As expected, 3-MA injection on day 3 improved the motor coordination as compared to saline or rapamycin. This effect was stronger when an additional injection of 3-MA was given at day 6. Overall, the results showed that in SMA, inhibiting autophagy protects MNs and extends lifespan by delaying disease progression.
This study is important from both clinical and basic science perspectives. It has paved the way for human trials to investigate the inhibition of autophagy as a promising approach for improving both the quality of life and the lifespan in SMA patients. Since inhibiting autophagy in MNs in SMA can trigger apoptosis, this study sheds light on the autophagy-apoptosis interplay that may underlie SMA pathogenesis.
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Jamshed Arslan, Pharm D.
University of Alabama at Birmingham, School of Medicine
Dr. Arslan studies cell signaling in mitochondrial defects in C. elegans and transgenic mice.
References
Piras, Antonio, et al. “Inhibition of Autophagy Delays Motoneuron Degeneration and Extends Lifespan in a Mouse Model of Spinal Muscular Atrophy.” Cell Death & Disease, vol. 8, 2017, n. pag. DOI: 10.1038/s41419-017-0086-4
