Necroptosis inhibition counteracts neurodegeneration, memory decline and key hallmarks of aging, promoting brain rejuvenation

Age is the main risk factor for the development of neurodegenerative diseases. In the aged brain, axonal degeneration is an early pathological event, preceding neuronal dysfunction, and cognitive disabilities in humans, primates, rodents, and invertebrates. Necroptosis mediates degeneration of injured axons, but whether necroptosis triggers neurodegeneration and cognitive impairment along aging is unknown. Here we show that the loss of the necroptotic effector Mlkl was sufficient to delay age-associated axonal degeneration and neuroinflammation, protecting against decreased synaptic transmission and memory decline in aged mice. Moreover, short-term pharmacologic inhibition of necroptosis in aged mice reverted structural and functional hippocampal impairment, both at the electrophysiological and behavioral level. Finally, a quantitative proteomic analysis revealed that necroptosis inhibition leads to an overall improvement of the aged hippocampal proteome, including a subclass of molecular biofunctions associated with brain rejuvenation, such as long-term potentiation and synaptic plasticity. Our results demonstrate that necroptosis contributes to the age-dependent brain degeneration, disturbing hippocampal neuronal connectivity, and cognitive function. Therefore, necroptosis inhibition constitutes a potential geroprotective strategy to treat age-related disabilities associated with memory impairment and cognitive decline.


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The current rise in human life expectancy is not precisely accompanied by an equivalent increase 44 in healthspan (Aburto et al. 2020  of axospinous synapses have been described in the aged hippocampus (Geinisman et al. 1992). The 52 reduced number of synaptic contacts is correlated with a decreased presynaptic fiber potential due 53 to a reduction of axons (Barnes & McNaughton 1980), contributing to the impaired synaptic plasticity 54 and cognitive deficits evidenced in aged organisms (Rosenzweig & Barnes 2003). In addition, white We recently demonstrated that mechanical and chemical-induced axonal degeneration is 114 regulated by necroptosis (Arrázola et al. 2019). To determine whether necroptosis is involved in age-115 associated AxD, we assessed necroptosis activation by the expression of the phosphorylated form of 116 MLKL (pMLKL) in the different hippocampal subfields, including the hilar axons of the DG and the 117 CA3-CA1 projecting Schaffer collateral axons (Fig 1f-k). The number of pMLKL positive hilar cells 118 increased in aged mice, as did pMLKL mean intensity (Fig 1f, h). Necroptosis activation was also 119 evidenced by an age-dependent translocation of pMLKL from the nucleus to the cytoplasm (Fig 1f, 120 magnified image), as previously described (Yoon et al. 2016). Axonal pMLKL staining was evaluated 121 in pan-axonal-NF positive hilar fibers of the DG (Fig 1i). Interestingly, pMLKL mean intensity 122 increased earlier in DG axons compared with dentate hilar somas throughout aging, reaching 123 significant differences in the old mice group (Fig 1h). Increased pMLKL signal was also observed in 124 other brain regions with defined axonal subfields, as axonal tracts in the striatum, the cerebellar white 125 matter and the ventral horn of the spinal cord, which also showed progressive AxD along aging (Fig   126   S3). The increase in pMLKL levels in the hilus was also accompanied by changes in the pattern of 127 pMLKL signal, from almost non-detected in adult mice axons, diffuse in the old group, to finally 128 become punctuated in axons of aged mice (Fig 1i, magnified image). These pMLKL aggregates have 129 been associated with MLKL oligomerization and its translocation to the plasma membrane, two key 130 steps for necroptosis execution (Samson et al. 2020). Same pMLKL pattern was observed by 131 immunohistochemistry against pMLKL in DG hilar cells and in the Schaffer collateral axons of CA3-132 CA1 circuit of aged mice (Fig 1k). These results indicate that necroptosis is activated during aging in 133 the hippocampus, a brain region that is considered one of the most vulnerable to the detrimental 134 effects of aging, affecting learning and memory (Spiegel et al. 2013).

