According to the World Health Organization (WHO) depression is the leading cause of disability worldwide with more than 300 million patients affected. Current antidepressants have a delayed onset of action and moreover, only two-thirds of patients suffering from depressive disorder respond to antidepressant drug treatment. The N-methyl-D-aspartate (NMDA) receptor antagonist ketamine offers promising perspectives for the treatment of major depressive disorder. Although ketamine demonstrates rapid and long-lasting effects, even in treatment-resistant patients, to date, the underlying mode of action remains elusive. Thus, the aim of this thesis was to investigate the molecular mechanism of ketamine and its major metabolites at clinically relevant concentrations by establishing an in vitro model based on human induced pluripotent stem cells (iPSCs)-derived neural progenitor cells (NPCs). As the pathophysiology of depression correlates with decreased adult neurogenesis, I aimed to investigate the molecular effects of ketamine on neural progenitor cell proliferation using a human-based iPSC-model. The findings from this thesis substantially contribute to an enhanced understanding of the molecular mode of action of ketamine as a novel signaling pathway involved in ketamine-induced effects was identified. Ketamine induced proliferation of human iPSC-derived NPCs and bioinformatic analysis of RNA-Seq data revealed significant upregulation of insulin-like growth factor2 (IGF2) and p11, a member of the S100 EF-hand protein family, which are both implicated in the pathophysiology of depression, 24 hours after ketamine treatment. In line with this, ketamine dependent proliferation was significantly impaired after IGF2 knockdown. Moreover, ketamine was able to enhance cAMP signaling in NPCs and both, cell proliferation as well as IGF2 expression, were reduced after protein kinase A (PKA)-inhibition. Noteworthy, the Nestin-expressing NPCs do not express functional NMDA receptors, suggesting that the proproliferative effect of ketamine in NPCs is NMDA receptor-independent. Furthermore, 24 hours post administration of ketamine (15 mg/kg) in vivo confirmed phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in the subgranular zone (SGZ) of the hippocampus in C57BL/6 mice. In conclusion, ketamine promotes proliferation of NPCs presumably by involving cAMP-IGF2 signaling.

The Dlx homeobox genes encode homeodomain transcription factors that are involved in multiple developmental aspects. In the brain, these genes take part in neuronal migration and differentiation, more precisely in the migration and differentiation of GABAergic neurons. Dysfunctions in the GABAergic system can lead to various pathological conditions, where impaired inhibitory function is one of the main causes of several neuropathies characterized by neuronal hyperexcitability. The Dlx genes are organized as bi-gene clusters and highly conserved cis-regulatory elements have been previously characterized to be fundamental for the regulation of Dlx expression in developing embryos of different vertebrates. The activity of these regulatory elements and the Dlx genes has been well studied in developmental stages of mice and zebrafish, but little is known about their activity in the adult brain. The extensive neurogenesis that takes place in the adult zebrafish brain provides an ideal platform for the visualization of mechanisms involving dlx genes during adulthood and their possible involvement in adult neurogenesis. Here we show novel information concerning the expression of dlx1a, dlx2a, dlx5a and dlx6a in the adult zebrafish brain and provide insight into the identity of cells that express dlx. We also demonstrate the involvement of dlx genes in brain regeneration and through lineage tracing, their fate determination in the adult zebrafish brain. Analyses in the adult zebrafish has revealed that all four dlx paralogs are expressed in the forebrain and midbrain throughout adulthood and expression is found in almost all areas presenting continuous proliferation. Most dlx-expressing cells present GABAergic neuronal identity in the adult forebrain where, in some areas they were identified as the Calbindin subtype. In some areas of the midbrain, especially within the hypothalamus, many dlxexpressing cell co-localized with a marker for neural stem cells. However, cells expressing dlx iii genes did not co-localize with markers for proliferating cells or for glia. Investigations during brain regeneration in response to injury in the adult zebrafish brain has revealed that dlx5a expression decreases shortly after lesion and that the dlx5a/6a bi-gene cluster, more specifically, dlx5a, is up regulated during the peak of regeneration response proposing a possible role for dlx during regeneration in adults. Studies of lineage tracing have shown the progeny of dlx1a/2a-expressing cells in adults are located within small clusters in different areas of the adult brain where they seem to become mature neurons. Our observations provide a better understanding about the role of dlx genes during adulthood, further contributing to the general knowledge of the molecular pathways involved in adult neurogenesis and regeneration in the zebrafish adult brain.

