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Neuroprotective actions of recombinant irisin in vitro. Because abnormal eukaryotic initiation factor 2α (eIF2α ) phosphorylation and inhibition of protein synthesis have been recently described as key mechanisms driving synapse damage and memory failure in AD models10,26,30–32, we examined the effects of recombinant irisin on phosphorylated eIF2α (eIF2α -P) and activating transcription factor 4 (ATF4) levels in cultured primary hippocampal neurons. Irisin prevented Aβ O-induced elevation in eIF2α -P and ATF4 (Extended Data Fig. 9a–c), as well as downregulation of de novo pro-tein synthesis in hippocampal neurons (Extended Data Fig. 9d,e). Control measurements revealed that total eIF2α immunoreactivity remained unchanged (not shown).We further found that recombinant irisin prevented dendritic spine loss in cultured hippocampal neurons exposed to Aβ Os (Extended Data Fig. 9f,g). Additional experiments determined that recombinant irisin reduced Aβ O binding to neurons (Extended Data Fig. 9h,i). Control binding studies revealed no direct interac-tion between Aβ Os and recombinant irisin (Extended Data Fig. 9j), ruling out the possibility that blockade of binding to neurons might be caused by sequestration of Aβ Os by recombinant irisin added to the medium. In addition, surface FNDC5/irisin and Aβ Os did not colocalize in dendrites of hippocampal neurons (Extended Data Fig. 9k). Results further showed that FNDC5/irisin overexpression reduced hippocampal soluble Aβ 42 levels in APP/PS1 M146L mice (Extended Data Fig. 9l), but not insoluble Aβ 42 in the hippocampus or cortex (Extended Data Fig. 9m–o).We found that recombinant irisin stimulated the cyclic AMP (cAMP)–protein kinase A (PKA)–cAMP responsive element-bind-ing protein (CREB) pathway in human cortical slices (Fig. 5a–c), a pathway that plays important roles in memory formation and has been found to be impaired in AD models33–35. Recombinant irisin further increased cAMP and phosphorylated CREB in mouse hip-pocampal slices (Fig. 5d,e). Irisin-induced CREB phosphoryla-tion was abolished by PKA inhibition with myristoylated protein kinase inhibitor (PKI) 14–22, a selective PKA inhibitor (Fig. 5e). We also found that PKA activity mediated protection against nuclear translocation of ATF4 induced by Aβ Os (Fig. 5f,g). Irisin further induced transient phosphorylation of extracellular signal-regulated kinase in cultured neurons (data not shown). The effect of recombinant irisin was similar to forskolin, a direct activator of adenylyl cyclase (data not shown). Taken together, these results provide initial clues into the mechanisms by which recombinant irisin affords neuroprotection in experimental models of AD.

FNDC5/irisin mediates the protective actions of physical exercise on synaptic plasticity and memory in AD models. From a trans-lational perspective, physical exercise could be a non-pharmacolog-ical strategy to increase hippocampal FNDC5/irisin in patients at risk of developing AD or in patients already exhibiting cognitive impairment. Thus, we initially tested whether a protocol of daily swimming (1 h per day, 5 days per week for 5 weeks) could protect mice from Aβ O-induced memory deficits and reduction in brain levels of FNDC5/irisin. Notably, exercised mice were protected from Aβ O-induced impairment in NOR memory both 24 h (Extended Data Fig. 10a) and 5 days (Extended Data Fig. 10b) following Aβ O infusion. Protection against Aβ O-induced memory deficits was also verified in the CFC paradigm (Extended Data Fig. 10c).We further found that this exercise protocol prevented Aβ O-induced reductions in FNDC5/irisin mRNA (Extended Data Fig. 10d) and protein (Extended Data Fig. 10e) in mouse hippo-campi. Moreover, consistent with a previous report18, hippo-campal levels of FNDC5/irisin (Extended Data Fig. 10e) and BDNF (Extended Data Fig. 10f,g) were higher in exercised mice compared to sedentary animals.We then asked whether brain FNDC5/irisin mediated the ben-eficial effect of physical exercise on synapse plasticity in APP/PS1 Δ E9 mice. We conducted an intracerebroventricular infusion of a lentiviral vector harboring FNDC5 shRNA and subsequently sub-jected mice to the exercise protocol. Results revealed that exercise improved LTP in APP/PS1 Δ E9 mice infused with an innocuous shRNA (targeting luciferase), but not in APP/PS1 Δ E9 mice exhib-iting downregulated brain FNDC5/irisin expression (Fig. 6a,b). Additionally, we observed that downregulation of brain FNDC5/irisin did not exacerbate LTP impairment in sedentary APP/PS1 Δ E9 mice (Fig. 6a,b).We next aimed to determine a potential role of peripheral FNDC5/irisin in the brain. We administered the AdFNDC5 vec-tor into the caudal vein of mice to induce peripheral FNDC5/iri-sin expression, as previously described18. Peripheral administration of