Amyotrophic lateral Sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of motor neurons in the central nervous system. To date, the etiopathogenetic mechanisms involved in the development of the disease are not well understood as well and effective therapy for this pathology has not been identified. Several mutations have been associated with the familial form of the disease, including mutations in the gene encoding the enzyme copper‑zinc superoxide dismutase 1 (Cu/Zn -SOD1) (Berdyński et al., 2022; Rosen et al., 1993). It has been identified >180 mutations in the SOD1 gene, including a point mutation G93A which results in a toxic gain of function of the encoded SOD1 protein (Kaur et al., 2016). Although the molecular mechanism underlying neurodegeneration remains controversial, several studies have revealed that SOD1-G93A mutation forms insoluble aggregates that interfere with normal cellular proteins or organelles inducing overproduction of ROS, mitochondrial dysfunction, endoplasmic reticulum stress, protein aggregation, and alteration in autophagy process (Bowling et al., 1993; Saxena et al., 2009). Accord, it has been previously described abnormal ROS accumulation and oxidative damage to proteins and lipids in the spinal cord and motor cortex of ALS patients as well as both in an in vitro and in vivo experimental model (Pedersen et al., 1998; Shaw et al., 1995). Moreover, it has been also reported that mutant SOD1 impairs the expression of Nrf2, a key modulator of protection against OS (Kirby et al., 2005; Wang et al., 2014). This transcription factor is involved in the antioxidant response since it counteracts ROS formation by translocating from the cytoplasm to the nucleus, where it induces the expression of genes encoding antioxidant enzymes (Taguchi et al., 2011).

By comparing the gene expression profile between motor cortex samples of ALS patients and SOD1 G93A mice, it has been selected a list of genes commonly dysregulated in both groups, including the adenylate cyclase-activating polypeptide 1 (ADCYAP1) gene, encoding pituitary adenylate cyclase-activating polypeptide (PACAP) (Morello et al., 2017). The latter is a neuropeptide widely expressed in the central and peripheral nervous system. It exerts a neuroprotective effect by binding to three receptors known as PAC1R, VIPAC-1, and VIPAC-2 (Arimura and Shioda, 1995; Shioda et al., 2006). Among them, PAC1R has a higher affinity for PACAP than VIP (Vaudry et al., 2009; Vaudry et al., 2000). The various biological effects exerted by PACAP are mediated by its binding to specific splicing variants of PAC1R which differently activate adenylate cyclase (AC)/protein kinase A (PKA), phospholipase (PLC)/protein kinase C (PKC) and mitogen-activated protein kinases/extracellular signal-regulated kinases 1 and 2 (MAPK/ERK1/2) signaling pathways. Furthermore, it has also been demonstrated that some protective effects of PACAP are mediated through the stimulation of an intracellular factor known as activity-dependent protein (ADNP) (D'Amico et al., 2023a, D'Amico et al., 2023b; Castorina et al., 2012; Nakamachi et al., 2006).

ADNP is a protein essential for brain development, cognitive function, and neuroprotection (Gozes et al., 2005; D'Amico et al., 2023a, D'Amico et al., 2023b; Zusev and Gozes, 2004). It was first described as a protein produced and secreted by astrocytes, which synthesize and secrete it following the stimulation by vasoactive intestinal peptide (VIP) (Pinhasov et al., 2003). Mutations in the ADNP gene have been associated with different types of neurological disorders, such as Alzheimer's disease, autism spectrum disorder, Parkinson's disease, stroke, and muscular dystrophy (Sragovich et al., 2017: Hacohen-Kleiman et al., 2020; Gozes and Shazman, 2022; Gozes et al. 2022; Arnett et al., 2018). ADNP is involved in a wide range of cellular processes, including neurite formation and maturation, and exerts a significant role in brain cell protection from damage caused by stress and aging. The cytoplasmic localization of ADNP through interaction with 14–3-3 proteins promotes neuronal morphogenesis, cortical connectivity, and calcium signaling, and they are also implicated in CaM/CaMKII/NRF2 signaling pathway in mesenchymal stem cells resulting in oxidative stress regulation (Yuan et al., 2022). Moreover, ADNP is directly implicated in muscle function (Kapitansky et al., 2020). It was observed that Adnp-deficient mice (Adnp+/−) displayed abnormal gene expression in various tissues, including the gastrocnemius muscle, tongue, and bladder. Notably, these variations in gene expression were influenced by the development stage and the sex of the mice. Additionally, the study revealed that intranasal administration of ADNP-derived peptide, known as NAP, restored normal gene expression patterns in response to ADNP deficiency (Kapitansky et al., 2020). The neuroprotective effects of NAP have been documented in both in vivo and in vitro models of neurodegeneration (Offen et al., 2000; Spong et al., 2001; Steingart et al., 2000; Gozes et al., 2004). Notably, NAP exhibited neuroprotective effects at subfemtomolar concentrations against the toxicities associated with tetrodotoxin, beta-amyloid peptide, N-methyl-d-aspartate, and the human immunodeficiency virus envelope protein (Bassan et al., 1999). Furthermore, it has been observed that the peptide can counteract various insults such as ischemia and traumatic brain injury and prevent cell death by reducing tau hyperphosphorylation and inhibiting caspase-3 activation (Idan-Feldman et al., 2012).

By using an in vitro model of ALS, we demonstrated that PACAP by binding to PAC1R prevented motor neuron death induced by serum deprivation through EGFR transactivation and induced MAPK/ERK1/2 survival signaling pathway (Maugeri et al., 2019a, Maugeri et al., 2019b, Maugeri et al., 2019c). This peptide is also able to counteract SOD1-G93A motor neuron degeneration by interfering with a hypoxia-induced autophagy process and by modulating the MAPK/ERK pathway (D'Amico et al., 2020). Furthermore, it reduced apoptotic cell death in human induced pluripotent stem cells (iPSC)-derived motor neurons (Bonaventura et al., 2018).

To date, it has not been demonstrated whether the peptide carries out a protective effect on SOD1-G93A motor neurons by counteracting OS induced by trophic factors deprivation. In a previous paper, it was demonstrated that in rats exposed to hypobaric hypoxia, ADNP prevented brain damage by counteracting OS and by modulating Nrf2 activation (Sharma et al., 2011). Based on the above-mentioned evidence, this study aimed to investigate whether the protective effect of PACAP on SOD1-G93A motor neurons deprived by trophic factors was also mediated by ADNP activation counteracting OS and modulating the intracellular translocation of Nrf2. Our findings revealed that PACAP treatment increased the expression of ADNP in SOD1-G93A motor neurons exposed to serum starvation. To study the protective effects of ADNP, we treated serum-deprived cells with a small synthesis fragment of ADNP, known as NAP. Here, we observed that NAP prevented cell death counteracting ROS formation and activating Nrf2 translocation from the cytoplasm to the nucleus. The present data suggested a trophic role of the PACAP/ADNP axis on motor neuron survival in ALS.