APC/CCdh1-Rock2 pathway controls dendritic integrity and memory.

Disruption of neuronal morphology contributes to the pathology of neurodegenerative disorders such as Alzheimer's disease (AD). However, the underlying molecular mechanisms are unknown. Here, we show that postnatal deletion of Cdh1, a cofactor of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase in neurons [Cdh1 conditional knockout (cKO)], disrupts dendrite arborization and causes dendritic spine and synapse loss in the cortex and hippocampus, concomitant with memory impairment and neurodegeneration, in adult mice. We found that the dendrite destabilizer Rho protein kinase 2 (Rock2), which accumulates in the brain of AD patients, is an APC/CCdh1 substrate in vivo and that Rock2 protein and activity increased in the cortex and hippocampus of Cdh1 cKO mice. In these animals, inhibition of Rock activity, using the clinically approved drug fasudil, prevented dendritic network disorganization, memory loss, and neurodegeneration. Thus, APC/CCdh1-mediated degradation of Rock2 maintains the dendritic network, memory formation, and neuronal survival, suggesting that pharmacological inhibition of aberrantly accumulated Rock2 may be a suitable therapeutic strategy against neurodegeneration.

Regulation of Bcl-xL-ATP Synthase Interaction by Mitochondrial Cyclin B1-Cyclin-Dependent Kinase-1 Determines Neuronal Survival.

The survival of postmitotic neurons needs continuous degradation of cyclin B1, a mitotic protein accumulated aberrantly in the damaged brain areas of Alzheimer's disease and stroked patients. Degradation of cyclin B1 takes place in the proteasome after ubiquitylation by the anaphase-promoting complex/cyclosome (APC/C)-cadherin 1 (Cdh1), an E3 ubiquitin ligase that is highly active in neurons. However, during excitotoxic damage-a hallmark of neurological disorders-APC/C-Cdh1 is inactivated, causing cyclin B1 stabilization and neuronal death through an unknown mechanism. Here, we show that an excitotoxic stimulus in rat cortical neurons in primary culture promotes cyclin B1 accumulation in the mitochondria, in which it binds to, and activates, cyclin-dependent kinase-1 (Cdk1). The cyclin B1-Cdk1 complex in the mitochondria phosphorylates the anti-apoptotic protein B-cell lymphoma extra-large (Bcl-xL), leading to its dissociation from the ß subunit of F1Fo-ATP synthase. The subsequent inhibition of ATP synthase activity causes complex I oxidative damage, mitochondrial inner membrane depolarization, and apoptotic neuronal death. These results unveil a previously unrecognized role for mitochondrial cyclin B1 in the oxidative damage associated with neurological disorders.

Weak neuronal glycolysis sustains cognition and organismal fitness.

The energy cost of neuronal activity is mainly sustained by glucose1,2. However, in an apparent paradox, neurons modestly metabolize glucose through glycolysis3-6, a circumstance that can be accounted for by the constant degradation of 6-phosphofructo-2-kinase-fructose-2,6-bisphosphatase-3 (PFKFB3)3,7,8, a key glycolysis-promoting enzyme. To evaluate the in vivo physiological importance of this hypoglycolytic metabolism, here we genetically engineered mice with their neurons transformed into active glycolytic cells through Pfkfb3 expression. In vivo molecular, biochemical and metabolic flux analyses of these neurons revealed an accumulation of anomalous mitochondria, complex I disassembly, bioenergetic deficiency and mitochondrial redox stress. Notably, glycolysis-mediated nicotinamide adenine dinucleotide (NAD+) reduction impaired sirtuin-dependent autophagy. Furthermore, these mice displayed cognitive decline and a metabolic syndrome that was mimicked by confining Pfkfb3 expression to hypothalamic neurons. Neuron-specific genetic ablation of mitochondrial redox stress or brain NAD+ restoration corrected these behavioural alterations. Thus, the weak glycolytic nature of neurons is required to sustain higher-order organismal functions.

Astrocytic mitochondrial ROS modulate brain metabolism and mouse behaviour.

To satisfy its high energetic demand1, the brain depends on the metabolic cooperation of various cell types2-4. For example, astrocytic-derived lactate sustains memory consolidation5 by serving both as an oxidizable energetic substrate for neurons6 and as a signalling molecule7,8. Astrocytes and neurons also differ in the regulation of glycolytic enzymes9 and in the organization of their mitochondrial respiratory chain10. Unlike neurons, astrocytes rely on glycolysis for energy generation9 and, as a consequence, have a loosely assembled mitochondrial respiratory chain that is associated with a higher generation of mitochondrial reactive oxygen species (ROS)10. However, whether this abundant natural source of mitochondrial ROS in astrocytes fulfils a specific physiological role is unknown. Here we show that astrocytic mitochondrial ROS are physiological regulators of brain metabolism and neuronal function. We generated mice that inducibly overexpress mitochondrial-tagged catalase in astrocytes and show that this overexpression decreases mitochondrial ROS production in these cells during adulthood. Transcriptomic, metabolomic, biochemical, immunohistochemical and behavioural analysis of these mice revealed alterations in brain redox, carbohydrate, lipid and amino acid metabolic pathways associated with altered neuronal function and mouse behaviour. We found that astrocytic mitochondrial ROS regulate glucose utilization via the pentose-phosphate pathway and glutathione metabolism, which modulates the redox status and potentially the survival of neurons. Our data provide further molecular insight into the metabolic cooperation between astrocytes and neurons and demonstrate that mitochondrial ROS are important regulators of organismal physiology in vivo.

Hippocampal neurons require a large pool of glutathione to sustain dendrite integrity and cognitive function.