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Due to the indispensable role of MLKL in executing necroptosis, we evaluated whether age-138 dependent AxD in the hippocampus was modified in aged Mlkl-knockout mice (Mlkl-KO) (Wu et al. 139 2013). Interestingly, AxD in pan-axonal NF-stained axons was significantly reduced in aged Mlkl-KO 140 mice at levels comparable with adult mice (Fig 2a, b). Moreover, non-pNF degenerated axons 141 profusely present in aged WT hippocampus, were almost undetected in aged Mlkl-KO mice, at levels 142 equivalent to younger WT mice (Fig 2a, c). As expected, we observed age-dependent AxD coupled 143 with neuroinflammation (Hwang et al. 2018) (Fig S4). The increased number of microglia in the 144 hippocampus of aged mice was prevented in aged Mlkl-KO mice, as it was the microglia overlapping 145 with degenerating axons (Fig S5), as previously described (Thadathil et al. 2021). Accordingly, 146 measurement of Iba1 mean intensity also indicated that aged Mlkl-KO mice present less microglia 147 activation than their WT littermates (Fig 2d, e). To confirm the contribution of necroptosis to the 148 inflammatory state of the aging brain, we measured the levels of several cytokines and chemokines 149 in hippocampal lysates and serum of adult versus aged WT and Mlkl-KO mice by Luminex High 150 6 Performance Assay (Table S1). Three of the twelve cytokines analyzed showed significant changes 151 under Mlkl deficiency in the hippocampus of aged mice. The levels of the pro-inflammatory cytokine 152 IL-12 decreased in aged Mlkl-KO hippocampus compared with aged WT mice, reaching levels 153 comparable with adult WT mice (Fig 2f). Interestingly, the anti-inflammatory cytokines IL-2 and IL-10 154 significantly increased in the hippocampus of aged Mlkl-KO mice (Fig 2g). Moreover, the systemic 155 pro-inflammatory profile also decreased in serum samples from aged Mlkl-KO mice (Fig S6). These 156 results indicate that necroptosis contributes to brain inflammation by modulating both pro-and anti-157 inflammatory cytokines.

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To further study whether necroptosis activation affects hippocampal function along aging, we first 161 performed electrophysiological recordings of the CA3-CA1 synapses to evaluate synaptic 162 transmission. Extracellular field-excitatory post-synaptic potentials (fEPSP) were registered in 163 hippocampal slices (Fig 2h). An age-dependent decrease in fEPSP slope was observed in WT mice.

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Nevertheless, in aged Mlkl-KO mice, the fEPSP slope was maintained at levels comparable to adult 165 WT mice (Fig 2i, j, k). To specifically evaluate whether axonal alterations contribute to an age-166 dependent decrease in synaptic transmission, we analyzed the facilitation index (fEPSP2/fEPSP1) 167 using a paired-pulse stimulation protocol (Fig 2l). The increased facilitation index observed in WT 168 aged mice indicates a decreased neurotransmitter release probability from the axonal compartment 169 (pre-synapse). Interestingly, the facilitation index of aged Mlkl-KO mice was comparable to adult WT 170 mice (Fig 2m). These results indicate that Mlkl deficiency delays the loss of synaptic strength in the 171 hippocampus inherent to brain aging, mainly preventing axonal function defects in aged mice.

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Since memory capabilities depend on proper hippocampal function and both are affected by age

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Aged Mlkl-KO mice travelled a larger distance and spent more time exploring in the target quadrant 180 Q4 compared to aged WT animals (Fig 2p,q), without significant changes in the mean swimming 181 speed between both groups (Fig S7), demonstrating that loss of Mlkl prevents learning and memory 182 loss associated with aging.

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Altogether, these results demonstrate that an age-associated increase in brain necroptosis 184 induces degeneration of axons in the hippocampus, thereby depressing synaptic transmission, and 185 impairing hippocampal-dependent functions, such as learning and memory in aged mice.

