Keywords

1 Introduction

Neurodegenerative diseases and stroke are considered worldwide one of the most important medical and economic problems in aging society. There is a firm consensus that understanding of molecular mechanisms of neuronal death is the rational way for designing clinically efficient neuroprotective agent(s). The key role of oxidative stress in acute and chronic brain damage has been firmly established. In the aging brain there is overproduction of reactive oxygen species (ROS) in conjunction with a reduction in the antioxidant defense. The brain is particularly vulnerable to oxidative stress because of its high oxygen consumption. The brain consumes about 20% of the body’s total oxygen due to its metabolic activity and high need for ATP. The brain is also rich in fatty acids which are prone to oxidative damage and has relatively poor antioxidant systems. Moreover, presence of free iron and other transition metals could also facilitate neurodegeneration in particular brain regions. The most common pathomechanism of neuronal damage involves excitatory amino acid–driven excitotoxicity, and dysregulation in calcium homeostasis, which are followed by perturbations in mitochondria function, activation of proteolitic enzymes, increased production of deleterious ROS, and decreased cell defense system. Cytoskeletal disruptions, protein aggregations, deficiency of neurotrophic factors, and inflammation also occur in neurodegenerative disorders. Further remarkable progress in biomedical sciences revealed several forms of cellular death with various morphological and biochemical characteristics, which may be roughly classified as unprogrammed and programmed cell deaths. Of them necrosis is described as an unprogrammed passive process triggered by disruption of normal ion fluxes and cell death begins within minutes of injury. Its main features are mitochondrial damage with rapid energy loss, swelling and vacuolation of cell body, rupture and dissolution of nucleus, and cell lysis, with inflammation injuring adjacent tissue. Identified so far programmed cell death forms include apoptosis, necroptosis, autophagy-dependent cell death, parthanatos, pyroptosis, and ferroptosis (Conrad et al. 2016, Fan et al. 2017). Apoptosis is an active process of programmed cell destruction associated with changes in gene expression and protein synthesis occurring hours to days after injury. It is characterized by condensation and fragmentation of nuclear chromatin (DNA laddering), compaction and shrinking of cell body, the loss of phospholipid asymmetry, and budding of apoptotic bodies and phagocytosis, without inflammation or damage to surrounding tissue. Besides its involvement in pathological processes, this form of cell death is also essential for proper embryologic, pre- and postnatal development. Autophagy-dependent cell death helps to maintain cell homeostasis via removing unnecessary or dysfunctional components and delivering them from cytoplasm to lysosomes for degradation. The cells which undergo autophagy show presence of cytoplasmatic vacuoles and there is lack of chromatin condensation. Parthanatos refers to a programmed cell death pathway triggered by excessive activation of poly (ADP-ribose) polymerase 1 (PARP1) and subsequent lethal depletion of nuclear NAD+. Ferroptosis is an iron-dependent form of regulated cell death associated with ROS accumulation and lipid peroxidation. Ferroptosis has been implicated in pathogenesis of cancer and neurodegenerative diseases. Since it is closely associated with oxidative stress, it can be prevented by inhibitors of lipid peroxidation, lipophilic antioxidants, and iron chelators (Conrad et al. 2016, Fan et al. 2017). Necroptosis (or necroptotic cell death) is the most widely studied form of nonapoptotic programmed cell death pathway characterized by necrotic cell morphological features, like the loss of plasma membrane integrity, swelling of cellular organelles, and lack of typical nuclear fragmentation (Vandenabeele et al. 2010). Apart from serving an important physiological role during development and adulthood (especially in the regulation of the immune system and inflammatory response), necroptosis has been reported to occur during acute injuries of various organs (brain, heart, lung, kidney, liver, intestine, and pancreas) suggesting that its inhibition can constitute a possible therapeutic intervention (Cao & Mu, 2020; Zhao et al. 2015). Moreover, together with other programmed forms of cell death, like apoptosis or autophagy, necroptosis has been associated with the pathogenesis of various neurodegenerative diseases (e.g., Alzhemier’s disease [AD], Parkinson’s disease [PD], amyotrophic lateral sclerosis [ALS], and Huntington’s disease [HD]) (Cao & Mu, 2020; Zhang et al. 2017). Since the aim of this chapter is to describe neuroprotective effects of the first-generation necroptosis inhibitor, necrostatin-1 (Nec-1), only some basic information on the mechanisms of necroptotic cell death pathway will be given just to enable a proper understanding of action of necroptosis-targeted compounds.

Necroptotic cell death is a caspase-independent process whose key players are two members of receptor-interacting serine/threonine-protein kinase (RIP) family, RIP1 and RIP3 (known also as RIPK1 and RIPK3). The molecular mechanisms and cellular signaling pathways involved in necroptosis are quite complex and some steps can be shared with other types of programmed cell death pathways (e.g., extracellular apoptosis) which are often regulated in a context- and cell-type-dependent manner (Vandenabeele et al. 2010, Vanden Berghe et al. 2015, Zhang et al. 2017, Zhao et al. 2015). One of the most widely investigated triggers of necroptosis is TNFα (tumor necrosis factor alpha) and its receptor TNFR1 which together initiate this cell death pathway (Fig. 1) in various cell types under conditions of deficient or blocked caspase-8 activity (usually achieved by the use of FADD-deficient cells or by pretreatment with pan-caspase inhibitor, z-VAD-fmk) (Degterev et al. 2005). The first step in this process involves autophosphorylation of RIP1 which subsequently phosphorylates RIP3 and physically interacts with the enzyme forming a supramolecular complex called necrosome (Vandenabeele et al. 2010). Next, the activated RIP3 phosphorylates mixed lineage kinase-like protein (MLKL), enabling the formation of MLKL oligomers that translocate to the plasma membrane and cause its permeabilization. The latter process promotes the release of DAMPs (Damage Associated Molecular Patterns) such as HMGB1 (high-mobility group box 1 protein) or mitochondrial DNA which under in vivo conditions can induce immune response and inflammation (Conrad et al. 2016, Vandenabeele et al. 2010, Zhao et al., 2015). Apart from TNFR also other death receptors, like Fas or TRAIL (TNF-related apoptosis-inducing ligand), have been reported in various in vitro studies to activate necroptosis under conditions of caspase deficiency (Vandenabeele et al. 2010). Moreover, among other potential inducers of necroptotic cell death, lipopolysaccharide (LPS), toll-like receptors (TLRs), bacteria and viruses, oxidative and genotoxic stress, excitotoxicity, and calcium overload have been listed. However, the detailed mechanisms of their contribution to the activation of this cell death route still remain obscure (Conrad et al. 2016). Some data from studies with transgenic models and enzymatic inhibitors suggest that RIP1, RIP3, or MLKL could be targeted for treatment of various inflammatory, degenerative, and infectious disorders (Cao & Mu, 2020; Conrad et al. 2016). However, due to the pleiotropic roles of RIP1 and RIP3 in other cellular processes including apoptosis it is difficult to define precisely the participation of necroptosis in multifactorial pathomechanism of neurodegenerative diseases (Degterev et al. 2019; Zhang et al. 2017). Undoubtedly, in recent years necroptosis has become one of the most studied form of cell death, and the inhibition of some elements of this signaling pathway could significantly reduce brain damage (Cao & Mu, 2020). This chapter aims to summarize results of the animal experimental studies which suggest the potential of Nec-1 as a clinically useful neuroprotectant. Data retrieved from PubMed (2005 – March 2021) showed the ability of Nec-1 to ameliorate acute and delayed neuronal cell death induced by hypoxia-ischemia, traumatic brain and spinal cord injury, and some neurodegenerative processes (summarized in Table 1).

Fig. 1
figure 1

Mechanisms of TNFα-induced cell death. Binding of TNFα to its receptor (TNFR1) triggers the assembly of complex I: TNFR1, TRADD (TNFR1-associated death domain), RIP1, TRAF2 (TNFR-associated factor 2), cIAPs (cellular inhibitor of apoptosis proteins) and CYLD (cylindromatosis). Under translational inhibition, TNFα stimulates the formation of a cytosolic complex IIa in which RIP1 disappears, whereas TRADD and FADD (FAS-associated death domain) interact in the presence of cFLIP (cellular FLICE-like inhibitory protein) and lead to the activation of caspase 8 (cas-8) and effector caspases (caspase 3 and caspase 7, Cas-3/7) and apoptosis. Under cIAPs inhibition complex IIb is formed (RIP1, RIP3, FADD and caspase 8) which induces RIP1-dependent apoptosis. Under conditions of caspase-8 inhibition or FADD deficiency, complex IIc (necrosome) is formed via association of RIP1 and RIP3 followed by a series of auto-and transphosphorylation of RIP1 and RIP3. Activated RIP3 phosphorylates and recruits mixed lineage kinase domain-like protein (MLKL) leading to the necroptosis. Phosphorylated MLKL translocates to the plasma membrane leading to pore formation with the release of DAMPs (damage-associated molecular patterns) such as HMGB1 (high-mobility group box 1) which induce inflammatory processes.

