Lactate, a metabolic end product of glycolysis in mammals, has emerged as an important energy substrate for the brain. In addition to its energetic role, lactate was shown to modulate the excitability of neurons, to have a neuroprotective role and to participate in long-term memory formation. One previous investigation from our group reported that lactate modulates 4 synaptic plasticity-associated genes and potentiates the activity of the N-Methyl-D-aspartic acid (NMDA) receptor, a major receptor type involved in glutamatergic neurotransmission. The current thesis aimed at first to extend these findings by examining genome-wide transcriptional responses to this metabolite in cortical neurons. Using ribonucleic acid(RNA) sequencing to evaluate expression changes in protein-coding genes, we found that lactate modulates robustly after 1h, 20 genes involved in the mitogen-activated protein kinase (MAPK) signaling pathway and in synaptic plasticity in a NMDA receptor activitydependent manner and that nicotinamide adenine dinucleotide, reduced (NADH), but not pyruvate, reproduces the modulatory effects of lactate on 70% of all differentially expressed genes. In a time course experiment, genes modulated after lactate treatment for 6h and 24h were also identified; these are involved in 9 signaling pathways including circadian rhythm, drug addiction, and retrograde endocannabinoid signaling. Bioinformatics analyses indicated CREB1 and CREM as candidate master regulators of gene expression and the modulatory effect of lactate was prevented by inhibitors of Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, indicating a role for this kinase in mediating lactate signaling. An examination of changes in dendritic spines’ morphology and density - a morphologicalcorrelate of synaptic plasticity – has shown that lactate modulated spine density changes induced by potassium chloride (KCl) and carbachol. An additional investigation described in this thesis indicated that different gut microbiota manipulations (germ-free, prebiotics, high-fat diet) regulated mRNA expression of genes involved in the Astrocyte-Neuron Lactate Shuttle (ANLS) - a metabolic cooperation mechanism between astrocytes and glutamatergic neurons. Overall, the results of this thesis help to establish a role for lactate as a signaling molecule in the brain, highlight mechanisms implicated in its signaling, and open new avenues for investigation of links between the gut microbiota and brain energy metabolism.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22338号 / 医博第4579号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邉 大, 教授 渡邊 直樹, 教授 髙橋 良輔 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Le bon fonctionnement cérébral dépend de la production et du positionnement appropriés des neurones, de la formation d’une connectivité neuronale adéquate et exige un équilibre strict de la formation de synapses excitatrice et inhibitrice. Des mutations sur des gènes qui sculptent et entretiennent normalement ces processus peuvent altérer les fonctions neuronales et conduire à divers troubles du développement neurologique chez l'homme, notamment des malformations du développement cortical (MDC). Les MDC sont des causes importantes de retard mental et représentent entre 20 et 40% des cas d'épilepsie résistante aux médicaments chez les enfants. Parmi les nombreux types de MDC, l'hétérotopie nodulaire périventriculaire (HNP) est la forme la plus répandue à l'âge adulte et elle est causée par une glie radiale défectueuse qui résulte en des nodules neuronaux ectopiques tapissant les parois des ventricules latéraux. Environ 88% des patients atteints d'HPN ont une épilepsie focale et la gravité des crises peut varier de légère à rare fréquence et de rémission sans besoin de médicaments antiépileptiques à insoluble. Plusieurs gènes ont été identifiés comme cause génétique de l'HPN chez des patients, notamment FLNA et NR2F1. Aucune corrélation entre l'étendue de l'HNP et la sévérité de l'épilepsie n'a été trouvée, ce qui suggère que des circuits aberrants dans le cortex cérébral normotopique plutôt que des nodules hétérotopiques pourrait expliquer la survenue de crises. Nous avons étudié l'impact in vivo de la perte de ces gènes lors de la dendritogénèse et de la synpatogénèse, ainsi que dans la morphologie et la maturation fonctionnelle des neurones corticaux / Proper brain function depends on the adequate production and positioning of neurons, the formation of correct neuronal connectivity, and requires a strict balance of excitatory and inhibitory synapse formation. Mutations in genes that normally carve and sustain these processes can alter neuronal functions and lead to various neurodevelopmental disorders in humans, including malformations of cortical development (MCD). MCDs are important causes of mental retardation and account for 20% - 40% of drug-resistant epilepsy cases in children. Among the many types of MCD, periventricular nodular heterotopia (PNH) is the most common form in adulthood and is caused by defective radial glia that result in ectopic neural nodules lining the walls of the lateral ventricles. About 88% of PNH patients have focal epilepsy and the severity of seizures can range from mild to rare frequency and remission without the need for antiepileptic drugs to insoluble. Several genes have been identified as a genetic cause of PNH in patients, including FLNA and NR2F1. No correlation between the extent of PNH and the severity of epilepsy was found, suggesting that aberrant circuits in the normotopic cerebral cortex rather than heterotopic nodules could explain the occurrence of seizures. We studied the in vivo impact of the loss of these genes during dendritogenesis and synpatogenesis, as well as in the morphology and functional maturation of cortical neurons.

