Necrostatin-1: a promising compound for neurological disorders

Necrostatin-1, a small molecular alkaloid, was identified as an inhibitor of necroptosis in 2005. Investigating the fundamental mechanism of Necrostatin-1 and its role in various diseases is of great significance for scientific and clinical research. Accumulating evidence suggests that Necrostatin-1 plays a crucial role in numerous neurological disorders. This review aims to provide a comprehensive overview of the potential functions of Necrostatin-1 in various neurological disorders, offering valuable insights for future research.

Frontiers in Cellular Neuroscience 02 frontiersin.orgpaper also introduces the "Toxicity of Necrostatin-1 in nervous system" and "Necrostatin-1 plays a neuroprotective role via other cell death pathways." These findings will offer valuable insights for future research.
Necroptosis plays an important role in various organs, such as the bone, brain, heart, kidney, skin, lungs, colon and so on.

Signaling pathway of Necrostatin-1
Necroptosis is a form of programmed necrosis that is independent of caspase regulation.When caspase is inhibited or not activated, necroptosis is activated (Zanetti and Weinlich, 2021).Previous studies have indicated that necrostatins are a class of compounds that inhibit RIP1.In normal and pathological conditions, necrostatins play an important role by inhibiting necroptosis or other pathways.In cells, necroptosis can be initiated by multiple upstream regulators, including TNF-α, FASL, APO-1 L, TRAIL, and IFN-α/β.Among them, TNF-α is the most important upstream regulator of necroptosis (Kearney et al., 2015;Pinci et al., 2022) (Figure 2).The binding of TNF-α to TNFR1 on the cell membrane stimulates different signaling pathways, including necroptosis, RIP1-dependent apoptosis (RDA), RIP1independent apoptosis (RIA), and nuclear factor kappa B (NF-κB).Meanwhile, RIPK1, RIPK3, and MLKL are important downstream regulators of necroptosis.The mechanism of necroptosis is related to the activation of RIP1, RIP3 and MLKL (Cao and Mu, 2021) (Figure 3).By interacting with the T-loop, necrostatins can potently inhibit RIP1 autophosphorylation.RIP1 phosphorylation leads to the recruitment of RIP3 to RIP1 and subsequent formation of RIP1-RIP3 complex.This complex induces the phosphorylation of MLKL, which forms small holes in the plasma membrane.Eventually, disruptions of the plasma membrane lead to cell death (Cao and Mu, 2021).Therefore, necrostatins efficiently blocks RIP1/RIP3/MLKL signal transduction by inhibiting RIP1 phosphorylation.Interestingly, Necrostatin-1 has no direct inhibitory effect on RIP3 and does not block its autophosphorylation.In addition, necrostatins may Main roles of necroptosis in various tissues.

Necrostatin-1 and other cell death pathways
Increasing studies show that Necrostatin-1 not only suppresses necroptosis but also inhibits other cell death pathways (ferroptosis, apoptosis, pyroptosis).Ferroptosis is caused by the iron-mediated accumulation of lipid peroxidation, which is distinct from apoptosis and necroptosis (Newton et al., 2024).Necrostatin-1 not only perform a critical role in necroptosis but also in ferroptosis and maintain significant cellular mechanism.Yuk et al. (2021) demonstrated that Necrostatin-1 blocked ferroptosis through a mechanism independent from RIP1 and IDO inhibition in Huh7 and SK-HEP-1 cells.Caspase-8 is an executor of apoptosis.The aggregation of caspase-8 can lead to self-activation and activation of exogenous apoptotic pathways.Meanwhile, they promote the degradation of RIP1/RIP3 and lead to the closure of necroptosis signaling pathways (Fritsch et al., 2019).Some studies have explored the role of Necrostatin-1 on brain injury and its relationship with cell death pathways.They found that Necrostatin-1 not only blocked the occurrence of necroptosis but also significantly inhibited the expression of caspase-3 (an apoptosis-associated protein) and beclin-1 (an autophagyassociated protein) (Wang et al., 2012).In addition, Necrostatin-1 attenuates caspase-1-dependent pyroptosis induced by the RIP1/ ZBP1 pathway in ventilator-induced lung injury (Shao et al., 2022).