Indiana University-Purdue University Indianapolis (IUPUI) / Traumatic brain injury (TBI) is a critical public health issue in the United States, affecting about 2.8 million people annually. Extensive cell death and neural degeneration directly and diffusively caused by the initial mechanical insult results in a wide range of neurological complications post-trauma. Learning and memory dysfunction is one of the most common complains. Hippocampal neuronal loss, together with other mechanisms, largely contributes to learning and memory impairment as well as other cognitive dysfunctions post-trauma. To date, no FDA-approved drug is available to target cell death or improve learning and memory following TBI. It is of great interest to develop alternative approaches targeting neural repair instead. Neural stem/progenitor cells (NSCs) in the adult hippocampus undergo life-long neurogenesis supporting learning and memory functions, thus hold great promise for post-traumatic neuronal replacement. The previous studies demonstrated that TBI transiently increase NSC proliferation. However, it is debated on whether TBI affects neurogenesis. The mechanism of TBI-enhanced NSC proliferation remains elusive. In the current studies, I have investigated post-traumatic neurogenesis after different injury severities, evaluated integration of post-injury born neurons, illustrated a molecular mechanism mediating TBI-enhanced NSC proliferation, proposed a de novo state of NSCs, and tested effects of a pharmacological approach on spatial learning and memory function recovery. My results demonstrated that post-traumatic neurogenesis is affected by injury severities, partially explained the pre-existing inconsistency among works from different groups. Post-injury born neurons integrate in neural network and receive local and distal inputs. TBI promotes functional recruitment of post-injury born neurons into neural circuits. Mechanistically, mechanistic target of rapamycin (mTOR) pathway is required primarily for TBI-enhanced NSC proliferation; NSCs feature a de novo alert state, in which NSCs are reversibly released from quiescence and primed for proliferation. Furthermore, my data demonstrated a beneficial role of ketamine in improving post-traumatic spatial learning possibly by activating mTOR signal in NSCs and/or promoting neuronal activity of post-injury born neurons. Together, my data support the feasibility of neurogenesis mediated neuronal replacement, provide a target for enhancing post-traumatic NSC proliferation and subsequent neurogenesis, and prove a potential pharmacological approach benefiting post-traumatic functional recovery in learning and memory. / 2021-11-04

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20756号 / 医博第4286号 / 新制||医||1024(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 伊佐 正, 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

Adult hippocampal neurogenesis, the generation of new neurons in the adult hippocampus, represents the most drastic form of ongoing plasticity in the human brain. When these adult-born neurons are a few weeks old, they have developed enough connections with surrounding hippocampal neurons to evoke meaningful change in network dynamics, but still have different morphological and physiological properties compared to developmentally generated neurons that render them more plastic. As such, and due to their location in the hippocampus, many have theorized that these new neurons play an important role in certain forms of learning memory as well as emotion. This dissertation outlines the first attempt to answer the question “what are new neurons in the hippocampus good for?” using human participants. Aerobic exercise is a lifestyle factor well-established from the animal literature to upregulate neurogenesis, while chronic stress is a known downregulator of neurogenesis. The second chapter of this thesis describes a study in which aerobic capacity and depression inventory scores demonstrated a significant positive correlation and a significant negative correlation with putative neurogenesis-dependent memory, respectively, in separate cohorts of healthy young adults. The third chapter outlines a study that expands on the one presented in the second by elucidating another potential role for neurogenesis in human cognition – remote memory. Finally, Chapter 4 describes a study investigating the utility of neurotrophins measured from peripheral blood as biomarkers for neurogenic activity in humans by examining how changes in their expression following chronic exercise predict changes in putative neurogenesis-dependent memory performance. These studies are the first to explicitly test and provide supporting evidence for the theoretical roles of adult hippocampal neurogenesis in humans. Taken together, these studies provide a strong foundation for how investigators and clinicians can indirectly quantify and test the function of adult-born neurons in the human brain. / Dissertation / Doctor of Philosophy (PhD) / New neurons are generated in the adult hippocampus throughout life. The hippocampus is a structure in the medial temporal lobe important for learning and memory as well as emotion. It is currently unknown what the contributions of newborn neurons are to these processes. This dissertation outlines the first attempt to answer the question “what are new neurons in the hippocampus good for?” using human participants. Aerobic exercise is a lifestyle factor well-known from research in rodents to positively influence the rate of birth of newborn neurons in the hippocampus, while long-term stress reduces the rate of birth. The second chapter of this thesis describes a study in which aerobic fitness and depression inventory scores demonstrated a significant positive correlation and a significant negative correlation with a memory test susceptible to high interference, respectively, in different populations of healthy young adults. The third chapter outlines a study that expands on the one presented in the second chapter by elucidating another potential role for neurogenesis in human cognition – long-term memory. Finally, Chapter 4 describes a study investigating the how measuring various proteins found in circulating blood may help us to understand how exercise influence the rate of birth of new hippocampal neurons in humans. These studies are the first to test and provide supporting evidence for the potential roles of newborn hippocampal neurons in humans. Taken together, these studies provide a strong foundation for how investigators and clinicians can indirectly quantify and test the role of adult-born neurons in the human brain.

