Erythropoietin (EPO), named after its role in hematopoiesis, is also expressed in mammalian brain. In clinical settings, recombinant EPO treatment has revealed a remarkable improvement of cognition, but underlying mechanisms have remained obscure. Here, we show with a novel line of reporter mice that cognitive challenge induces local/endogenous hypoxia in hippocampal pyramidal neurons, hence enhancing expression of EPO and EPO receptor (EPOR). High-dose EPO administration, amplifying auto/paracrine EPO/EPOR signaling, prompts the emergence of new CA1 neurons and enhanced dendritic spine densities. Singlecell sequencing reveals rapid increase in newly differentiating neurons. Importantly, improved performance on complex running wheels after EPO is imitated by exposure to mild exogenous/inspiratory hypoxia. All these effects depend on neuronal expression of the Epor gene. This suggests a model of neuroplasticity in form of a fundamental regulatory circle, in which neuronal networks-challenged by cognitive tasks-drift into transient hypoxia, thereby triggering neuronal EPO/EPOR expression. 1 1234567890():,;E rythropoietin (EPO) is a hypoxia-inducible growth factor in mammalian kidney, named after its role in hematopoiesis 1,2 . Unexpectedly, both EPO and its receptor (EPOR) were later detected in the brain, where they are upregulated by injury conditions. High-dose recombinant human (rh) EPO, a drug in clinical use for anemic patients, exerts neuroprotective and neuroregenerative effects that are independent of the hematocrit, which is mechanistically unexplained 3-8 . Moreover, rhEPO improves cognitive function and reduces gray matter loss in a range of neuropsychiatric conditions 9-13 . Even in healthy mice, rhEPO treatment improves cognition, which is associated with enhanced hippocampal long-term potentiation [14][15][16] . Surprisingly, rhEPO increases the number of mature hippocampal pyramidal neurons without underlying effect on cell proliferation or cell death 17 . This effect is mediated in neurons mainly by JAK-STAT, PI3K/AKT/PKB, Ras-MEK, and ERK1/2, as well as NF-κB; pathways widely comparable to the hematopoietic system [18][19][20] . This raises the question whether the expression of EPO and its receptor serves a physiological function in the nervous system, and what could be the triggering factors of EPO expression under physiological conditions. ResultsGeneration of pyramidal neurons in adult mice and amplification by rhEPO. First, we developed a method to directly label and quantify newly generated neurons in the hippocampal cornu ammonis (CA) field of adult mice. This was possible by permanently labeling all mature pyramidal neurons present at P27 using a tamoxifen-inducible reporter gene in NexCreERT2::R26R-tdT mice (Fig. 1a, b) 21 . Thus, all neurons differentiating and maturing after termination of the tamoxifen-induced Cre recombination lack tdTomato, but can be positively identified by Ctip2, a specific marker of pyramidal neurons, thereby revealing adult 'neurogenesis' independent of DNA synt...

Physical activity and cognitive challenge are established non-invasive methods to induce comprehensive brain activation and thereby improve global brain function including mood and emotional well-being in healthy subjects and in patients. However, the mechanisms underlying this experimental and clinical observation and broadly exploited therapeutic tool are still widely obscure. Here we show in the behaving brain that physiological (endogenous) hypoxia is likely a respective lead mechanism, regulating hippocampal plasticity via adaptive gene expression. A refined transgenic approach in mice, utilizing the oxygen-dependent degradation (ODD) domain of HIF-1α fused to CreERT2 recombinase, allows us to demonstrate hypoxic cells in the performing brain under normoxia and motor-cognitive challenge, and spatially map them by light-sheet microscopy, all in comparison to inspiratory hypoxia as strong positive control. We report that a complex motor-cognitive challenge causes hypoxia across essentially all brain areas, with hypoxic neurons particularly abundant in the hippocampus. These data suggest an intriguing model of neuroplasticity, in which a specific task-associated neuronal activity triggers mild hypoxia as a local neuron-specific as well as a brain-wide response, comprising indirectly activated neurons and non-neuronal cells.

Highlights d Microglia transiently respond to EPO by apoptosis, followed by abridged proliferation d Reduction of microglia allows undisturbed fast differentiation of immature neurons d Microglial and pyramidal EPOR are critical for neurodifferentiation in CA1 on EPO d EPO acts as regulator of neuronally expressed IL-34 and CSF1R-dependent microglia

PrefaceExecutive functions, learning, attention, and processing speed are imperative facets of cognitive performance, affected in neuropsychiatric disorders. In clinical studies on different patient groups, recombinant human (rh) erythropoietin (EPO) lastingly improved higher cognition and reduced brain matter loss. Correspondingly, rhEPO treatment of young rodents or EPO receptor (EPOR) overexpression in pyramidal neurons caused remarkable and enduring cognitive improvement, together with enhanced hippocampal long-term potentiation. The ‘brain hardware upgrade’, underlying these observations, includes an EPO induced ~20% increase in pyramidal neurons and oligodendrocytes in cornu ammonis hippocampi in the absence of elevated DNA synthesis. In parallel, EPO reduces microglia numbers and dampens their activity and metabolism as prerequisites for undisturbed EPO-driven differentiation of pre-existing local neuronal precursors. These processes depend on neuronal and microglial EPOR. This novel mechanism of powerful postnatal neurogenesis, outside the classical neurogenic niches, and on-demand delivery of new cells, paralleled by dendritic spine increase, let us hypothesize a physiological procognitive role of hypoxia-induced endogenous EPO in brain, which we imitate by rhEPO treatment. Here we delineate the brain EPO circle as working model explaining adaptive ‘brain hardware upgrade’ and improved performance. In this fundamental regulatory circle, neuronal networks, challenged by motor-cognitive tasks, drift into transient ‘functional hypoxia’, thereby triggering neuronal EPO/EPOR expression.

ObjectivesNon-alcoholic fatty liver disease (NAFLD) can persist in the stage of simple hepatic steatosis or progress to steatohepatitis (NASH) with an increased risk for cirrhosis and cancer. We examined the mechanisms controlling the progression to severe NASH in order to develop future treatment strategies for this disease.DesignNFATc1 activation and regulation was examined in livers from patients with NAFLD, cultured and primary hepatocytes and in transgenic mice with differential hepatocyte-specific expression of the transcription factor (Alb-cre, NFATc1c.a. and NFATc1Δ/Δ). Animals were fed with high-fat western diet (WD) alone or in combination with tauroursodeoxycholic acid (TUDCA), a candidate drug for NAFLD treatment. NFATc1-dependent ER stress-responses, NLRP3 inflammasome activation and disease progression were assessed both in vitro and in vivo.ResultsNFATc1 expression was weak in healthy livers but strongly induced in advanced NAFLD stages, where it correlates with liver enzyme values as well as hepatic inflammation and fibrosis. Moreover, high-fat WD increased NFATc1 expression, nuclear localisation and activation to promote NAFLD progression, whereas hepatocyte-specific depletion of the transcription factor can prevent mice from disease acceleration. Mechanistically, NFATc1 drives liver cell damage and inflammation through ER stress sensing and activation of the PERK-CHOP unfolded protein response (UPR). Finally, NFATc1-induced disease progression towards NASH can be blocked by TUDCA administration.ConclusionNFATc1 stimulates NAFLD progression through chronic ER stress sensing and subsequent activation of terminal UPR signalling in hepatocytes. Interfering with ER stress-responses, for example, by TUDCA, protects fatty livers from progression towards manifest NASH.