Toxicity of Necrostatin-1
Although numerous studies have shown that Necrostatin-1 plays a neuroprotective role, there is evidence to support that Necrostatin-1 may damage the nervous system.In rotenone-induced PD model, Necrostatin-1 abolished necroptosis but did not prevent toxicity (Ye et al., 2023).Most likely, Necrostatin-1 activates a switch between cell death pathways.We think that Necrostatin-1 induces apoptosis and necroptosis by inhibiting mitophagy and promoting the accumulation of mitochondrial damage.Autophagy and necroptosis play an important role in most neurodegenerative diseases.Goodall et al. described a strong interaction between necrosome components and autophagy-related proteins.The knockdown of Necrostatin-1 abrogates this interaction and promotes apoptosis (Goodall et al., 2016).The inhibitory effect of Necrostatin-1 on autophagy has been reported in 6-hydroxydopamine treated neurons (Wu et al., 2015).Additionally, RIP1 knockdown upregulated autophagy, while Necrostatin-1 was shown to downregulate autophagy (Yonekawa et al., 2015).By inhibiting mitophagy, Necrostatin-1 affects mitochondrial morphology and mitochondrial clearance, which could enhance the effect of any Parkinsonian toxin (Alegre-Cortés et al., 2020).These different research results indicate that the underlying mechanism among Necrostatin-1, necroptosis and apoptosis is a complicated network, which is why Necrostatin-1 exhibits different effects in the nervous system.

Necrostatin-1 and inflammation
Neurodegenerative diseases are a large group of neurological disorders characterized by neuronal loss, including Alzheimer's disease (AD), Parkinson's disease (PD), and others (Dugger and Dickson, 2017).Although these neurodegenerative diseases have different pathogenetic mechanisms, inflammation plays a crucial role in their progression.Inflammation is the body's defensive response to stimuli, and there is a mutually reinforcing effect between necroptosis and inflammation (Pasparakis and Vandenabeele, 2015).Necroptosis eventually leads to the release of cellular contents, causing an inflammatory response.Simultaneously, inflammation induces necroptosis via pro-inflammatory mediators (Kearney et al., 2015).Therefore, inhibiting necroptosis has great potential for treating neurodegenerative diseases by reducing inflammation.RIP1, a key target of necroptosis, promotes inflammatory responses via necroptotic cell death.In addition to inducing necroptotic cell death, RIP1 can also directly induce inflammation by producing pro-inflammatory cytokines, independent of cell death (Ofengeim and Yuan, 2013).As an inhibitor of RIP1, Necrostatin-1 exhibits significant anti-inflammatory effects in various inflammatory diseases, including hepatitis, pneumonia, and arthritis (Zhou et al., 2013;Jhun et al., 2019).Apoptosis of neutrophils is necessary for the resolution of inflammation.Necrostatin-1 is not only an inhibitor of necroptosis but also a promoter of neutrophil apoptosis, inhibiting the development of inflammation (Jie et al., 2016).Indoleamine 2,3-dioxygenase (IDO), a rate-limiting enzyme of tryptophan  10.3389/fncel.2024.1408364Frontiers in Cellular Neuroscience 04 frontiersin.orgcatabolism, plays a crucial role in inflammation.Necrostatin-1 is also an inhibitor of IDO (Vandenabeele et al., 2013), suppressing inflammation through this mechanism in addition to necroptosis inhibition.Neuroinflammation is responsible for generating and sustaining the sensitization of nociceptive neurons that lead to chronic pain.Liang et al. found that Necrostatin-1 ameliorates neuropathic pain by inhibiting neuroinflammation (Liang et al., 2019).
6 Necrostatin-1 and reactive oxygen species Reactive oxygen species (ROS), highly reactive chemical substances, have long been studied in nervous system diseases (Singh et al., 2019).ROS, as regulators of mitochondrial dynamics, regulate neuronal development and function.However, a dramatic increase in ROS levels leads to cell structure damage under harmful conditions (Singh et al., 2019).Relevant studies indicate that the generation of ROS is probably RIP1-dependent (Jantas and Lasoń, 2021).ROS can increase the expression of RIP1/RIP3 and improve the stability of the RIP1-RIP3 complex (Chauhan et al., 2017).Glutamate, an important neurotransmitter, plays a crucial role in various neurological diseases.In HT-22 cells, Necrostatin-1 inhibits glutamate-induced oxytosis by increasing cellular glutathione (GSH) and reducing ROS (Xu et al., 2007).Additionally, Necrostatin-1 suppresses the phosphorylation of ERK1 and ERK2 after glutamate treatment (Zhang et al., 2013).CoCl2-induced neurotoxicity is associated with ERK1/2 phosphorylation and ROS production, which inhibit cell differentiation and lead to cell death.Chen R. et al. (2018) found that Necrostatin-1 inhibits CoCl2-induced neurotoxicity by decreasing ROS production and ERK1/2 phosphorylation.In H 2 O 2 -induced SH-SY5Y cell lines, Necrostatin-1 reduces oxidative stress-induced cell damage by inhibiting cathepsin D (Jantas et al., 2020).In peripheral nerve injury (PNI) and spinal cord injury (SCI) rat models, Necrostatin-1 can reduce ROS and inflammation (Yu et al., 2023).Further studies indicate that Necrostatin-1 not only inhibits necrosis by inhibiting RIP1/RIP3/MLKL but also inhibits apoptosis by activating Bcl-2 (Wang et al., 2014).