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Table 1 Neuroprotective effects of Necrotstatin-1 in animal models
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2 Characterization of Necrostatin-1

Nec-1 was the first identified necroptosis inhibitor among the tested library of ~15.000 compounds in a phenotypic screen employing human monocytic U937 cell line exposed to TNFα+z-VAD-fmk (Degterev et al. 2005). In parallel, other chemicals (5-(1H-Indol-3-ylmethyl)-2-thiohydantoins and 5-(1H-indol-3-ylmethyl)hydantoins) named necrostatins have been designed and selected in a SAR (structure-activity relationship) study as potent inhibitors of necroptotic cell death induced by TNFα in FADD-deficient Jurkat T cells (Teng et al. 2005). Although various derivatives of Nec-1 as well other inhibitors of necroptosis have been synthesized and investigated in screening necroptotic cellular models, only some of them have been tested in animal studies. Nevertheless, Nec-1 remains the most often used research tool to study the role of necroptosis in various physiological and pathological conditions (Cao & Mu, 2020; Conrad et al. 2016).

2.1 Mechanisms of Necrostatin-1 Action

Nec-1 (5-(1H-Indol-3-ylmethyl)-(2-thio-3-methyl) hydantoin or methyl-thiohydantoin-tryptophan) is a small-molecule compound (MW = 259.33 g/mol, Fig. 2a). It has been found to be a potent inhibitor of necroptosis without evoking any cytotoxic effects up to 100μM which was superior to some other tested compounds from 5-(1H-indol-3-ylmethyl)-2-thiohydantoin group (Teng et al. 2005). Later on, Nec-1 and its analogues have been identified as potent allosteric inhibitors of kinase domain of RIP1 (Degterev et al. 2008) and description of the crystal structure of this enzyme facilitated further rational design and development of RIP1 inhibitors (Xie et al. 2013). Nec-1 inhibited TNFα-induced necroptotic cell death with an EC50 = 490 nM (Teng et al. 2005, Degterev et al., 2005) and inhibited RIP1 kinase activity in in vitro assay with EC50 = 182 nM (Degterev et al. 2008), suggesting a good correlation between these two processes. Because of its ability to stop RIP1 autophosphorylation and consequently to prevent its interaction with RIP3 and subsequent formation of necrosome and MLKL activation, Nec-1 for many years has been regarded as a potent inhibitor of RIP1-RIP3-MLKL signaling (Degterev et al., 2008, Takahashi et al. 2012). However, being a RIP1 inhibitor, Nec-1 apart from necroptosis could also inhibit RIP1-dependent apoptosis (Cao & Mu, 2020; Conrad et al. 2016).

Fig. 2
figure 2

Chemical structure of chosen members of Necrostatins family: (a) Necrostatin-1 (Nec-1); (b) Necrostatin-1 stable (Nec-1s, 7-Cl-O-Nec-1, Nec-2); (c) Necrostatin-1 inactive (Nec-1i). (Source: https://pubchem.ncbi.nlm.nih.gov)

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In order to verify Nec-1 specificity as a necroptosis inhibitor, its two analogs, namely Nec-1 stable (Nec-1 s, 7-Cl-O-Nec-1, Nec-2) (Fig. 2b) and Nec-1 inactive (Nec-1i) (Fig. 2c), were investigated in parallel with the parent drug (Degterev et al., 2005). They were chosen in a wide structure-activity relationship (SAR) study which revealed that elimination of methyl group in the hydantoin moiety of Nec-1 reduced its antinecroptotic activity (Nec-1i). Interestingly, an addition of chlorine group to the phenyl ring and substitution of sulfur to oxygen in the hydantoin moiety (Nec-1 s) improved ten times Nec-1 inhibitory effect on RIP1 activity (EC50 = 18 nM) (Degterev et al. 2008) and 2–10 times increased its protective potency in cellular models of necroptosis (Degterev et al. 2005, Teng et al. 2005). However, such differences in the potency of action of Nec-1 derivatives not always have been observed in vivo at least after their direct administration to the brain. For example, in the mouse brain ischemia model (MCAO, middle cerebral artery occlusion) an intracerebroventrical (i.c.v.) administration of Nec-1, 7-Cl-O-Nec-1 and Nec-1 s (2–4μl of 4 mM) to a similar extent reduced infarct volume and improved neurological score (Degterev et al., 2005).

Besides inhibiting RIP1 kinase, Nec-1 is also a potent inhibitor of indoleamine 2,3-dioxygenase (IDO) (EC50 = 116μM) which may contribute to its mechanism of action in inflammatory and neurodegenerative processes (Zhang et al. 2017, Zhao et al. 2015). Takahashi et al. (2012) compared Nec-1, Nec-1 s, and Nec-1i with respect to their effect on IDO activity and on necroptotic cell death inhibition, and found that Nec-1 was identical in terms of the chemical structure to methyl-thiohydantoin-tryptophan (MTH-Trp), an inhibitor of IDO. They found that both Nec-1 and Nec-1i inhibited human IDO activity with similar potency (EC50 ~ 116μM) as did 1-methyl-tryptophan (1-MT, EC50 ~ 143μM), an IDO inhibitor, whereas Nec-1 s was devoid of such activity (Takahashi et al. 2012). Although the IDO inhibitor 3-MT was not protective in the mouse cellular necroptosis assay (L929 cells exposed to recombinant human TNFα), Nec-1i was only 10× less potent than Nec-1 or Nec-1 s and was equipotent at high concentrations in the inhibition of necroptotic cell death (Takahashi et al. 2012). It is in contrast to findings from human cell–based models of necroptosis where Nec-1i was ineffective (Degterev et al., 2005, Teng et al. 2005). Moreover, in vivo high doses (6 mg/kg) of Nec-1, Nec-1i, and Nec-1 s given intravenously (i.v.) prevented the mortality associated with TNFα-induced systemic inflammatory response syndrome (SIRS) equally well, but, paradoxically, low doses of Nec-1 or Nec-1i (0.6 mg/kg) sensitized mice to TNFα-induced mortality. The abovementioned data question the relevance of using Nec-1i as a negative control for necroptosis in in vitro and in vivo studies (Takahashi et al. 2012) suggesting that species differences and/or route of administration could influence Nec-1i effects. Since Nec-1 s did not exhibit low-dose toxicity in SIRS model, it has been suggested to be preferentially used in animal studies (Takahashi et al. 2012). Since inflammation significantly contributes to the development and progression of age-related neurodegenerative diseases (Fan et al. 2017, Zhang et al. 2017), the additional activity of Nec-1 on IDO could be rather favorable when considering Nec-1 as an efficient neuroprotectant.

2.2 Pharmacokinetics of Necrostatin-1

Pharmacokinetics and bioavailability of Nec-1 in Sprague-Dawley rat plasma has been evaluated by Geng et al. (2017) using LC-MS/MS method. It seems that Nec-1 remains stable in plasma in different processing and storage environmental conditions. The plasma concentration of Nec-1 was determined in rats who received a single dose via intravenous and oral route (5 mg/kg), respectively. A two-compartment model fitted the concentration-time profile of the Nec-1 with Cmax 1733 μgL-1 and t1/2 1.8 h for intravenous route, and Cmax 648 μgL-1 and t1/2 1.2 h for oral route, respectively. The results showed that absolute bioavailability of Nec-1 was 54.8% (Geng et al. 2017). In an analytical study using the original HPLC-Q-TOF method, Mikuš et al. (2018) measured Nec-1 s plasma concentrations in Wistar rats 50 min after i.v. bolus dose (0.37 mg/kg) administration which was ca. 105 ng/ml (377 nM). However, it is difficult to compare pharmacokinectic properties of Nec-1 and Nec-1 s since there are no studies where both compounds would be tested under the same experimental conditions. Data on BBB (blood-brain barrier) crossing by Nec-1 are rather limited. In the early study on necroptosis, Teng et al. (2005) reported that Nec-1 after i.v. administration (dose 1 mg/kg) in CD1 mice had rather moderate pharmacokinetic characteristics (Cmax 873 ngL-1 and t1/2 67 min) with significantly higher removal rate in females, but it readily entered the CNS (after 30 min 0.74μM brain concentration) showing a good brain-to-plasma ratio (2.4). However, the compound was metabolically unstable and was relatively quickly cleared from the body (within 180 min) (Teng et al. 2005).