The brain is capable of plasticity, so that the structural and functional loss that are caused by cortical injury may recover. Neuroinflammatory response can greatly influence post-injury recovery by modulating synaptic plasticity. In our previous work, mesenchymal derived exosomes were found to promote functional recovery by converting microglia from a pro-inflammatory state to an anti-inflammatory state in aged rhesus monkeys after cortical injury in the primary motor cortex. In the present project, we demonstrated the effects of exosomes on synaptic changes and synapse-microglia interactions after lesion in the same monkeys. To further investigate the effects of modulating neuroinflammation on synaptic changes after injury, we also investigated dietary curcumin, an anti-inflammatory substance, in a separate group of monkeys. Both treatments showed an effect as neuroinflammatory modulators that reduced the density of microglial markers, Iba- 1/P2RY12. However, the cortical injury induced synaptic loss was reversed by the exosome treatment, whereas the other anti-inflammatory treatment, curcumin, did not show the same effect. Our results are consistent with previous study that cortical injury induced synaptic loss and microglia activation. Exosomes can both reduce inflammation and synapse loss after injury, but curcumin only showed anti-inflammatory effects. Overall, these data suggested that exosomes and curcumin had different mechanisms of how to modulate inflammation and synaptic properties to promote recovery after cortical injury.

One of the earliest symptoms of Alzheimer’s disease (AD) and age-related cognitive decline is topographical disorientation or impairment to spatial navigation. Furthermore, aging and AD are associated with cortical gray-matter thinning, particularly in the medial temporal and posterior cingulate regions, which have been associated with spatial navigation. Aerobic exercise has been well-established as a beneficial intervention to curtail the neurodegenerative effects of aging. This study aims to explore the relationship between cardiorespiratory fitness (CRF), and two markers of AD and cognitive aging, virtual navigation ability and cortical thickness of the entorhinal, parahippocampal and retrosplenial regions. Cross-sectional data utilized in this study was collected from 23 healthy older adults (60-80 years). Measures included in our analyses consisted of estimated VO2max, T1-weighted structural MR images, and behavioral performance on a virtual navigation task, measured as numbers of objects located during recall. Cortical thickness of the regions of interest (ROIs) was determined by processing T1-weighted MR images in FreeSurfer. We hypothesized that greater CRF would correlate with improved virtual navigation performance and greater cortical thickness of ROIs. Our analyses did not reveal statistically significant relationships between CRF and navigation performance or CRF and cortical thickness. However, Pearson’s correlations found right retrosplenial cortical (RSC) thickness and navigation performance to be significantly related. Multiple regression models of right RSC thickness and navigation performance were performed controlling for age, sex, education and task version. These analyses revealed that greater right RSC thickness predicted navigation performance. Additionally, this model showed that older age predicts decline in navigation performance. Our findings did not survive multiple comparisons correction; nonetheless, the results provide promising insight to the relationship between cortical thickness and navigation performance in healthy aging. Further cross-sectional and longitudinal investigations with a larger sample size are required to assess the impact of CRF and exercise on cortical thickness and navigation abilities in healthy aging. Understanding these relationships would contribute to the expansive body of literature that has linked CRF and exercise to neuroprotective mechanisms in the aging brain.

Indiana University-Purdue University Indianapolis (IUPUI) / Chronic kidney disease (CKD) currently affects ~37 million Americans and causes substantially increased risk of skeletal fracture and fracture-related mortality. Current methods to treat CKD-related bone loss remain alarmingly ineffective. Skeletal fragility in CKD is predominately driven by deteriorations in cortical bone, highlighted by significant cortical porosity development. It is hypothesized that cortical porosity is largely driven by chronically high levels of parathyroid hormone (PTH), which alters the balance of bone remodeling in favor of rampant osteoclast activity and bone resorption. Restricting cortical bone deterioration and the development of cortical pores is likely essential to improve CKD patients’ bone health and reduce their fracture risk. The goal of this series of studies was to answer the following key questions: (1) to what degree do bisphosphonates, an approved pharmacological agent used in metabolic bone disease, accumulate in the skeleton of animals with CKD; (2) can smaller and more frequent doses of bisphosphonates alter skeletal accumulation and improve cortical architecture and the mechanical integrity of bone; (3) can non-bisphosphonate pharmacological interventions more specifically affect cortical bone deterioration. Utilizing epi-fluorescence and two-photon microscopy, our results show that bisphosphonates accumulate more in rats with renal impairment and fractionating bisphosphonates lowered skeletal accumulation irrespective of disease state. Further, studies in both rat and mouse models of CKD demonstrated different bisphosphonate treatments alone do not recover declines in cortical microarchitecture or mechanical properties in CKD. These findings demonstrate that a single intervention is not sufficient in managing CKD-induced bone alterations. Utilizing individual pore tracking analysis, we demonstrated cortical pores can be modulated with therapeutic interventions and can infill, despite the presence of CKD. Potent suppression of PTH led to significant pore infilling while more subtle reductions in PTH, via a calcimimetic, had less striking effects on bone. Calcimimetics mitigated cortical microarchitecture deterioration and reduced the rate of cortical pore expansion. Overall, these findings highlight the importance of PTH management for treating cortical deterioration in CKD. Although bisphosphonates can be utilized in ways that reduce skeletal accumulation, they appear to need co-therapies to reduce skeletal fragility associated with CKD.

