Amyotrophic lateral sclerosis (ALS) is a neurodegenerative condition that involves the targeted degeneration of motor neurons. The precise pathogenetic mechanisms are still largely unclear. In this study, we utilized the SOD1-G93A mouse model of ALS to investigate the effects of folic acid (FA), an important factor involved in homocysteine metabolism. Our results indicated that a little FA prolonged the lifespan of the SOD1-G93A mice and medium and high-dose FA significantly shortened the lifespan of the SOD1-G93A mice. Furthermore, we observed that an overdose of FA significantly elevated inflammation levels in the cerebellum, as evidenced by increased concentrations of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), cluster of differentiation 68 (CD68), cluster of differentiation 86 (CD86), and monocyte chemoattractant protein-1 (MCP-1), facilitating microglial activation. The low-dose FA effects were the opposite of the overdose FA. Additionally, high-dose FA increased the levels of phosphorylated p65 (p-p65)/p65, thereby promoting the NF-κB signaling pathway. Conversely, high-dose FA inhibited the expression of total superoxide dismutase (T-SOD) while increasing the level of malondialdehyde (MDA) in the cerebellum; these effects were not observed in muscle tissue. Moreover, high-dose FA elevated the levels of Bax2/Bcl-2. Concurrently, high-dose FA induced increased apoptosis in SOD1-G93A mice, as demonstrated by TUNEL staining. Notably, the brain iron content of SOD1-G93A mice remained unchanged following FA treatment. These findings provide evidence that an overdose of FA supplementation may exert pro-inflammatory effects by promoting the NF-κB pathway, oxidative stress, and apoptosis in the context of ALS.
Peroxynitrite (ONOO-) is a reactive nitrogen species implicated in oxidative injury and impaired calcium handling. We examined whether ONOO- inactivates the cardiac sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) and whether heat shock protein 70 (Hsp70) preserves SERCA2a function during ONOO- exposure. Human embryonic kidney (HEK-293) cells were co-transfected with SERCA2a and either empty pMT2 vector (S2a-pMT2) or Hsp70 (S2a-Hsp70). Cells were untreated or exposed to ten nominal 250 μM additions of ONOO-, delivered once per minute for 10 min. Hsp70 abundance was greater in S2a-Hsp70 cells, although the magnitude of this difference varied by treatment (interaction, p = 0.0015). SERCA2a abundance was unaffected by transfection condition or ONOO- exposure. Co-immunoprecipitation showed that Hsp70 and SERCA2a were present in the same protein complex under the conditions examined. ONOO- reduced maximal SERCA2a activity by approximately 20% in S2a-pMT2 cells (p = 0.03), whereas activity was preserved in S2a-Hsp70 cells. ONOO- also increased SERCA2a-associated reactive carbonyl content by approximately 80% in S2a-pMT2 cells (p = 0.01), with no increase in S2a-Hsp70 cells. SERCA2a 3-nitrotyrosine content and FITC-binding capacity did not differ among groups. These findings show that Hsp70 overexpression preserves SERCA2a activity during acute ONOO- exposure and that this protection is associated with reduced SERCA2a carbonyl accumulation.