Apart from low metabolic stability, another important drawback of Nec-1 as potential clinical therapeutic is related to its low solubility in water. Usually for in vitro studies, stock solutions of this compound are prepared in DMSO (>10 mg/kg) (Degterev et al. 2008, Jantas et al. 2020) whereas for in vivo studies DMSO, ethanol, or PEG400 are used (Dionísio et al., 2019, Geng et al. 2017, Mikuš et al. 2018, Nikseresht et al. 2015). This poor physicochemical property of Nec-1 requires the usage of relevant vehicles for control groups (percentage of the solvent can reach 1–3% in vitro and up to 20% in vivo), which by itself could affect the outcome in particular experimental models of human pathologies (Geng et al. 2017, Mikuš et al. 2018).

3 Neuroprotection by Necrostatin-1 in Animal Models

3.1 Brain Ischemia

During brain ischemia (stroke) occurs the breakdown of neuronal cellular integrity by ionic imbalance, energetic collapse, excitotoxicity, and increased ROS production. These factors lead to neuronal cell damage executed via rapid nonspecific necrosis (cell lysis) in ischemia core and by programmed cell death pathways (apoptosis or necroptosis) in the penumbra region. Moreover, the injury-induced neuroinflammatory processes could worsen the extent of brain damage and neurological deficits evoked by this life-threatening episode (Mehta et al. 2007). The first evidence that necroptosis contributes to delayed ischemic brain injury in vivo in mice through a mechanism distinct from apoptosis and that it offers a new therapeutic target for stroke with an extended window for neuroprotection was provided by Degterev et al. (2005). In mice exposed to transient focal cerebral ischemia (MCAO) Nec-1, 7-Cl-Nec-1 and Nec-1 s but not Nec-1i (i.c.v. 2–4 ul of 4 mM) given before and after (during infusion and reperfusion) occlusion to similar extent (~30%) reduced infract volume and improved neurological deficits (Degterev et al. 2005). Further tested compound, 7-Cl-Nec-1 was also effective when given 2 and 6 h after MCAO and its administration was associated with the inhibition of autophagy induced during reperfusion time. Interestingly, the pan-caspase inhibitor z-VAD-fmk was protective when given 2 but not 6 h after occlusion but it synergized with Nec-1 in protection at both time points (Degterev et al. 2005). These data encouraged further animal research aiming to prove neuroprotective potency of Nec-1 and its derivatives for stroke management. In line with the studies postulating that the combination therapy rather than monotherapy may be required for efficient neuroprotection in brain ischemia, Xu et al. (2010) evaluated protective effects of combined treatment with Gly(14)-humanin (i.c.v. 0.1μg) and Nec-1 (i.c.v. 2.6μg) in the mouse transient MCAO model (75 min occlusion and 24 h reperfusion). They showed that administration of humanin or Nec-1 alone 4 h after MCAO moderately reduced cerebral infarct volume (~12%) but combined treatment with both agents to a higher extent decreased lesion size (up to 20%) and most importantly improved also neurological score (Xu et al. 2010).

Using the rat model of global cerebral ischemia (four-vessel occlusion) followed by reperfusion (I/R model), Yin et al. (2015) found that Nec-1 (i.c.v. 0.1 and 1μg) administered 1 h before ischemia in a dose-dependent manner protected hippocampal CA1 neurons which was accompanied by a decrease in expression and nuclear translocation of RIP3, increased NAD+ level, and attenuated cathepsin B (Yin et al. 2015). In a similar experimental model, Yang et al. (2017a) showed that 1 h pretreatment with Nec-1(i.c.v. 1μg.) 5 days after induction o I/R prevented locomotor deficit, relieved anxiety behavior, and improved cognitive functions in rats. Moreover, Nec-1 protected neurons in the hippocampal CA1 region against ischemic damage through newly identified RIP3 nuclear substrate DAXX (death associated protein) which appeared to play an important role in I/R-induced neuronal death (Yang et al. 2017a). In another study, Ni et al. (2018) in the permanent MCAO model in rats found that Nec-1 (i.c.v. 24 nM) pretreatment (10 min before MCAO) or RIP1 knockdown (shRNA RIP1 given 5 days before MCAO) were protective and attenuated neurological deficits. This beneficial effect of Nec-1 was evidenced in the ischemic cortex by the reduced infarct volume (~10%), improved neuronal (MAP-2) and astrocyte (GFAP) survival, decreased RIP1–RIP3 interaction, and attenuated level of autophagy (LC3II) and lysosomal (cathepsin B, lysosomal membrane permeability) markers. Moreover, they confirmed protective effects of Nec-1 in the mouse pMCAO model where this compound was effective (~15% reduction of infarct volume) in pretreatment (10 min before MCAO) and posttreatment (6 h after MCAO) schedules (Ni et al. 2018).

Ischemic stroke, apart from neuronal and astrocyte damage, can also cause a severe injury to white matter, especially to oligodendrocyte precursor cells (OPCs) and premature oligodendroglia. Chen et al. (2018) showed that Nec-1 (i.c.v. 0.04 mg/kg) injected to mice 1 h before the onset of 60-min MCAO followed by 24 h reperfusion, 21 days after injury significantly promoted OPC survival, alleviated white matter injury, and improved cognitive function. It was accompanied by a decrease in RIP1, RIP3, and pMLKL expression in the subventricular zone and corpus callosum (Chen et al. 2018). In another study, a novel analogue of Nec-1, DTIO was tested in the mouse and rat brain ischemia models (Li et al. 2018). In the mouse permanent MCAO model, DTIO (i.v. 5 and 10 mg/kg, given 3 h after the occlusion) reduced brain damage by about 30% and attenuated neurological deficits. The same group tested the DTIO (10 mg/kg) effect in the rat transient MCAO model (90 min MCAO) where this compound was given i.v. 3 h after MCAO and starting from day 2 it was administrated intraperitoneally (i.p.) once a day for 28 days. At these two end points DTIO significantly reduced infarct volume (~20% at 24 h) and improved neurological performance without influencing cerebral blood flow. Moreover, DTIO inhibited ischemia-induced release of pro-inflammatory cytokines (TNFα, IL-1β, and IL-6) in the cerebral cortex 12 h and 7 days after the induction of permanent or transient ischemic episodes, respectively. Moreover, in a rat model DTIO prevented the formation of glial scar as found 7 days after MCAO by evaluation of expression of astrocytic markers (GFAP, phosphacan and neurocan) (Li et al. 2018).

A relationship between sirtuin 1 (Sirt1), a member of histone deacetylase class III, and the necroptosis signaling pathway and its downstream events has been explored by administration of ex-527 (selective and potent inhibitor of Sirt1) and Nec-1 in the rat model of focal cerebral ischemia (Nikseresht et al. 2019). They demonstrated that 60 min pretreatment with Nec-1 (i.c.v. 1 μg) reduced the infarct volume of ischemic brains to a significantly higher extent (~35%) than the inhibitor of Sirt1 (10μg, ex-527) did. Although both agents reduced RIP3 and MLKL in the prefrontal cortex and hippocampus, Nec-1 did not affect Sirt1 expression. Moreover, both agents improved survival rate, but only Nec-1 attenuated stroke-associated neurological deficits (Nikseresht et al. 2019). A direct in vivo evidence that phosphorylated RIP1 (Ser 166) plays an important role in the initiation of RIP3/MLKL-dependent necroptosis in the pathogenesis of ischemic stroke in the rodent brain was provided by Deng et al. (2019). In the rat MCAO model they found that Nec-1 but not Nec-1i (i.c.v. 1.5μl of 20 mM) given 30 min before occlusion reduced infract volume by about 40% and attenuated ischemia-associated neurological deficits. It was accompanied with a decrease in the number of p-RIP1-positive neurons and amelioration of p-RIP1, RIP3, MLKL, p-MLKL, and mature IL-1β expression, suggesting a direct interplay between RIP1 and inflammation after I/R (Deng et al. 2019).