Humanin (HN) is an anti-apoptotic peptide that suppresses neuronal cell death induced by Alzheimer's disease, prion protein fragments, and serum deprivation. Recently, we demonstrated that Gly14-HN (HNG), a variant of HN in which the 14th amino acid serine is replaced with glycine, can decrease apoptotic neuronal death and reduce infarct volume in a focal cerebral ischemia/reperfusion mouse model. In this study, we postulate that the mechanism of HNG's neuroprotective effect is mediated by the PI3K/Akt pathway. Oxygen-glucose deprivation (OGD) was performed in cultured mouse primary cortical neurons for 60 min. The effect of HNG and PI3K/Akt inhibitors on OGD-induced cell death was examined at 24 h after reperfusion. HNG increased cell viability after OGD in primary cortical neurons, whereas the PI3K/Akt inhibitors wortmannin and Akti-1/2 attenuated the protective effect of HNG. HNG rapidly increased Akt phosphorylation, an effect that was inhibited by wortmannin and Akti-1/2. Mouse brains were injected intraventricularly with HNG before being subjected to middle cerebral artery occlusion (MCAO). HNG treatment significantly elevated p-Akt levels after cerebral I/R injury and decreased infarct volume. The protective effect of HNG on infarct size was attenuated by wortmannin and Akti-1/2. Taken as a whole, these results suggest that PI3K/Akt activation mediates HNG's protective effect against hypoxia/ischemia reperfusion injury.

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Socioeconomic status (SES) is a key predictor of a multitude of health and functional outcomes, and growing up in low SES contexts is associated with poor neurocognitive outcomes and mental health problems across the lifespan. Recent studies of the links between SES and brain development suggest that associations between low SES and poor neurocognitive and health outcomes are likely accounted for by impacts on neurodevelopment. Low SES is associated with structural brain development, including reduced cortical thickness in frontal regions relevant to higher-level cognitive functions. However, understanding of how the impacts of SES on neurodevelopment contribute to developing psychopathology is limited. The study of how neurodevelopmental processes may contribute to depression is of particular interest given numerous neural correlates of depression, including reduced cortical thickness in frontal regions. Whereas both SES and depression independently have demonstrated associations with grey matter maturation, no studies to date have examined how the associations between SES, depression, and cortical structure interrelate. The current study addresses this gap in the literature by examining associations between SES at both household and neighborhood levels, depression symptoms, and cortical structure in adolescence. Furthermore, exploratory analyses investigated specific pathways of SES effects on depression symptoms and cortical structure through exposure to psychosocial stress. Data were drawn from an ongoing prospective longitudinal study of reward function development. Participants included 232 youth ages 9-13 (57.03% female; 46.59% White, 39.36% African American, 11.24% Multiracial, 2.81% Other; 11.24% Hispanic) and their primary caregivers who completed assessments of depression symptoms and stress at baseline and a 9-month follow-up, as well as an MRI assessment between baseline and follow up. Regression analyses examined associations between 1) SES to cortical structure, 2) SES to depression symptoms, 3) baseline depression symptoms and cortical structure, and 4) cortical structure to depression symptoms at follow-up. Structural equation models examined indirect effects of 1) SES on depression symptoms through cortical structure, 2) SES on cortical structure through depression symptoms, and 3) SES on cortical structure and depression symptoms through experiences of psychosocial stress. Neighborhood SES was positively associated with mean cortical thickness, and household SES was inversely associated with depression symptoms at follow up, controlling for baseline symptoms. No indirect effects were identified. Findings suggest that different aspects of SES may confer unique risks for neural and psychosocial development in early adolescence, such that SES of the neighborhood appears to have global effects on neurodevelopment that are not mediated by mood or proximal stress, whereas SES of the household appears to be associated with increasing mood symptoms and heightened stress experiences in early adolescence. / Psychology

The nucleosome remodeling and deacetylase (NuRD) complex makes use of Chromodomain Helicase DNA binding protein 3 (CHD3) to remodel chromatin. Of the three CHD proteins NuRD may incorporate (CHD3/4/5); the function of CHD3 within the brain is the only one to have not been studied through gene knockout. Chd3 mutations in humans cause a neurodevelopmental disorder (SNIBCPS) characterized by intellect and speech deficits, but limited work has been done to describe a phenotype in mice. We have generated mice with forebrain specific deletion of Chd3 to characterize its impact on embryonic and postnatal cortical development. To analyze the consequence of Chd3 ablation on cortical lamination, layer-specific staining was performed and showed a decreased number of cells in layers II-IV. Neuronal birthdating has demonstrated that this is due to a defect in migration, causing the cells to be retained in the lower layers. Behaviour testing has also indicated defect in fear learning and memory in heterozygote males. These initial indications of defects in cortical development and behavioural deficits in the Chd3 mutant mice suggest that the animals are a good first model of the SNIBCP syndrome.