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To further explore the role of necroptosis in brain aging, we use the RIPK3 inhibitor, GSK'872 189 (Salvadores & Court 2020;Yang et al. 2017). Diffusion pumps were filled with vehicle or GSK'872, 190 and intraperitoneally implanted to continuously diffuse the inhibitor in 23-month-old mice for 28 days

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and comparable with those observed in adult animals (Fig 3b and 2c). Moreover, decreased 197 microglia activation was also observed, indicating that a short-term treatment with GSK'872 is capable 198 of reverting one of the main signs of brain inflammation associated with aging (Fig 3c, d).

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Since RIPK3 is involved in pathways that regulate cytokines secretion (Orozco & Oberst 2017) 200 we performed Luminex High Performance Assay to detect changes in a pool of selected cytokines 201 (Tables S1). Interestingly, the hippocampus of GSK'872 treated mice showed a pattern of cytokine 202 levels highly similar to those observed in untreated adult mice (Fig 3e). The analysis reveals that 203 RIPK3 inhibition mainly reduces pro-inflammatory cytokines, such as TNF-α, IL-6, IL-12, and IFN-γ, 204 reaching youthful-like cytokine levels equivalent to adult mice (Fig 3f).

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At the functional level, electrophysiological recordings in the hippocampus demonstrated 208 equivalent fEPSP between aged mice treated with GSK'872 and untreated adult mice. Average traces 209 showed significant differences between aged vehicle-treated mice versus those that received the 210 RIPK3 inhibitor (Fig 3g, h). In addition, facilitation index was significantly lower in the hippocampus 211 of aged mice treated with GSK'872 compared with the aged-vehicle mice (Fig 3i, j), showing that late 212 and short-term necroptosis inhibition can revert the loss of synaptic strength in aged mice.

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Remarkably, RIPK3 inhibition significantly improved learning in aged mice (Fig 3k). Memory 214 assessment indicated that aged mice with GSK'872 treatment spent more time in the target quadrant 215 and travelled larger distance in Q4 compared with aged vehicle-treated mice (Fig 3l-n). Thus, RIPK3 216 inhibition is capable to recover aged mice from learning and memory impairment.

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These results demonstrate that a short-term systemic administration of GSK'872, in a late phase 218 of the lifespan of mice, can revert key hallmarks of brain aging, including AxD, neuroinflammation, 219 and age-associated memory impairment, proposing the inhibition of necroptosis as an attractive 220 therapeutic target to improve memory in the elderly.

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In order to elucidate the specific molecular alterations underpinning our necroptosis-inhibitory 224 approaches toward reducing aging phenotypes, we employed a state-of-the-art single-shot, label free 225 quantitative proteomic approach. Hippocampus of adult and aged WT mice, aged Mlkl-KO mice, and 226 aged GSK'872-treated mice were subjected to single-shot label-free mass spectrometry (see 227 workflow in Fig S9a), obtaining a high degree of coverage of the proteome with almost 7,000 proteins 228 detected (Fig S9b).

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After relative expression ratios were calculated and expression profile clustering were performed, 230 we identified subsets of proteins exhibiting opposing directionality in expression between the "normal"  Fig S11a) and in brain rejuvenation (Bouchard & 240 Villeda 2015; Wyss-Coray 2016) (Fig 4b and Fig S11b). Most of the molecular cascades belonging 241 to these pathways are typically associated with neurodegeneration and/or neuronal aging, including 242 molecular cascades involved in synaptic mechanisms, senescence and cellular homeostasis (Fig   243   4b).