Some brain ischemia studies brought new findings about new regulators of necroptosis. For example, Li et al. (2019) showed an interplay between TRAF2 and RIP3 and MLKL in the mouse transient MCAO model. The TRAF2 expression was induced by MCAO at 24 h after reperfusion in cortical and striatal neurons and microglia. Interestingly, only striatal knockdown of TRAF2 increased the infarct size and cell death which was associated with microglial activation and increased expression of pro-inflammatory cytokines (TNFα, IL-1β and CD32). In this model, Nec-1 (i.c.v. 3μl of 4 mM) given just before occlusion reduced the neurological deficits induced by ischemia and decreased the number of necrotic (PI-positive) cells in the striatal regions in wild type and TRAF2−/− mice (Li et al. 2019). Mu et al. (2021) in the rat transient MCAO model showed that i.p. injection of Nec-1 but not Nec-1i (0.5μl/g body weight of the 4 mM stock solution right after occlusion, repeated at 2 h after the onset of reperfusion and once every next 12 h) significantly reduced the infarct area (by about 30%) and increased the survival of neurons. Moreover, this effect was accompanied by the inhibition of AIF nuclear translocation induced by I/R (Mu et al. 2021). Nec-1 may also provide neuroprotection in ischemic stroke through reduction of microglial neuroinflammation. To this end, Jiao et al. (2020) showed in the transient MCAO model in mice that recombinant human thioredoxin-1 (rhTrx-1, a putative RIP1 inhibitor, i.v. 10 mg/kg, given just after occlusion) significantly reduced the infarct volume (~15%) and improved neurological functions. It was associated with changes in microglia polarization (decrease in M1 and increase in M2 phenotypes) and attenuation of inflammatory cytokines (TNFα and IL-1β) (Jiao et al. 2020).

3.2 Neonatal Hypoxia-Ischemia

Neonatal hypoxia-ischemia (NHI) is a trigger of necrosis and a continuum of hybrid cell death programs where excitotoxicity, oxidative injury, and inflammation are thought to play the prominent role in histopathological and behavioral consequences of this acute brain damage. NHI is often modeled in rodents by unilateral carotid artery ligation followed by 45–180 min hypoxia which can produce moderate-to-severe injury in cortical, striatal, and hippocampal brain regions in immature rodents (usually mice or rats at postnatal day 7, P7). The first study evidencing the neuroprotective efficacy of Nec-1 in NHI was carried out by Northington et al. (2011). They showed that a single i.c.v. injection of Nec-1 (0.1μL of 80μmol) in P7 C57B6 mice directly after NHI (45 min hypoxia) significantly decreased injury in the forebrain and thalamus at P11 (4 days after NHI) and P28 (3 weeks after NHI) and this effect was more pronounced in males. Further biochemical study performed 3 and 24 h after NHI revealed that Nec-1 inhibited RIP1-RIP3 complex formation, attenuated oxidative stress markers, and attenuated NFκB activity and inflammatory response (TNFα, IL-1β, IL-6, IL-12) (Northington et al. 2011). In the next study, the same group further explored biochemical processes associated with neuroprotective action of Nec-1 by investigation of mitochondrial dysfunction in neurons and astrocytes after NHI in P7 male and female mice (Chavez-Valdez et al. 2012). Nec-1 immediately after NHI (3–24 h) regardless of gender attenuated oxidative stress and mitochondrial injury as evidenced by prevention of nitric oxide (NO•) production, iNOS (inducible nitric oxide synthase) activation, attenuation of glutathione oxidation, upregulation of HIF-1α (hypoxia inducible factor-1 alpha) and BNIP3 expression, decline in mitochondrial complex-I activity, decrease in ATP levels, and mitochondrial structural pathology in astrocytes and in neurons. Moreover, Nec-1 at P11 decreased astrogliosis (GFAP level) induced by NHI (Chavez-Valdez et al. 2012). The next report from the same team showed that in both sexes NHI acutely (24 h) increased BDNF levels in the forebrain but not in the diencephalon which was followed by the delayed decrease (96 h) and late recovery (21 day). Nec-1 prevented the decrease in BDNF levels in the forebrain while increased its levels in the diencephalon. At the level of Bdnf gene, Nec-1 induced its expression after 96 h in the diencephalon in both sexes whereas in the forebrain region only in male mice. Moreover, only in female mice, NHI increased p75ntr and truncated TrkB levels which were attenuated by Nec-1 but in male mice Nec-1 preserved BDNF levels and TrkB activation. Finally, Nec-1 attenuated hippocampal cell damage after NHI in male and female mice, however, in the thalamus only in the former ones. Thus, it has been concluded that the regional differences in BDNF levels may be a consequence of injury severity after NHI, but sexual differences in response to Nec-1 may represent a differential thalamo-cortical preservation or alternatively off-target regional effect of Nec-1 (Chavez-Valdez et al. 2014). Importantly, Nec-1 helped verify the hypothesis that the ER stress is a prominent feature of delayed neuronal death via programmed necrosis after neonatal NHI. Nec-1 attenuated biochemical markers of ER stress (PERK and eIF2α phosphorylation, GADD43 and unconventional XBP-1 splicing) after NHI as well as rescued ER morphological changes induced by NHI at delayed stages (Chavez-Valdez et al. 2016).

A still unsolved problem is to what extent necroptosis and apoptosis can overlap and which elements of these two death signaling pathways are critical for their interaction in hypoxic cells. Askalan et al. (2015) studied the role of apoptotic and necroptotic processes in two models of NHI in P7 rats inducing mild-moderate (90 min) or severe (180 min) hypoxia. It was observed that 7 days after injury in severe hypoxia model there were more lesions with higher rate of apoptotic (caspase-3 and AIF dependent) and necroptotic (RIP3) cell death. Moreover, in males, but not in females, apoptosis was more profound in rats exposed to the mild-moderate hypoxia as compared to results obtained in the severe NHI model. These findings justify further the evaluation of Nec-1 or other necroptosis inhibitors in NHI by employing models of various severities and highlight the need for continuation of research on both sexes. Another interesting point is that hypoxia-ischemia combined with inflammation is an important cause of neonatal encephalopathy which affects up to 1% of newborns and over a longer period of time leads to cerebral palsy. It can be modeled in animals by treatment with LPS and exposure to hypoxia-ischemia. In the study by Savard et al. (2015), the induction of necroptotic (increased RIP3) and apoptotic (caspase-3 activation) processes was observed in P12 rat pups treated for 4 h with LPS (200μg/kg i.p.) followed by 1.5 h hypoxia-ischemia. In that model, the authors uncovered a new paradigm of neuronal self-injury orchestrated by neuronal synthesis of IL-1β and MMP-9 and observed the protective effect of systemically administered IL-1 receptor antagonist (IL-1Ra) (Savard et al. 2015). It is expected that Nec-1 should be beneficial also in such model, since reports from brain ischemia models showed modulating effects of Nec-1 on inflammatory response and MMP-9 activity.

3.3 Intracranial Hemorrhages

Intracranial hemorrhages comprise intracerebral hemorrhage (ICH), intraventricular hemorrhage, and subarachnoid hemorrhage (SAH) and are life-threatening neurological diseases characterized by high mortality and morbidity. They can occur as a result of head injury or spontaneously, usually from a ruptured cerebral aneurysm. Unfortunately, to date, limited therapeutic options are available for management of intracranial hemorrhages (Fang et al. 2020). Since many studies showed involvement of nonapoptotic forms of programmed cell death including necroptosis and neuroinflammation in the pathophysiology of intracranial hemorrhages, some research has been done on Nec-1 effects in ICH and SAH animal models. The first study addressing this issue was conducted by Chang et al. (2014) who used the mouse ICH model induced by unilateral intrastriatal infusion of type IV collagenase. They showed that i.c.v. injection of Nec-1 (2.6μg) 15 min before ICH reduced the injury volume and number of PI-positive cells and prevented changes induced by ICH in autophagy (LC3-II, Beclin-1, p62)- and apoptosis (Bcl-2 protein level and decreased cleaved caspase-3)-associated proteins at 24 and 72 h after ICH. These data showed that a cross talk existed among necroptosis, apoptosis, and autophagy after ICH and Nec-1 to some extent could suppress the activation of these cell death pathways (Chang et al. 2014). In another study employing the same ICH model King et al. (2014) demonstrated that Nec-1 and Nec-1i (i.c.v. 2–4μl of 4 mM) administrated at the time of injury completely reduced hematoma size at 72 h. However, only Nec-1 improved neurological outcome in this model which was associated with the reduced number of PI-positive cells, preserved BBB permeability, and decreased astrogliosis and brain edema (King et al. 2014). Moreover, Su et al. (2015) demonstrated that Nec-1 pretreatment improved neurological function and attenuated brain edema in mice after ICH via reduction of RIP1-RIP3 interaction and PI-positive cells, and inhibited microglia activation and expression of pro-inflammatory genes (TNFα and IL-1β).