AdFNDC5 rescued NOR memory defects in Aβ O-infused mice (Fig. 6c). We next evaluated plasma levels of FNDC5/irisin in this group of mice and found unaltered levels in Aβ O-infused animals (Fig. 6d). As expected, plasma levels of FNDC5/irisin were increased in animals that received AdFNDC5 intravenously (Fig. 6d). Moreover, while hippocampal levels of FNDC5/irisin were decreased in Aβ O-infused mice, intravenous injection of AdFNDC5 resulted in increased hippocampal FNDC5/irisin levels and pre-vented the decrease triggered by Aβ Os (Fig. 6e). These results suggest that peripheral FNDC5/irisin can either reach the brain or trigger an increase in brain FNDC5/irisin. They further dem-onstrate that peripheral FNDC5/irisin affords protection against Aβ O-induced memory impairment.Our second approach to investigate the role of peripheral FNDC5/irisin in synaptic plasticity and memory in AD models consisted of neutralizing peripheral FNDC5/irisin. To this end, we carried out intraperitoneal injections of anti-FNDC5 antibodies in either sedentary or exercised Aβ O-infused mice. This approach has been previously described to attenuate irisin-induced expression of pro-thermogenesis genes in mice16. Interestingly, intraperito-neal administration of anti-FNDC5 blocked the protective actions of physical exercise against the impairments in synaptic plasticity and memory induced by Aβ Os (Fig. 6f,g). We further noted that intraperitoneal administration of the anti-FNDC5 antibody per se impaired synaptic plasticity and performance in the NOR test (Fig. 6f,g). We next aimed to evaluate the effects of exercise and peripheral administration of anti-FNDC5 on hippocampal FNDC5/irisin levels. In accordance with the results in Extended Data Fig. 10, we found that exercise prevented the decrease in hippocampal levels of FNDC5/irisin triggered by Aβ Os (Fig. 6h). Interestingly, we observed that administration of anti-FNDC5 led to a decrease in hippocampal FNDC5/irisin levels in exercised Aβ O-infused mice (Fig. 6h). Collectively these results indicate that peripheral irisin may reach the brain and mediate the neuroprotective actions of exercise in synaptic plasticity and memory in AD.Lastly, we tested whether the approach to neutralize peripheral FNDC5/irisin using an anti-FNDC5 antibody would block the ben-eficial effects of exercise on memory in APP/PS1 Δ E9 mice. Our results showed that peripheral administration of the anti-FNDC5 antibody prevented the protective actions of exercise in APP/PS1 Δ E9 mice in the NOR test (Fig. 6i). Collectively, these results impor-tantly demonstrate that FNDC5/irisin mediates the positive effects of exercise on synaptic plasticity and memory.

AD is a neurological disorder that primarily affects memory, with no cure to date. A variety of potential underlying mechanisms have been proposed to explain the pathogenesis of AD10,36. Considerable evidence indicates that memory impairment in AD is caused by synapse failure and loss2,37,38. Thus, therapies aimed at restoring or preserving synapse function and cognition are highly warranted.Irisin was originally discovered as an exercise-induced myokine that shifts the adipose metabolism toward a thermogenic profile16,39. There has been some debate as to the nature and identity of FNDC5/irisin, and to its functional relevance in humans40. While concerns have been expressed regarding the lack of specificity of anti-irisin antibodies41, sensitive approaches have been successfully employed to confirm the identity of irisin and to measure its circulating levels in humans17,20. Three complementary lines of evidence indicate the specificity of the anti-FNDC5 antibody and ELISA kit used in the current work: (1) FNDC5/irisin levels detected by ELISA showed the expected increase or decrease, respectively, when FNDC5/irisin was overexpressed by use of an adenoviral vector (in both brain and circulation) or when it was knocked down in the brain using a len-tiviral vector; (2) surface labeling of FNDC5/irisin by anti-FNDC5 in cultured neurons was predictably reduced in neurons infected by a lentiviral vector harboring shRNA to knock down FNDC5/irisin; (3) mass spectrometry analysis identified peptides contained within FNDC5 in excised gel bands that were immunolabeled by the anti-FNDC5 antibody. By coupling immunodetection and mass spectrometry, we offer initial evidence that FNDC5/irisin is present in multiple forms with distinct apparent molecular weights in the brain, suggesting it may undergo posttranslational modifications and/or exist in different aggregation states. Indeed, putative glyco-sylation sites are present in FNDC5/irisin21, and previous reports have suggested that shifts in electrophoretic mobility of FNDC5/irisin are due to glycosylation16,20.An interesting study demonstrated that FNDC5/irisin is induced via PGC-1α in the mouse brain and promotes