New models in which aging-related neurodegeneration more closely resembling the combination of pathologies that develop in aging humans, are needed. The fish Nothobranchius, which naturally develops such pathologies over the course of its short lifespan, is one such model. This review compares the lifespans and pathologies of different Nothobranchius strains to those of current vertebrate models of aging. Furthermore, existing data pertaining to neurodegeneration in these fish is discussed in the context of their reported neuropathologies, along with open questions related to mammalian chronopathologies. Specifically, the evidence for a Parkinson’s disease-like pathology is discussed. Neurogenesis and age-related changes therein are discussed in the context of siRNA and neurodegeneration. We also discuss changes in the expression of neuropeptide Y in relation to the brain-gut axis and how these change with age. Age-related behavioral changes are discussed, along with the assays used in their evaluation. Genetic discoveries are outlined and discussed with a view on DJ-1/NRF2 signaling in N. furzeri, and insights gained from comparative genomics and siRNA studies. Finally, research focus areas are highlighted, and a case is made for the utility of these fish in the study of aging-related neurodegeneration, and to screen for environmental risk factors of aging-related neuropathology.

Reptiles exhibit neurogenesis throughout the brain during adulthood. However, very few studies have quantified telencephalon-wide neurogenesis in adulthood, and no studies have performed these investigations in snakes. Quantifying neurogenesis in the adult snake is essential to understanding class-wide adult neurogenesis and providing insight into the evolution of this trait. The thymidine analog 5-bromo-2’-deoxyuridine (BrdU) was used to quantify cell proliferation, migration, and survival in the ball python (Python regius). First, to determine the proper dose of BrdU for injection we subcutaneously injected 50mg/kg, 100mg/kg, and 250mg/kg into 15 adult male P. regius. We found the 250mg/kg dose marked significantly more cells than the 50mg/kg dose, but not the 100mg/kg dose. Then we subcutaneously injected 100mg/kg BrdU into 15 juvenile male P. regius at 3 different time points (2 days, 2 weeks, 2 months) prior to sacrifice to quantify proliferation, migration, and survival of cells in several telencephalic subregions. After sectioning and immunohistochemical staining, we found proliferation to be highest in the accessory olfactory bulb (AoB), retrobulbar regions (AD, AV), dorsal ventricular ridge (DVR), and dorsolateral amygdala/lateral amygdala (DLA/LA). Of the proliferating cells, the proportions of cells that migrated after 2 weeks were highest in the ventral lateral region (VL), anterior medial and lateral cortices (aMC, aLC), and anterior NS (aNS). After 2 months, the highest proportional survival was in the AoB, aLC, aMC, aNS, DVR, and ventral medial regions (VM). Regions involved in long-term functions like spatial memory may require less proliferation and longer survival, while regions involved in short-term functions undergo more proliferation with higher relative attrition.

