Normal neuronal structure and maintenance of neuritic processes is essential for cognitive activity and adequate motor coordination in the brain [1]. Decreased neuritic processes is an early hallmark of neurodegenerative diseases, such as Alzheimer's disease or amyotrophic lateral sclerosis [2], and is enhanced by oxidative damage and reactive oxygen species (ROS) generation. Thus, antioxidant molecules are essential to maintain normal brain function [3].

Vitamin C is an essential micronutrient with two chemical forms, ascorbic acid (AA), its reduced form, and dehydroascorbic acid (DHA), its oxidized form. Physiologically, vitamin C is mainly found as AA, which, among other functions, acts as an antioxidant in the central nervous system [[4], [5], [6]].

Vitamin C homeostasis is maintained through its recycling between neurons and astrocytes. Once AA is taken up by neurons via SVCT2 transporters [7,8], it is intracellularly oxidized to DHA, which is released to the extracellular fluid through DHA transporter, GLUT3 [9], and taken up by astrocytes via GLUT1 [10], where is intracellularly reduced to AA, thus allowing for constant vitamin C concentrations in brain parenchyma [[11], [12], [13]]. However, under pathophysiological conditions, characterized by an increase in oxidant species in the brain, extracellular DHA generation may occur [12], allowing for its accumulation inside neurons.

Different reports have shown the effects of high levels of DHA. For instance, we have previously described that treating cortical neurons with increasing DHA concentrations significantly lowers reduced glutathione (GSH) levels and glycolytic rates [14]. Similar results were observed in KRAS and BRAF mutant colorectal cancer cells exposed to DHA, where metabolomic analyses also suggested the inhibition of the enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [15]. DHA has also been associated with cell death, with N2a and HN33.11 cells showing low cell viability when treated with DHA in oxidative stress conditions [16]. Furthermore, combined treatment of AA and hydrogen peroxide induced cell death with activation of receptor-interacting serine-threonine protein kinase-1 (RIPK1) [17], a scaffolding protein relevant for inflammatory and cell death response [[18], [19], [20], [21]].

RIPK1 is part of the Ser/Thr receptor-interacting protein kinase family, which has seven members [19,22]. RIPK1 is composed of an N-terminal kinase domain, an intermediate domain containing a RIP homotypic interaction motif (RHIM), and a C-terminal death domain [18,20,21]. To date, the only RIPK1 substrate described is itself. While several autophosphorylation sites have been described [18,23,24], induction of cell death has only been reported with phosphorylation on Ser161 and Ser166 [25,26].

Once a ligand binds to its death-domain containing receptor (e.g., TNFα to TNFR1), a multiprotein complex called complex I assembles, where RIPK1 acts as a scaffold protein and is inhibited through different ubiquitination and phosphorylation events [[27], [28], [29], [30], [31], [32], [33]]. Under these conditions, the canonic NF-κB pathway is activated, inducing the expression of pro-survival and proinflammatory genes [34,35]. However, disassembly of complex I allows for the formation of cytosolic complex IIa, where procaspase-8 is activated, cleaving RIPK1 and inducing RIPK1-independent apoptosis [36]. However, if RIPK1 inhibition is inhibited, procaspase-8 is activated and recruited, forming complex IIb and inducing a RIPK1-dependent apoptosis [18]. Finally, under conditions in which caspase-8 is inhibited or absent, RIPK1 is activated, recruiting RIPK3 and MLKL effector protein, which translocate and generate pores in the plasma membrane, activating necroptosis [36].

Recent studies from our group using NE have shown that prolonged AA treatment induces significant loss of cellular neurites, which is prevented in the presence of a DHA recycling cell, such as astrocytes [37]. This is also important in vivo, since inhibition of GLUT1 in astrocytes inhibits neurite formation in the cerebral cortex [38]. However, whether these effects are associated with RIPK1 activation is still unknown.

In this work, we evaluated the effect of DHA accumulation on neurites in NE and the role of RIPK1 under these conditions. Increasing DHA concentrations led to shortening of cellular processes, which was associated with early RIPK1 phosphorylation. This effect was partially prevented by treatment with a specific RIPK1 inhibitor, Necrostatin-1s, and completely rescued by a pan-caspase inhibitor, zVAD-FMK. Overall, these results suggest that RIPK1- and caspase-dependent mechanisms underly the impact of DHA.