Regarding in vivo models of SAH, Chen et al. (2017) demonstrated an increase in expression of necroptosis proteins (RIP1, RIP3, and MLKL) during brain injury (after 3–48 h) evoked by endovascular perforation (endovascular filament model) in rats. Further, they found that Nec-1 (i.c.v. 200μg; given 30 min after injury) partially attenuated brain swelling, reduced the lesion volume, reduced the number of PI-positive cells, and improved neurologic outcomes. These effects were accompanied by reduced expression of necroptosis proteins, inhibited NFκB nuclear translocation, and reduced release of pro-inflammatory cytokines (TNFα and IL-1β). In the next study, these findings were confirmed using a wider range of methods and measured parameters. The beneficial effect of 1 h pretreatment with Nec-1 was confirmed by reduction in lesion size and ventricle volumes (MRI method), which ameliorated albumin leakage and the degradation of tight junction proteins (ZO-1, occludin and claudin-5). Nec-1 inhibited the production of the pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and reduced expression and activity of MMP-9 (Chen et al. 2019). In another study, Zhou et al. (2017) using the SAH rat model studied interactions between the RIP1-RIP3-DRP1 pathway and NLRP3 inflammasome. Nec-1 or mitochondrial division inhibitor (Mdivi-1) administered i.p. 1 h after SAH alleviated brain edema and neurological deficits measured after 24 h. It was accompanied by reduced RIP1, RIP3, phosphorylated DRP1, and NLRP3 inflammasome (Zhou et al. 2017). Further in vivo study in rats where SAH was induced by a double injection of autologous blood (200 μl) into the cisterna magna, Nec-1 (i.c.v. 3μg) alleviated brain edema and improved neurobehavioral scores. Apart from decreasing the hippocampal expression of RIP1 and RIP3, Nec-1 improved synaptic connectivity (PSD-95, Neurexin-1 and Neuroligin-1) and activated CREB-BDNF pathway (Yang et al. 2019a). Collectively, the data clearly indicate that Nec-1 could be protective in experimental animal models of intracranial hemorrhages engaging various mechanisms and underline the necessity of development of new RIP1 inhibitors with better pharmacokinetic properties for clinical purposes.

3.4 Traumatic Brain and Spinal Cord Injuries

A growing body of evidence indicates that necroptosis together with apoptotic and autophagic cell death play an essential role in the mechanism of neuronal death after traumatic brain (TBI) and spinal cord injury (SCI). Direct mechanical trauma to the brain or to the spinal cord causes the primary injury and sets off a complex of toxic mediators, cytokines, and free radicals which induce the secondary injury executed by apoptotic and nonapoptotic forms of cell death. The first report demonstrating that Nec-1 could be protective in TBI was published by You et al. (2008). They showed in the controlled cortical impact (CCI) model in mice that pre-(5 min) and post-(5–15 min but not 30 min) treatment with Nec-1 (i.c.v. 4μl of 4 mM) but not with Nec-1i reduced histopathological and functional deficits in mice lasting up to 35 days after injury. It was associated with reduced lesion (by about 30–40% in the pretreatment schedule), decreased number of PI-positive cells in the cortex and hippocampus, and attenuated neuroinflammation and astrogliosis without influence on caspase-3 level. These results suggested that Nec-1 might be a novel therapeutic agent with pleiotropic action which could be considered for the treatment of head injury and other acute CNS disorders (You et al. 2008). Using the same TBI model, Zhu et al. (2014) demonstrated an increase in Akt and mTOR signaling in cortical and hippocampal regions 24 h after CCI. Pretreatment with Nec-1 s (i.c.v. 4μl of 4 mM) further increased hippocampal Akt and improved place learning, and these beneficial effects were reduced by rapamycin or Akt inhibitor (Zhu et al. 2014). Finally, Bao et al. (2019) showed in CCI TBI model that Nec-1 (i.c.v. 6μl of 25 mM solution) and melatonin but not the caspase inhibitor z-VAD-fmk reduced cortical lesion volume, attenuated brain edema, and improved motor ability and learning and memory. It was accompanied with alleviated necroptotic markers (RIP1, RIP3, and MLKL) in the hippocampus and cortex and reduced the number of TUNEL-positive cells without affecting caspase-3-positive ones. Moreover, Nec-1 and melatonin reduced HMGB1 and RAGE levels, NFκB activation, and neuroinflammatory proteins (IL-1β, IL-6, and NLRP3) in both studied brain regions. They also found that TNFα-induced protein 3 (TNFAIP3) was indispensable for regulating and controlling necroptosis and inflammation after CCI, and that the lack of TNFAIP3 A20 led to aggressive necroptosis and attenuated the anti-necroptotic effects of Nec-1 and melatonin (Bao et al. 2019). In another type of TBI model, namely the closed head TBI model (a weight-drop model), Wang et al. (2012) aimed to explore the effect of Nec-1 on apoptosis and autophagy. They demonstrated in mice that 15 min pretreatment with Nec-1 (i.c.v. 2.6μg) attenuated TBI-induced cortical and hippocampal injury after 24 h and it was associated with reduced levels of autophagic (LC3II, p62, and Beclin-1) and apoptotic (caspase-3 and Bcl-2) markers (Wang et al. 2012). In another study employing the same type of TBI but this time in rats, Mu et al. (2021) demonstrated a beneficial effect of Nec-1 (i.c.v. 60μg) given just after surgery which was evidenced by attenuation of oligodendrocyte cell damage, diminution of APP (amyloid precursor protein) level, and protection of axons and myelin in the brain stem 2–6 days after injury. Moreover, Nec-1 administration resulted in a significant decrease in the BNIP3-positive oligodendrocytes suggesting an involvement of mitochondrial mechanisms in myelin protection mediated by Nec-1 (Mu et al. 2021).

Regarding the protective potential of Nec-1 in SCI, the first study was conducted by Wang et al. (2014a). In the rat SCI model (mechanical injury), Nec-1 given intrathecally (i.t. 1–50μg) 20 min before injury and 12–24 h after SCI in a dose-dependent manner attenuated the extent of lesion. Moreover, Nec-1 mitigated cell death (TUNEL-positive cells), reduced necroptotic (RIP1, RIP3 and MLKL) and apoptotic (caspase-3, Bax/Bcl-2) markers, regulated oxidative stress parameters (ROS, MDA, and SOD), and inhibited release of pro-inflammatory cytokines (TNFα, IL1β, and IL-6). Moreover, it alleviated pathological changes and improved blood supply in the spinal cord trauma which was observed up to 7 weeks after injury (Wang et al. 2014a). In the next study in the same SCI model, it was evidenced that Nec-1 improved mitochondrial functions as observed 12–24 h after injury. Nec-1 reduced the elevated mitochondrial Ca2+, preserved the decreased MMP, increased ATP production, promoted the activity of mitochondrial complex I, inhibited cytochrome c release, maintained the normal mitochondrial ultrastructure, increased mitochondrial biogenesis (Tfam, cytochrome b), and regulated their fusion (Mnf1, Mnf2) and fission (Fis1) (Wang et al. 2015). The next study explored the relationship between necroptosis and ER stress. The authors revealed that Nec-1 pretreatment reduced ultrastructural damage to the neuronal and astrocyte ER and mitochondria and inhibited neuronal expression of ER stress-related genes and proteins (Grp78, CHOP, XBP1) (Wang et al. 2017). In the SCI model (a laminectomy at T9 and compression with a vascular clip) in female mice, Nec-1 (i.t. 4μl of 4 mM) given 5 min after injury decreased PI-positive cell counts, alleviated tissue damage, increased the number of surviving neurons, and improved long-term functional recovery (Liu et al. 2015). However, Nec-1 did not show antiapoptotic properties (no changes in TUNEL-positive cells and in caspase-3), which is in contrast to observations made in the rat SCI model (Wang et al. 2014a). These discrepancies could be probably due to different Nec-1 doses, animal sexes, or species used in particular experiments. Another study performed in male mice showed that microglia/macrophages played a key role in inflammation after SCI and these immune cells were eliminated by necroptosis (Fan et al. 2015). Nec-1 given after injury and 2x/day for 5 days (i.v. 7.8 mg/kg) decreased the number of PI-positive microglia cells at day 5. Moreover, it was demonstrated that ER was involved in necroptosis of microglia/macrophages as measured by double and triple staining with cell-specific (Iba1 or CXCR4), necroptotic (MLKL), and ER stress (GRP78) markers (Fan et al. 2015). Finally, the beneficial effects of Nec-1 were confirmed in female mice where Nec-1 (i.p. 5 mg/kg) administered immediately after SCI and continued for a longer period of time (7–28 days) improved locomotor function and reduced spinal cord edema at day 7. These effects were associated with reduced RIP1, pRIP3, ROS, MDA, and increased level of ATP, MMP, SOD, and GSH observed at day 7 after SCI (Wang et al. 2019a) Interestingly, similar protective efficacy and mechanisms of action (apart from the influence on RIP1) were found for the RIP3 inhibitor GSK’872 (i.p. 2 mg/kg) (Wang et al. 2019a). Thus, results of the experimental studies strongly suggest that Nec-1 may have therapeutic potential for patients with acute brain or spinal cord injuries.

3.5 Retina Injuries

The retina is one of the most intensively studied parts of the CNS first of all to understand common blinding diseases (e.g., age-related macular degeneration, glaucoma, anterior ischemic optic neuropathy, and diabetic retinopathy traumatic optic neuropathy) and also as a convenient model for examination of neurodegeneration induced by detrimental physical and ischemic factors. The first study investigating whether necroptosis contributes to retinal neuronal damage and functional impairment was carried out by Rosenbaum et al. (2010) in the rat model of retinal ischemia. They found that animals subjected to raised intraocular pressure for 45 min and receiving intravitreal injections of Nec-1 (2μl of 4 mM) during and 2 h after I/R showed a significant preservation in thickness and histoarchitecture of the inner retina and functional improvement compared with vehicle-treated controls, as observed 7 days post-injury. Its short-term effect (2–6 h) was connected with the inhibited autophagy (LC3) and decreased number of PI-positive but not TUNEL- or caspase-3-positive cells. Nec-1i serving as negative control was not protective in this study suggesting RIP1-dependent effects of Nec-1 in this model (Rosenbaum et al. 2010). These data were confirmed by the next study employing the mouse model of retinal I/R injury where Nec-1 (i.p. 2μg/g, 1 h before and continued once daily up to day 7) significantly improved neuronal cell survival in the ganglion cell layer. This effect was accompanied with attenuation of expression of proinflammatory genes (Tnf, Il1b, Cybb, Nos2, Ccl2, Ccl5, Cxcl10, Icam1) (Dvoriantchikova et al. 2014). Additionally, Kim et al. (2017) provided evidence that TNFα produced by astrocytes early after retinal I/R was responsible for necroptotic cell death of RGCs and showed that Nec-1 given intrevitreally (2μl of 0.1μg/ml) just after I/R via decreased RIP1 activation and improved the retina histopathology at all its layers. Finally, Do et al. (2017) showed that also another RIP1 inhibitor RIC (i.p. 10 and 30 mg/kg, given immediately after ischemia induction) was protective in the rat retina I/R model by increasing neuronal survival and attenuation of TUNEL-positive cell count in ganglion cell layer as found 3 days after I/R. Interestingly, RIC was also protective in retinal epithelial cell damage induced by single i.p. injection of 50 mg/kg of sodium iodate which models the pathogenesis of dry age-related macular degeneration. In this model, RIC administrated in a single dose (i.p. 20–30 mg/kg) or repeated doses (i.p. 330μg/ml, twice daily for 10 days starting at 3 days before injury) in eye drops prevented RPE loss and preserved retinal morphology without evoking cytotoxicity suggesting its putative clinical efficacy (Jang et al. 2019).

The induction of necroptosis was also observed in physical models of retina damage. Trichonas et al. (2010) was the first to demonstrate the protection of Nec-1 in the retinal detachment (RD) model. Although they showed in mice an increase in expression of RIP3 3–5 days after RD, intraretinal (i.r.) administration of Nec-1 (200μM) at the time of surgery was not protective. However, when it was combined with the apoptosis inhibitor, z-VAD-fmk, a partial prevention of photoreceptor loss was observed 3 days after injury as evidenced by the decreased number of TUNEL-positive cells, increased outer nuclear layer (ONL) thickness, reduced oxidative stress, prevented AIF translocation, and attenuated number of CD11-positive microglia/macrophages (Trichonas et al. 2010). In the next study, Dong et al. (2012) showed the protective effects of pre- and post-(6 h) treatment with Nec-1 (i.r. 400μM) but not with Nec-1i in the rat RD model as demonstrated by the improved photoreceptor survival and functional outcome. It was associated with the inhibition of necroptotic (pRIP1, PI-positive cells) but not apoptotic processes (Dong et al. 2012). In the next study of those authors using the same experimental model, they additionally showed an involvement of autophagy inhibition in Nec-1-mediated photoreceptor protection as confirmed by ameliorated LC3II expression and autophagosome count (Dong et al. 2014). Necroptosis has been reported to be the major cell death pathway also in degenerating rod photoreceptors. In the P23H rhodopsin mutant rats and mice, a model representing one of the most common forms of retinal degeneration, upregulation of RIP1, RIP3 and activation of microglia and inflammasome (NLRP3/caspase-1/IL-1β) were found in P45 and/or P120 animals. In this genetic model of retinal degeneration, subcutaneous injection of Nec-1 s (15 mg/kg) once daily starting from P21 significantly preserved retina morphology and function which were superior to the effect mediated by antioxidant NAC (150 mg/kg). This protective effect of Nec-1 s was associated with inhibition of RIP1, RIP3, NFκB, ROS, DNA fragmentation, microglia activation, and inflammasome induction (Viringipurampeer et al., 2016). Nec-1 s was also tested in traumatic optic neuropathy induced by a blunt ocular injury in rats (Thomas et al. 2019). An intravitreal injection of Nec-1 s (5μl of 3.6 mM) just after injury and at 7 day did not improve histological and functional parameters evaluated at day 14. However, it prevented RGC loss at the center of the impact site as well as improved RGCs survival in primary cultures (Thomas et al. 2019). The presented data on Nec-1-mediated protection in various animal models of retina damage support further studies on the development of more clinically relevant RIP1 inhibitors for the of acute and chronic retinal pathologies.

3.6 Neurodegenerative Diseases

3.6.1 Alzheimer’s Disease

In AD, neuronal degeneration in specific brain areas causes a dysfunction in learning and memory processes, and the pathology is associated with extracellular aggregation of amyloid-β (Aβ) and intarcellular hyperphosphorylation of tau. Apart from apoptotic processes, also necroptosis has been evidenced to be involved in the pathogenesis of AD (Caccamo et al. 2017), however there are rather limited in vivo studies on the effectiveness of Nec-1 in AD. In the study on APP/PS1 double transgenic mice, Nec-1 (i.p. 6.25 mg/kg, twice a week) given to 4-month-old animals and continued twice a week to 7 months (7 weeks of administration) significantly improved working spatial memory of the mutants which achieved the performance level observed in wild-type animals (Yang et al. 2017b). Moreover, Nec-1 reduced cortical and hippocampal level of β-amyloid (Aβ) oligomers, plaques, hyperphosphorylated tau, and tau aggregation without affecting Aβ production. These promising prophylactic effects of Nec-1 at the behavioral level were also demonstrated in the model of acutely induced memory deficits in ICR mice. Nec-1 (i.v. 6.25 mg/kg) given twice a week, starting a week before i.c.v. Aβ(1–42) injection and continued for the next week, significantly ameliorated memory deficits and histopathological features in these animals (Yang et al. 2017b). Further study using APP/PS1 double transgenic mice showed that Nec-1 also reduced necroptotic and apoptotic markers in the cortex of aged mutant mice (4 weeks treatment from 8 to 9 months) by decreasing the levels of p-RIP3 and Bax and increasing the levels of Bcl-2 (Yang et al. 2019b).