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In order to explore the contribution of necroptosis in age-associated neuronal dysfunction, we 245 evaluated several biological functions affected by normal aging in our proteomic analysis. These  (Fig 4c). Remarkably, key molecular and cellular functions associated with neurodegeneration, 251 neuronal integrity and function, showed a clear reversion in the context of pathway analysis in 252 necroptosis-targeted aging in comparison with normal aging (Fig 4c). This analysis demonstrated 253 that the inhibition of necroptosis supports proper brain function in aged animals, improving key

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Among the cascades elucidated in our proteomic analysis, those classified as synaptic 258 mechanisms showed the highest predicted z-score (Fig 4b). Synaptic long-term potentiation (LTP) is 259 a key process directly related with learning and memory, and early impaired during aging (Lynch et (Fig 5b). We therefore analyzed hippocampal synaptic plasticity by studying LTP magnitude in the 264 CA3-CA1 transmission. By the usage of a high-frequency stimulation protocol, we found that LTP 265 induction was compromised in aged WT mice when compared to adult WT mice (Fig 5c-e).
266 Surprisingly, adult Mlkl-KO mice presented a higher LTP magnitude than the control WT group and 267 the loss of Mlkl in aged mice restored LTP induction and maintenance beyond adult WT mice 268 potentiation. Similar to KO experiments, we detected a reduction in LTP magnitude when we 269 compared adult WT mice with aged vehicle-treated mice. Surprisingly, only one month of GSK'872 270 treatment in aged mice was capable to improve LTP magnitude, reaching adult-like levels (Fig 5f-h).

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As a correlate of learning and memory improvement, hippocampal long-term synaptic plasticity also 272 impacts dendritic spine remodeling (Engert & Bonhoeffer 1999). We observed that the inhibition of 273 necroptosis in aged Mlkl-KO mice protects from the loss of spines in CA1 neurons of the hippocampus 274 along aging (Fig 5i, j). Altogether, these results demonstrate that inhibition of necroptosis either by 275 genetic knock-out of Mlkl or by pharmacologic RIPK3 inhibition improved or restore synaptic plasticity 276 in aged mice (functionally and morphologically), a synaptic process that is crucial to support brain

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The accumulation of senescent cells in aged tissue, including the brain, is one of the most  , Fig 6a, b). We therefore evaluated cell senescence by SA-  (Fig 6c), but it was significantly lower in 293 aged Mlkl-KO mice (Fig 6c, d). A reduced SA-βgal activity was also observed in the hippocampus of 294 10 aged mice treated with GSK'872 compared with vehicle-treated animals (Fig 6e, f), reinforcing the 295 data obtained from the proteomic analysis, which overall indicates that age-related activation of 296 necroptosis contributes to the development of key pathological changes that are involved in brain

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Increased pMLKL levels in aged mice suggests that necroptosis could be also considered as a 321 biomarker of aging progression. Of note, aged Mlkl-KO mice presented a youthful phenotype in the 322 hippocampus, reaching levels of AxD comparable with those observed in younger mice (Fig 2).

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As a chronic inflammatory condition, aging also influences the inflammatory status of the brain  Mlkl-KO mice only IL-12 decreased, reaching levels comparable with adult mice (Fig 2). IL-12 is 327 produced in the brain by microglia and required for IFN-γ and TNF-α production, two master 328 inflammatory cytokines. The increased expression of IL-12 in the brain has been associated with 329 spontaneous neurological disorders in aged mice (Hofer et al. 2004). By contrast, the IL12-KO mouse 330 11 exhibits lower levels of microglia activation and reduced neurodegeneration in an excitotoxicity-331 mediated injury model (Chen et al. 2004). Additionally, increased levels of the anti-inflammatory 332 cytokines, IL-2 and IL-10, were specifically detected in aged Mlkl-KO mice (Fig 2), suggesting that

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Quantitative proteomic analysis demonstrated that about 7,000 proteins changed their expression 360 profile as a consequence of aging (Fig 4 and Fig S9), of which 2,516 shown to be upregulated and   (Fig 4 and Fig S11), including 373 those elucidated from the proteomic analysis and experimentally evaluated, such as synaptic 374 plasticity and neuronal senescence (Fig 5 and Fig 6). Moreover, other interesting pathways were 375 also demonstrated to be positively modulated by necroptosis inhibition (Fig S12-S15). These

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Images were taken with Nikon Eclipse E200 optic microscope with 4X and 10X objective 450 magnification. ImageJ software was used to process the images. Positive area for SA-βgal activity 451 was measured and representative images are shown.