Ischemic stroke and ischemia/ reperfusion
Ischemic stroke (IS) often results in injury to oligodendroglia.Oligodendrocyte precursor cells (OPCs) are more vulnerable to cerebral ischemia than other mature oligodendroglia.Necrostatin-1 significantly promotes oligodendrocyte precursor cell survival and reduces white matter damage after cerebral ischemia (Chen et al., 2018a) through the RIPK1/RIPK3/MLKL signaling pathway (Deng et al., 2019).Necrostatin-1 also provides neuroprotection in neonatal hypoxiaischemia (HI) by preserving mitochondrial function (Chavez-Valdez et al., 2012).Cerebral ischemia/reperfusion (I/R) induces selective neuronal injury in the CA1 region of the hippocampus.In cerebral I/R rats, Necrostatin-1 improves locomotive ability and relieves anxious behavior while decreasing the death rate of neurons through the RIP3/ DAXX signaling pathway (Yang et al., 2017).Traumatic brain injury (TBI) is a leading cause of cerebral I/R injury.In a TBI mouse model, You et al. found that Necrostatin-1 has anti-inflammatory effects (You et al., 2008), while Wang et al. found that Necrostatin-1 inhibits autophagy and apoptosis (Wang et al., 2012).These results suggest that Necrostatin-1 may have therapeutic potential for IS and cerebral I/R.

Parkinson's disease
PD is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta.Several types of cell death, including apoptosis, autophagy-induced cell death, and necrosis, have been implicated in PD progression.In PD models, Necrostatin-1 prevents rotenone-induced necroptosis by affecting mitochondrial morphology (Alegre-Cortés et al., 2020) and exerts a protective effect on dopaminergic neurons by decreasing the expression of cathepsin B and increasing the expression of Bcl-2 (Wu et al., 2015;Jantas and Lasoń, 2022).

Epilepsy
Epilepsy is a common, highly debilitating neurological disease characterized by the abnormal discharge of brain neurons.Necrosis and apoptosis are the major forms of neuronal death post-epilepsy.In an epileptic mouse model, Necrostatin-1 significantly decreases damage to hippocampal tissue and downregulates apoptosis/ necroptosis-related proteins such as cleaved-caspase-3, Bax, RIP1, RIP3, and MLKL (Lin et al., 2020).A 40 μM concentration of Necrostatin-1 has an optimal effect (Lin et al., 2020), and inhibition of necroptosis may prolong seizure latency (Guan et al., 2021).