In chemical AD-like mouse model induced by a single i.c.v. aluminum administration (AlCl3. 2μl of 2 mM), co-treatment with Nec-1 (i.c.v. 2μl of 4–8 mM) significantly improved learning and memory (Morris water maze task) and decreased the extent of neuronal cell damage in the hippocampal CA3 region observed 20 days after injection of the compounds. Moreover, Nec-1 reduced necrotic (pRIP1), apoptotic (caspase-3), autophagy (LC3II), and AD-related (Aβ and tau) proteins. However, when Nec-1 was given post-Al exposure (2–8 h) no protective effects were found (Qinli et al. 2013). These protective effects of Nec-1 were also confirmed in the chronic Al-based model (0.5% AlCl3 given for 5 days). It was found that Nec-1 reduced Al-induced learning and memory impairment and decreased Al-evoked neuronal cell damage as measured 14 days after the last injections. Moreover, Nec-1 attenuated Al-induced changes in RIP1, caspase-3, and LC3II. The neuroprotective effect of Nec-1 was stronger than that mediated by the caspase inhibitor z-VAD-fmk, and combination of them resulted in synergistic action (Hao et al. 2020).

The factors indirectly linked with mild cognitive impairment and development of AD include neuroinflammation and diabetes. In the model of spatial memory impairment in rats induced by neuroinflammation (i.c.v. LPS 15μg), co-treatment with Nec-1 (i.c.v. 1μl 10μM) attenuated cognitive deficits measured by spontaneous alternations in the Y-maze test. This effect was associated with the reduced level of necroptosis (RIP1, RIP3), apoptosis (Bax/Bcl2, caspase-8, and caspase-3), oxidative stress (MDA), and increased antioxidant enzymes (SOD, GSH, and catalase) in the hippocampus and frontal cortex (Nikseresht et al. 2015). In the rat model of prediabetes (20 weeks of high-fat diet, HFD) which is characterized by cognitive decline and brain pathology, Nec-1 given from week 13 (s.c. 1.65 mg/kg/day) improved cognitive function, synaptic plasticity, and brain mitochondrial function and reduced hyperphosphorylated Tau. Moreover, it attenuated HFD-induced necroptosis (pRIP1, RIP3), oxidative stress, and microglia without alteration in insulin sensitivity (Jinawong et al. 2020). It has been shown that Nec-1 could be also beneficial in improving cognitive performance in the aged animals. Duan et al. (2018) using the accelerating aging mouse model induced by D-galactose (i.p. 25 mg/kg), administrated for 2 months and subjected to anesthesia and partial hepatectomy, demonstrated that Nec-1 (i.p. 6.25 mg/ml) given 1 h before surgery strikingly mitigated cognitive impairment and prevented neuronal loss and microglia activation in the DG hippocampal region. These effects were accompanied with decreased RIP1 level, improved synaptic plasticity (increase in Glu1A expression), and alleviated neuroinflammation (IL-1α, IL-1β, and TNFα) (Duan et al. 2018). All the above studies performed in various AD-like animal models provide substantial evidence that Nec-1 and its analogues could be promising candidates for the development of a neuroprotective drug for mild cognitive impairment (MCI) and AD.

3.6.2 Parkinson’s Disease

The most significant pathological feature of PD is the progressive loss of dopaminergic neurons in the pars compacta of the substantia nigra (SNpc). In addition, PD has been also associated to neurodegeneration of other neuronal cell types including olfactory, cortical, and autonomic peripheral neurons. All these processes contribute to motor, cognitive, psychiatric, and peripheral symptoms in this chorea. Mitochondrial dysfunction and increased ROS level are thought to be the main causes of neuronal death in PD (Oñate et al., 2020). Only a few studies are available on the neuroprotective effects of Nec-1 or its stable analogue Nec-1 s in animal PD models. In the study by Iannielli et al. (2018) mice were injected daily with MPTP (i.p. 20 mg/kg; 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) for five consecutive days, while Nec-1 (i.c.v. 1μg/day) or Nec-1 s (i.p. 10 mg/kg/day) administration started 3 days after the first injection of MPTP and was continued for 3 weeks. Histological and biochemical analysis performed on day 24 showed comparable protective effects of both compounds evidenced by increased number of TH-positive neurons (by about 27%, the unbiased stereological sampling method) in the substantia nigra pars compacta (SNpc) and increased TH immunolabeling in DA striatal fibers (15% recovery, optical densitometry) when compared to the MPTP group. Moreover, Nec-1 s reduced intracellular oxidative stress level induced by MPTP injections as confirmed by immunohistochemistry for 4-hydroxynonenal (4-HNE) in TH + -nigral DA neurons (Iannielli et al., 2018). However, in another study employing the mouse MPTP model, Nec-1 s treatment (i.p. 10 mg/kg/day) initiated 1 h after a single MPTP (i.p. 40 mg/kg) injection and continued for the next 30 days was not protective, however, some tendency toward the increase in TH+ densitometry in SNpc could be noticed (Dionísio et al., 2019). It is not excluded that used various image analysis systems for calculation of TH+ cells in SNpc (quantitative vs. semiquantitative in Iannielli’s and Dioniso’s studies, respectively), animal numbers and/or different schedule of MPTP administration was responsible for the lack of a significant protection by Nec-1 s in the latter study. Nevertheless, these investigators reported that MPTP-induced dopaminergic neurodegeneration in the SNpc was attenuated in RIP3 knockout mice as compared with the wild-type mice (Dionísio et al. 2019). Lin et al. (2020) also used the mouse MPTP model to study the mechanisms of necroptosis and the effects of Nec-1. The used acute intoxication model involving the cumulative MPTP (i.p. 20 mg/kg) administration every 2 h up to 8 h (5 doses) led to the increased expression of necroptosis-related proteins (RIP1, RIP3, and MLKL), which were associated with DA neuronal loss in the SNpc. Treatment with Nec-1 (i.p. 1.65 mg/kg/day) initiated 12 h before application of MPTP and continued up to 21 days ameliorated DA neuronal loss in the SNpc (unbiased stereology of TH-positive neurons) and increased striatal DA level. The similar protective effects against MPTP were observed in RIP3−/− and MLKL−/− mice when compared to the wild-type ones where also the decreased level of pro-inflammatory cytokines (TNFα, IL-1β, and IL-6) was found suggesting the interplay between necroptosis and neuroinflammation in this PD model (Lin et al. 2020). Oñate et al. (2020) showed an induction of necroptosis (RIP3 and MLKL) in postmortem brain tissue from PD patients and in the 6-OHDA (6-hydroxydopamine) PD model (4μg/μl, single injection to right striatum) in mice. Genetic ablation of necroptosis mediators (MLKL−/− and RIP3 −/− mice) as well as pharmacological inhibition of RIP1 by Nec-1 s (i.p. 8 mg/kg/day, 3 days before and 7 days after surgery) decreased DA neuronal loss in the SNpc, prevented striatal denervation, attenuated axonal loss, and improved animals motor performance. Finally, it has been shown that in mice treated with MPTP (i.p. 30 mg/kg for 5 days) there was an increase in necroptosis in TH-positive SNpc neurons which negatively correlated with expression of miR-425. Further molecular evaluation identified RIP1 as miR-425 target and intracerebral administration of miR-425 mimics (AgomiR-425) attenuated necroptosis activation and dopaminergic neuron loss as well as improved motor performance after MPTP administration in mice (Hu et al. 2019). All these results highlight the potential neuroprotective utility of Nec-1 or its analogues in preventing PD progression.