BDNF expression18. Extending those previous findings and arguing for a physiologi-cal role of FNDC5/irisin in the human brain, we show that ex vivo human adult cortical slices express FNDC5/irisin and respond to exogenous recombinant irisin by activating the cAMP–PKA–CREB memory pathway42. Our in vitro findings further show that irisin blocks Aβ O binding to neurons and prevents Aβ O-induced eIF2α -P and inhibition of protein synthesis. We and others have recently described these events as essential for synapse and memory failure in AD models, thereby pinpointing a potential downstream pathway by which irisin preserves memory10,26,30. We note that the cellular receptor(s) for irisin remain(s) to be identified, which limits current knowledge on downstream signaling mechanisms. The identifica-tion of the irisin receptor(s) and the detailed signaling mechanisms triggered in the periphery and in the brain are needed in the field.The reduced brain and CSF levels of FNDC5/irisin in AD patients and in animal models reported in this study support the notion that defective brain hormonal signaling in AD impacts mechanisms related to memory formation and brain function3–5. It is noteworthy that we found a positive correlation between age and CSF irisin in control, non-demented individuals, but not in MCI and AD patients, suggesting that the increase in brain FNDC5/irisin with aging may be part of an endogenous mechanism to cope with the many challenges faced by the aging brain.Irisin was reduced in the CSF of AD patients, but not in plasma, indicating a specific decrease in the CNS. Our finding that hippo-campal FNDC5/irisin is reduced in moderate-to-late AD, but not in MCI, suggests that decreased FNDC5/irisin is not a likely cause of early cognitive impairment in AD, but may contribute to memory ARTICLESNATURE MEDICINEdefects as disease progresses. Additional studies measuring irisin levels in the CSF during healthy aging and in patients with other neurological disorders are anticipated and will help to determine when irisin levels decrease during the course of AD.Blockade of either brain or peripheral FNDC5/irisin in mice impaired LTP and NOR memory, implicating FNDC5/irisin in physiological memory processes. However, future studies are warranted to address the precise physiological roles of brain and peripheral FNDC5/irisin in the formation and consolidation of dif-ferent types of memories.It is noteworthy that peripheral administration of AdFNDC5 led to increases in hippocampal FNDC5/irisin and protected mice against memory impairment induced by Aβ Os. Most importantly, peripheral FNDC5/irisin is implicated in the preservation of hippo-campal FNDC5/irisin levels, synaptic plasticity, and memory in AD mice. Collectively, these results provide mechanistic information on the beneficial actions of FNDC5/irisin in the brain and suggest that a cross talk between peripheral and central FNDC5/irisin influ-ences synaptic plasticity and memory in mice.Physical exercise has been previously shown to induce mem-ory-related events in the brain43–45 and has been proposed as an approach to reduce the risk of AD, potentially bringing about signif-icant benefits to subjects with MCI and early AD46–49. Many efforts to identify the endogenous molecules responsible for the beneficial effects of exercise are underway. Brain PGC-1α and BDNF28,50, as well as peripheral cathepsin B and β -hydroxybutyrate51,52, have been described as important molecules acting as mediators of exercise-induced neuroprotection. The results presented in this study add FNDC5/irisin to this list.Our findings suggest that FNDC5/irisin could comprise an attractive novel therapy aimed to prevent dementia in patients at risk, as well as delay its progression in patients at the later stages, including those who can no longer exercise. Many patients with dementia are disabled due to other age-related conditions or comorbidities (for example, arthritis, heart disease, obesity, visual problems, depression) that preclude them from engaging in regu-lar physical exercise. Therefore, the development of alternative approaches that build on the beneficial effects of exercise in the brain may benefit those patients.In conclusion, our results demonstrate that FNDC5/irisin lev-els are reduced in human AD brains and CSF and in AD mouse models, and that boosting either brain or peripheral FNDC5/irisin levels attenuates synaptic and memory impairments in AD mouse models. We further show that FNDC5/irisin is a novel mediator of the beneficial effects of exercise on synapse function and memory in AD models. Bolstering brain FNDC5/irisin levels, either pharmaco-logically or through exercise, may thus constitute a novel therapeu-tic strategy to protect and/or repair synapse function and prevent cognitive decline in AD.

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