PACAP Modulates the Autophagy Process in an In Vitro Model of Amyotrophic Lateral Sclerosis.

Agata Grazia D’Amico,Salvatore Saccone,Dora Reglodi,Velia D’Agata,Concetta Federico,Sebastiano Cavallaro,Grazia Maugeri

doi:10.3390/ijms21082943

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of complex etiology leading to motor neuron degeneration. Many gene alterations cause this pathology, including mutation in Cu, Zn superoxide dismutase (SOD1), which leads to its gain of function. Mutant SOD1 proteins are prone to aberrant misfolding and create aggregates that impair autophagy. The hypoxic stress is strictly linked to the disease progression since it induces uncontrolled autophagy activation and the consequent high rates of cell death. Previously, we showed that pituitary adenylate cyclase-activating polypeptide (PACAP) exerts neurotrophic activity in cultured mSOD1 motor neurons exposed to serum deprivation. To date, no studies have examined whether the protective effect of PACAP on mSOD1 cells exposed to hypoxic insult is mediated through the regulation of the autophagy process. In the present study, we used the neuroblastoma-spinal cord-34 (NSC-34) cell line, stably expressing human wild type or mutant SOD1 G93A, to represent a well characterized in vitro model of a familial form of ALS. These cells were exposed to 100-µM desferrioxamine mesylate salt for 24h, to mimic the hypoxic stress affecting motor neurons during the disease progression. Our results showed that PACAP treatment significantly reduced cell death and hypoxia-induced mSOD1 accumulation by modulating the autophagy process in G93A motor neurons, as revealed by the decreased LC3II and the increased p62 levels, two autophagy indicators. These results were also confirmed by evaluating the vacuole formation detected through light chain 3 (LC3) immunofluorescence. Furthermore, the PACAP effects on autophagy seem to be mediated through the activation of the MAPK/ERK signaling pathway. Overall, our data demonstrated that PACAP exerts an ameliorative effect on the mSOD1 motor neuron viability by modulating a hypoxia-induced autophagy process through activation of MAPK/ERK signaling cascade.

Highlights

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of complex etiology, characterized by a motor neuron degeneration in the spinal ventral horn, cerebral cortex and brainstem nuclei leading to muscle weakness [1,2].It is classified in two different forms: a sporadic and a familial form [3]
Our results showed that pituitary adenylate cyclase-activating polypeptide (PACAP) treatment significantly reduced cell death and hypoxia-induced mutant SOD1 (mSOD1) accumulation by modulating the autophagy process in G93A motor neurons, as revealed by the decreased LC3II and the increased p62 levels, two autophagy indicators
The results suggest that PACAP prevented mSOD1 motor neuron death through the regulati of cellular homeoTsotbaesttiesr bchyaramctoerdizue lthaetienffgectthofePAaCuAtoPpinhAaLgSymportoorcneesusroensdmegeendeiraattieodn, ibnythMe pAresPeKnt/ERK signali cascade activasttuidoyn.we investigated its involvement in the autophagy process by using an in vitro model of neuroblastoma-spinal cord-34 (NSC-34) cells stably bearing a human Cu/Zn superoxide dismutase1

Summary

Introduction

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of complex etiology, characterized by a motor neuron degeneration in the spinal ventral horn, cerebral cortex and brainstem nuclei leading to muscle weakness [1,2]. It is classified in two different forms: a sporadic (sALS) and a familial (fALS) form [3]. Since the cytoplasm of the motor neurons in ALS patients and mutant SOD1 (mSOD1) animals contains characteristic aggregates including autophagy-related protein LC3II, it has been hypothesized that the aberrant induction of the autophagy process was implicated in disease pathogenesis [7]. It has been demonstrated that the overexpression of mSOD1 induced an increase in the autophagic activity in an in vitro model of ALS [8]

Methods

Results

Discussion

Conclusion