3.6.3 ALS and Other Neuropathies

Some data from preclinical studies and also clinical observations have suggested that necroptosis is involved in the pathogenesis of ALS (Re et al. 2014). Promising studies performed by Ito et al. (2016) using optineurin knockout mice (OPTN−/−) and SOD1(G93A) transgenic mice showed that axonal degeneration which preceded motor neuron loss was correlated with the activation of RIP1and RIP3. Similar observations were made in samples from human ALS patients suggesting necroptosis as a central player in progressive axonal degeneration. In 3 months-old OPTN−/− mice, Nec-1 s (oral dosing, 2.5–5 mg/day, for 1 month starting from 8 weeks of age) blocked demyelination in the spinal cord, prevented axonal loss and swelling, reduced the number of TUNEL-positive cells in the lumbar spinal cord, and improved motor performance in these animals. Similar protective effects of Nec-1 s were observed in SOD1(G93A) mice where additionally the RIP1 inhibitor delayed the onset of motor dysfunctions (Ito et al. 2016). However, recently the role of necroptosis in ALS was questioned. Dermentzaki et al. (2019) showed that constitutive deletion of RIP3 in SOD1(G93A) transgenic mice did not improve behavioral or neuropathological deficits. Moreover, no protection on myelin damage has been found in RIP3-silenced SOD1(G93A) and OPTN−/− mice. In addition, Wang et al. (2020) using SOD1(G93A) mice did not observe any beneficial effect of MLKL genetic ablation on disease onset, progression, and survival suggesting that motor neuron degeneration and activation of neuroinflammatory cells (astrocytes and microglia) are independent of MLKL expression. Moreover, while RIP1 accumulation occurred in the spinal cord of SOD1G93A mice in late-stage disease, RIP3 and MLKL expression levels were undetectable which is in contrast to results obtained by Ito et al. (2016). Thus, more preclinical investigations preferably using other models of ALS are needed to determine if necroptosis plays a critical role in the pathogenesis of ALS.

Necroptosis has been shown to be involved in various peripheral nerve injuries. Liang et al. (2019) found that in a rat sciatic nerve chronic constriction injury model there was a time-dependent increase in necroptosis-related proteins (RIP1 and RIP3) in the spinal cord which correlated well with the changes in mechanical and thermal allodynia observed from day 1 to 21 after surgery. Nec-1 (i.p. 200–400μg/kg/day for 21 days) reduced peripheral nerve injury–related behavioral symptoms and it was associated with attenuation of necroptosis (RIP1, RIP3), reduction in the number of PI-positive cells, inhibition of NFκB activity, and decreased level of pro-inflammatory cytokines (TNFα, IL-1β, and substance P) (Liang et al. 2019). In the model of auditory neuropathy induced by ouabain, Wang et al. (2014b) observed the activation of necroptotic cell damage of the spiral ganglion cells (SGCs) which led to hearing loss. Nec-1 (10μl of 3 mM) given 10 min after ouabain (10μl of 10 mM) partially decreased SGC loss of the cochlear axis which was associated with reduction in RIP3 expression but it did not prevent hearing loss. However, a synergistic effect was observed when Nec-1 was combined with z-VAD-fmk (10μl of 3 mM) which led to almost complete inhibition of SGC death and significantly improved hearing function (Wang et al. 2014b). In their next study, using a similar model of auditory neuropathy, RIP3 ablation protected rats from ouabain-induced hearing damage and loss of function, while overexpression of RIP3 enhanced ouabain-induced injury that could be partially reversed by Nec-1. It was suggested that targeting RIP3 might prevent SGC death in clinical practice, and eventually might be helpful in the treatment of sensorineural hearing loss (Wang et al. 2021). Finally, in ex vivo model of mechanical sciatic and optic nerve injury, Nec-1 (100μM) given at the time of injury strongly delayed axonal degeneration after transection and also preserved their electrophysiological activity. It was associated with reduction in axonal mitochondrial fragmentation, and the inhibitor of mitochondrial fission Mdivi was protective in the model with similar efficacy as Nec-1 (Arrázola et al., 2019). Altogether, these results clearly support the potential clinical utility of Nec-1 and its derivatives in the treatment of various neuropathies.

3.6.4 Other Neurodegenerative Diseases

Nec-1 was demonstrated to be beneficial in the genetic Huntington’s disease (HD) model, R6/2 transgenic mice (Zhu et al. 2011). Nec-1 (i.c.v.) administrated in osmotic pumps from 5 weeks of age, at week 11 helped to maintain the body weight and motor functions and was observed to significantly delay the disease onset (21.5%) but had a modest effect on survival of mice. It was postulated that, since Nec-1 treatment may revert necroptosis to apoptosis, concomitant treatment with both apoptosis and necroptosis inhibitors might be more efficient in the treatment of HD disease than each of these inhibitors given alone (Zhu et al. 2011). Moreover, an effectiveness of Nec-1 was demonstrated in the animal model of Niemann-Pick disease, type C1 (NPC1) which is characterized by progressive neuronal dysfunction, including cerebellar ataxia and dementia (Cougnoux et al. 2016). Treatment of Npc1-mutant mice with Nec-1 (i.p. 90 and 180 mg/kg) given every second day starting from the third week of age for the next 4 weeks significantly delayed cerebellar Purkinje cell loss and progression of neurological symptoms and increased lifespan (by about 10–14 days). No effect in the measured parameters was found for Nec-1i suggesting the involvement of RIP1-dependent mechanism of Nec-1 action in this genetic model of cerebral neurodegeneration (Cougnoux et al. 2016). Nec-1 was also considered for putative treatment of multiple sclerosis (MS). In experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, Nec-1 after repeated intrathecal injection (1.65 mg/kg) from day 2 every 3 days for 15 days decreased the number of lesions and inflammatory cell infiltrates in the spinal cord tissues and attenuated production of pro-inflammatory cytokines (TNFα, IL-1β and INFγ) in the EAE mice. In addition, it increased pDrp1/Drp1 and reduced necroptotic (pMLKL/MLKL) and apoptotic (Bax/Bcl-2, Bim) markers in the spinal cord tissue of EAE mice (Wang et al. 2019b).

Summing up the paragraph dedicated to neurodegenerative diseases, it is worth mentioning that DNL104, a selective and brain-penetrant inhibitor of RIP1, is in clinical development for AD and ALS (I phase). It was tested in healthy volunteers to investigate its safety and tolerability, pharmacokinetic profile in plasma and cerebrospinal fluid, and pharmacodynamic effects of RIP1 inhibition in peripheral blood mononuclear cells. DNL104 was well tolerated in the single-ascending dose and during the dosing period of the multiple ascending group. However, post-treatment liver toxicity was observed in 37.5% of subjects in the latter group which was assessed to be drug related. The authors also demonstrated that DNL104 led to RIP1 kinase inhibition, and this was not associated with CNS toxicities which supports development of CNS-penetrant RIP1 inhibitors in the future (Grievink et al. 2020).

4 Conclusions

In a critical appraisal of Nec-1 as a putative neuroprotectant some pros and cons should be taken into account. There is a substantial body of evidence suggesting that necroptosis contributes to the mechanism of neuronal damage related to hypoxia-ischemia, traumatic CNS injury, neuropathies, and neurodegenerative diseases. However, this programmed nonapoptotic cell death appears to be biochemically and functionally linked with several alternative cell death signaling pathways, and the relationship between various death pathways is extremely complex. Furthermore, although RIP1 is the key element in the concept of necroptosis, some data suggest that it can be involved also in non-necroptotic cell damage. Indeed, it has been found that RIP1 and RIP3 not only mediate necroptosis but also are involved in apoptosis and inflammatory signaling. Nec-1 is a potent inhibitor of RIP1, and most of the experimental studies used this compound in order to gain evidence that necroptosis is involved in neuronal damage under study and that Nec-1 may be proposed as a novel neuroprotectant in humans. Indeed, Nec-1 and its analogues like Nec-1 s display in in vivo models moderate neuroprotective effects, which are statistically significant, but rarely robust enough to be enthusiastic about them. In other words, Nec-1 when given alone failed to provide full neuroprotection in most tested experimental models and is more efficient in pre-treatment than in the clinically more desired post-treatment schedule. Some encouraging results have been obtained when Nec-1 was used in combination with an antiapoptotic agent, and this strategy appears to be more promising at least in some models on neuronal cell injury. Surprisingly, some neuroprotective effects of Nec-1 have been ascribed to the inhibition of apoptosis, instead of necroptosis. Some other shortcomings of this compound as a candidate for a neuroprotective medicine have to be mentioned. Regarding physical properties and pharmacokinetics, its solubility in water is limited (requires DMSO) and after systemic administration its half-time is short. Although i.c.v. or intrathecal administration of Nec-1 attenuated neuronal damage to various extent in some experimental animal models, it is a hardly acceptable route of drug administration to patients. While remaining a highly valuable experimental tool in untangling the complex relationships between various signaling neuronal death pathways and elucidating molecular mechanism of CNS pathologies, it is rather not likely to be considered a candidate for a neuroprotective drug. Further development of novel necroptosis inhibitors with more favorable pharmacokinetic characteristics, therapeutic efficacy, and safety profiles is required.

5 Cross-References