Other RIP inhibitors in neurological disorders
Increasing evidence suggest that RIP inhibitors play an important role in neurological pathologies.Necroptosis-associated RIP inhibitors include RIP1 inhibitors and RIP3 inhibitors (Figure 4  and Table 1).Besides the Necrostatin-1, Necrostatin-1 s is another important RIP1 inhibitor.Preeti et al. (2023) want to evaluate the neuroprotective effect of Necrostatin-1 s in the type-2 diabetes mellitus model.They found that Necrostatin-1 s mitigates cognitive decrement.Further, Necrostatin-1 s reduced tau and amyloid oligomer load.In the periventricular leukomalacia model, the expression level of RIP1 was drastically increased.Necrostatin-1 s greatly ameliorated cerebral ischemic injury and long-term neurobehavioral abnormalities, exhibiting a reduction of cerebral infarct size and neuronal loss (Sun et al., 2024).In addition, Kartik et al. (2023) found that Necrostatin-1 s significantly improve the survival of dopaminergic neurons in the PD mouse model.Other RIP1 inhibitors such as GSK772, PK68, GSK095, and GSK547 were not reported to improve nerve damage.GSK872 is a widely used RIPK3 inhibitor.Similar to Necrostatin-1 s, GSK872 improves various nerve damage such as retinal neuroinflammation, neurodegeneration, SCI, hydrocephalus and so on (He et al., 2021;Liu et al., 2021;Huang et al., 2023).Necrosulfonamide is a specific MLKL inhibitor.In a transient middle cerebral artery occlusion (tMCAO) rat model, necrosulfonamide reduces infarction volume and improves neurological deficits (Zhou et al., 2023).Besides the neuroprotective effects of tMCAO, necrosulfonamide also ameliorates SCI and intracerebral hemorrhage injury (Wang et al., 2018;Zhang et al., 2022).Interestingly, necrosulfonamide increased cleaved PARP-1 levels, indicating the protective effects of necrosulfonamide is not related to apoptosis (Zhou et al., 2017).

Application of Necrostatin-1
Beyond treating various diseases, Necrostatin-1 plays a crucial role in plastic surgery, preservation, transplantation, and inhibition of drug toxicity.Plastic surgery failure is a challenge for the medical cosmetology industry.Increasing research shows that Necrostatin-1 can treat various I/R injuries, such as those affecting the heart, lung, kidney, and skeletal muscle.In flap surgery, I/R injury is considered the primary problem.Liu et al. (2019) found that Necrostatin-1 has a protective effect against I/R injury in a skin flap model.These results suggest that Necrostatin-1 could be a promising novel strategy in plastic surgery.Cryopreservation of spermatogonial stem cells (SSCs) is important for preserving the lineages of valuable livestock and producing transgenic animals.As a potential cryoprotectant, Necrostatin-1 improves the cryopreservation efficiency of SSCs (Jung et al., 2020).Jo et al. (2015) also found that Necrostatin-1 improves the survival of mouse oocytes.Numerous studies show that Necrostatin-1 promotes the maturation, development, and graft function of neonatal porcine islets (Lau et al., 2020a(Lau et al., ,b, 2021)), providing an effective strategy for the future application of islet grafts (Qin et al., 2022).Emerging evidence suggests that Necrostatin-1 has potential radical scavenging activities (Ushijima and Monzaki, 2023).Ning et al. found that Necrostatin-1 can decrease cisplatin-induced nephrotoxicity by inhibiting oxidative stress (Ning et al., 2018).Takemoto et al. discovered that Necrostatin-1 ameliorates acetaminophen-induced hepatotoxicity by inhibiting ROS (Takemoto et al., 2014).These results suggest that Necrostatin-1 has some benefit in alleviating drug toxicity.Interestingly, Necrostatin-1 can mitigate and treat radiation-induced damage in mice (Huang et al., 2016).

Discussion
In this review, we explored the mechanisms and roles of Necrostatin-1 in various neurological disorders (Table 2).Meanwhile, we propose that Necrostatin-1 has great clinical potential in the treatment of these disorders.In addition to treating various diseases, Necrostatin-1 plays an important role in plastic surgery, preservation, transplantation, and inhibition of drug toxicity.Nevertheless, there are still many questions regarding Necrostatin-1 that need to be addressed.First, Necrostatin-1 has a short half-life, which may affect its application.Second, it remains unclear whether Necrostatin-1 can affect one or multiple RIP1-dependent pathways in various neurological disorders.These findings suggest that the mechanism of

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TABLE 2
Role of Necrostatin-1 in neurological disorders.