Inhibition of autophagy delays motoneuron degeneration and extends lifespan in a mouse model of spinal muscular atrophy

Abstract

Spinal muscular atrophy (SMA) is a recessive autosomal neuromuscular disease, due to homozygous mutations or deletions in the telomeric survival motoneuron gene 1 (SMN1). SMA is characterized by motor impairment, muscle atrophy, and premature death following motor neuron (MN) degeneration. Emerging evidence suggests that dysregulation of autophagy contributes to MN degeneration. We here investigated the role of autophagy in the SMNdelta7 mouse model of SMA II (intermediate form of the disease) which leads to motor impairment by postnatal day 5 (P5) and to death by P13. We first showed by immunoblots that Beclin 1 and LC3-II expression levels increased in the lumbar spinal cord of the SMA pups. Electron microscopy and immunofluorescence studies confirmed that autophagic markers were enhanced in the ventral horn of SMA pups. To clarify the role of autophagy, we administered intracerebroventricularly (at P3) either an autophagy inhibitor (3-methyladenine, 3-MA), or an autophagy inducer (rapamycin) in SMA pups. Motor behavior was assessed daily with different tests: tail suspension, righting reflex, and hindlimb suspension tests. 3-MA significantly improved motor performance, extended the lifespan, and delayed MN death in lumbar spinal cord (10372.36 ± 2716 MNs) compared to control-group (5148.38 ± 94 MNs). Inhibition of autophagy by 3-MA suppressed autophagosome formation, reduced the apoptotic activation (cleaved caspase-3 and Bcl2) and the appearance of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive neurons, underlining that apoptosis and autophagy pathways are intricately intertwined. Therefore, autophagy is likely involved in MN death in SMA II, suggesting that it might represent a promising target for delaying the progression of SMA in humans as well.

Pharmacological c-Jun NH2-Terminal Kinase (JNK) Pathway Inhibition Reduces Severity of Spinal Muscular Atrophy Disease in Mice

“…According to the literature (Branchu et al, 2013 ; Biondi et al, 2015 ; Genabai et al, 2015 ), these data confirm that JNK-signaling pathway is activated in SMA MNs and is probably involved in neuronal cell death. Indeed, confirming previous observations (d’Errico et al, 2013 ; Piras et al, 2017 ), here we report the reduction in the density of MNs in SMA spinal cord compared to WT group (WT PBS = 1,651.14 ± 90.3 cells/mm 3 ; SMA PBS = 891.22 ± 82.5 cells/mm 3 ; Mann-Whitney test, p = 0.0043; Figures 2A,A′,B ). As a consequence of neurodegeneration, proximal muscles of SMA mice are early affected by atrophy (Valsecchi et al, 2015 ; Boido et al, 2018 ): indeed, quadriceps fibers showed reduced area (WT PBS = 578.4 ± 80.8 μm 2 ; SMA PBS = 297.7 ± 31.7 μm 2 ; unpaired t -test, p = 0.043; Figures 2C,C′,D ), perimeter (WT PBS = 90.8 ± 7.1 μm; SMA PBS = 76.2 ± 1.9 μm; unpaired t -test, p = 0.036; Figure 2E ), maximal (WT PBS = 32.4 ± 1.7 μm; SMA PBS = 28.04 ± 0.78 μm; unpaired t -test, p < 0.001) and minimal Feret’s diameter (WT PBS = 22.8 ± 1.4 μm; SMA PBS = 16.7 ± 0.85 μm; unpaired t -test, p < 0.001; Figure 2F ).…”

Blocking p62-dependent SMN degradation ameliorates spinal muscular atrophy disease phenotypes

“…We observed that this defect was accompanied by increased levels of p62 and ubiquitinated proteins and that an acute decrease in SMN protein levels produced the same results. Our findings agree with those of recent studies that reported autophagy alterations upon SMN downregulation in cell lines (50,67,68) and answer the previously unresolved question of why autophagy is dysfunctional in SMA and explain the nature of the interplay between SMN and autophagy activity. We postulate that, as a consequence of SMN deficiency-induced autophagy impairment, ubiquitinated proteins and p62 accumulate.…”

Stasimon Contributes to the Loss of Sensory Synapses and Motor Neuron Death in a Mouse Model of Spinal Muscular Atrophy

“…Recent studies have shown that Stasimon is an ER-resident, six-transmembrane-pass protein that is essential for mouse embryonic development (Van Alstyne et al, 2018b) and functions in autophagy (Moretti et al, 2018;Morita et al, 2018;Shoemaker et al, 2019). There have also been reports of autophagy dysregulation in SMA, but there is disagreement about whether this pathway is inhibited (Custer and Androphy, 2014;Periyakaruppiah et al, 2016;Rodriguez-Muela et al, 2018) or stimulated (Gonç alves et al, 2018;Piras et al, 2017) by SMN deficiency. Therefore, although autophagy inhibition has been shown to ameliorate the phenotype of SMA mice by blocking p62-mediated SMN degradation (Rodriguez-Muela et al, 2018), it remains to be established whether autophagy defects directly contribute to SMA pathology.…”

“…Stasimon Deficiency Is Sufficient to Induce p53 S18 Phosphorylation Recent studies have shown that Stasimon is required for autophagy in cultured mammalian cells (Moretti et al, 2018;Morita et al, 2018;Shoemaker et al, 2019). There have also been reports of autophagy dysregulation in SMA (Custer and Androphy, 2014;Gonç alves et al, 2018;Periyakaruppiah et al, 2016;Piras et al, 2017;Rodriguez-Muela et al, 2018). Therefore, we sought to investigate the possible involvement of Stasimon-dependent autophagy defects in SMA pathogenesis.…”