Loss of brain glutathione has been associated with cognitive decline and neuronal death during aging and neurodegenerative diseases. However, whether decreased glutathione precedes or follows neuronal dysfunction has not been unambiguously elucidated. Previous attempts to address this issue were approached by fully eliminating glutathione, a strategy causing abrupt lethality or premature neuronal death that led to multiple interpretations. To overcome this drawback, here we aimed to moderately decrease glutathione content by genetically knocking down the rate-limiting enzyme of glutathione biosynthesis in mouse neurons in vivo. Biochemical and morphological analyses of the brain revealed a modest glutathione decrease and redox stress throughout the hippocampus, although neuronal dendrite disruption and glial activation was confined to the hippocampal CA1 layer. Furthermore, the behavioral characterization exhibited signs consistent with cognitive impairment. These results indicate that the hippocampal neurons require a large pool of glutathione to sustain dendrite integrity and cognitive function.

The MDM2-p53 pathway is involved in preconditioning-induced neuronal tolerance to ischemia.

Brain preconditioning (PC) refers to a state of transient tolerance against a lethal insult that can be evoked by a prior mild event. It is thought that PC may induce different pathways responsible for neuroprotection, which may involve the attenuation of cell damage pathways, including the apoptotic cell death. In this context, p53 is a stress sensor that accumulates during brain ischemia leading to neuronal death. The murine double minute 2 gene (MDM2), a p53-specific E3 ubiquitin ligase, is the main cellular antagonist of p53, mediating its degradation by the proteasome. Here, we study the role of MDM2-p53 pathway on PC-induced neuroprotection both in cultured neurons (in vitro) and rat brain (in vivo). Our results show that PC increased neuronal MDM2 protein levels, which prevented ischemia-induced p53 stabilization and neuronal death. Indeed, PC attenuated ischemia-induced activation of the p53/PUMA/caspase-3 signaling pathway. Pharmacological inhibition of MDM2-p53 interaction in neurons abrogated PC-induced neuroprotection against ischemia. Finally, the relevance of the MDM2-p53 pathway was confirmed in rat brain using a PC model in vivo. These findings demonstrate the key role of the MDM2-p53 pathway in PC-induced neuroprotection against a subsequent ischemic insult and poses MDM2 as an essential target in ischemic tolerance.

Neovascularization and functional recovery after intracerebral hemorrhage is conditioned by the Tp53 Arg72Pro single-nucleotide polymorphism.

Intracerebral hemorrhage (ICH) is a devastating subtype of stroke that lacks effective therapy and reliable prognosis. Neovascularization following ICH is an essential compensatory response that mediates brain repair and modulates the clinical outcome of stroke patients. However, the mechanism that dictates this process is unknown. Bone marrow-derived endothelial progenitor cells (EPCs) promote endothelial repair and contribute to ischemia-induced neovascularization. The human Tp53 gene harbors a common single-nucleotide polymorphism (SNP) at codon 72, which yields an arginine-to-proline amino-acidic substitution (Arg72Pro) that modulates the apoptotic activity of the p53 protein. Previously, we found that this SNP controls neuronal susceptibility to ischemia-induced apoptosis in vitro. Here, we evaluated the impact of the Tp53 Arg72Pro SNP on vascular repair and functional recovery after ICH. We first analyzed EPC mobilization and functional outcome based on the modified Rankin scale scores in a hospital-based cohort of 78 patients with non-traumatic ICH. Patients harboring the Pro allele of the Tp53 Arg72Pro SNP showed higher levels of circulating EPC-containing CD34+ cells, EPC-mobilizing cytokines - vascular endothelial growth factor and stromal cell-derived factor-1α - and good functional outcome following ICH, when compared with the homozygous Arg allele patients, which is compatible with increased neovascularization. To assess directly whether Tp53 Arg72Pro SNP regulated neovascularization after ICH, we used the humanized Tp53 Arg72Pro knock-in mice, which were subjected to the collagenase-induced ICH. The brain endothelial cells of the Pro allele-carrying mice were highly resistant to ICH-mediated apoptosis, which facilitated cytokine-mediated EPC mobilization, cerebrovascular repair and functional recovery. However, these processes were not observed in the Arg allele-carrying mice. These results reveal that the Tp53 Arg72Pro SNP determines neovascularization, brain repair and neurological recovery after ICH. This study is the first in which the Pro allele of Tp53 is linked to vascular repair and ability to functionally recover from stroke.

γ-Glutamylcysteine detoxifies reactive oxygen species by acting as glutathione peroxidase-1 cofactor.

Reactive oxygen species regulate redox-signaling processes, but in excess they can cause cell damage, hence underlying the aetiology of several neurological diseases. Through its ability to down modulate reactive oxygen species, glutathione is considered an essential thiol-antioxidant derivative, yet under certain circumstances it is dispensable for cell growth and redox control. Here we show, by directing the biosynthesis of γ-glutamylcysteine-the immediate glutathione precursor-to mitochondria, that it efficiently detoxifies hydrogen peroxide and superoxide anion, regardless of cellular glutathione concentrations. Knocking down glutathione peroxidase-1 drastically increases superoxide anion in cells synthesizing mitochondrial γ-glutamylcysteine. In vitro, γ-glutamylcysteine is as efficient as glutathione in disposing of hydrogen peroxide by glutathione peroxidase-1. In primary neurons, endogenously synthesized γ-glutamylcysteine fully prevents apoptotic death in several neurotoxic paradigms and, in an in vivo mouse model of neurodegeneration, γ-glutamylcysteine protects against neuronal loss and motor impairment. Thus, γ-glutamylcysteine takes over the antioxidant and neuroprotective functions of glutathione by acting as glutathione peroxidase-1 cofactor.