Alzheimer's disease (AD), marked by a serious and progressive decline in cognitive abilities, is a severely debilitating disease that is becoming an increasing concern with our aging population. Defects in neurogenesis have been shown to exist in AD and aggravate the neuropathology and cognitive deficits associated with the disease. In this study, I aimed to characterize the cellular and molecular defects of neurogenesis in the triple transgenic mouse model of AD (3xTG). To do so, I first performed a detailed immunohistochemistry characterization using neurogenic markers that were quantified and analyzed in the hippocampus of control and 3xTG mice. This analysis not only revealed an overall decrease in the pool of neural stem and progenitor cells (NSPCs) in 3xTG brains, but also defects in proliferation, differentiation and a loss within the neuroblast, immature neuron and mature neuron populations. Subsequent immunohistochemistry analysis of two molecular targets, Hopx and LPAR1, involved in NSC maintenance and proliferation respectively, revealed their dysregulation in 3xTG brains, providing some indication of molecular defects underlying this loss. The neurosphere assay was next employed to assess cell-autonomous defects and fewer neurospheres were formed from cultured 3xTG NSPCs, suggesting a defect in NSPC pool expansion that is intrinsic to 3xTG NSPC function. Molecular characterization of these cultured NSPCs via qPCR revealed the upregulation of mitochondrial and fatty acid oxidation genes in 3xTG NSPCs, suggesting not only a dysregulation of metabolic functions, but also an acclimation to oxidative stress conditions. Interestingly, 3xTG NSPCs formed larger and more neurospheres when grown in galactose medium - which is used to simulate oxidative stress - relative to the control, confirming an adaptative response to oxidative stress conditions. Further characterization of these cellular defects and underlying molecular mechanisms can reveal novel therapeutic strategies for AD.

This experiment explored whether a long-term aerobic exercise program may induce significant structural and functional changes in the hippocampus, an area of the brain that is important for spatial navigation and memory formation. Based on existing rodent studies, we hypothesize that exercise will cause a shift to allocentric processing, away from a less robust egocentric learning strategy. It is possible that exercise-induced relief of chronic stress, which contributes to improved hippocampal function, will increase reliance on allocentric spatial navigation. Neurogenesis, which occurs in the dentate gyrus region of the hippocampus, is another indicator of hippocampal function that may influence this shift to allocentric learning. The current study examines whether six weeks of aerobic exercise enhances allocentric processing in healthy young adults. Forty-nine young adults (35 female; age range 18-29 years) were randomly assigned to one of three groups: 1) High intensity interval training group, 2) Moderate intensity training group, or 3) Non-exercising control group. Hippocampus-dependent memory was assessed before and after the intervention on a Virtual Reality Water Maze task, and a high interference memory task, the Mnemonic Similarity Task (MST) which may be dependent on hippocampal neurogenesis. Levels of chronic stress and depression were measured using the Beck Depression Inventory II. It was expected that exercise would improve spatial memory performance on the water maze task, and that performance would improve in proportion to enhanced fitness levels. This improvement in spatial memory performance was expected to correlate with the two indicators of hippocampal function that were assessed in the current study—chronic stress and performance on the high interference memory task. Six weeks of regular aerobic exercise resulted in a 21.5% improvement in spatial memory performance on the water maze task, indicating improved hippocampus-mediated spatial memory function. Improvements displayed by high intensity exercisers were greater than those observed in the moderate intensity exercisers, suggesting that higher intensity exercise may be more effective in enhancing hippocampal function. Importantly, low responders to exercise exhibited a 30% improvement in water maze performance, suggesting that even minor fitness improvements can lead to significant cognitive gains. Chronic stress and depression, and performance on the MST were not significantly associated with changes in spatial memory performance; however trends observed may offer some explanation to the aforementioned changes in spatial memory. Findings from the current study have important implications for treatment options in populations that are currently, or at risk of suffering from impaired hippocampal function. / Thesis / Master of Science (MSc)

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24985号 / 生博第514号 / 新制||生||68(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 北島 智也, 教授 見学 美根子, 教授 今吉 格 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM