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The mammalian neocortex comprises a diverse population of excitatory neurons that perform distinct roles in the neocortical circuit. These neurons are born from a heterogeneous population of progenitor cells during embryonic development and it is increasingly being recognised that individual progenitors can impart specific functional characteristics to their offspring. For example, clonally-related sister neurons in mouse neocortex are biased to form gap junctions with one another early in development, to form synaptic connections with one another as they mature, and to show similar response properties in the adult. This highlights a fundamental role for neuronal lineage in the formation of precise neocortical connectivity. However, the extent to which neuronal phenotype is determined by lineage, and the process by which this arises, is not fully understood. Nor is it known whether similar lineage relationships exist in the human neocortex. Deciphering neocortical lineage relationships has been limited by the techniques available to identify and manipulate clonally-related neurons. In this thesis I have developed novel molecular tools for the identification and manipulation of clonally-related neurons and refined their use by in utero surgery in mice. I first developed a retrovirus encoding Cre recombinase and demonstrated that this can be successfully combined with reporter mice to capitalise on optogenetics for functional studies. In an effort to establish a reliable and unequivocal method of identifying clonally-related neurons, I then constructed a retrovirus encoding genetically-distinct RNA barcodes. I confirmed that the RNA barcode could be reliably retrieved from single neurons and used to determine clonal relationships in mouse neocortex and in an in vitro model of human neocortex derived from induced pluripotent stem cells (iPSCs). By comparing the dendritic morphology of barcoded mouse neocortical neurons, I was able to demonstrate that the dendritic arbor of clonally-related neurons is more similar than control neurons derived from different progenitors. This may contribute to specific patterns of synaptic connectivity amongst clonally-related neurons. Within the iPSC system, I demonstrate the utility of the retroviral RNA barcode and revealed that clonally-related human neocortical cells exhibit a higher probability of being gap-junction coupled. These studies advance our understanding of lineage relationships in neocortical excitatory neurons in mouse and provide the first evidence that human neocortical clones exhibit similar functional relationships to those observed in the rodent neocortex.

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Adult neurogenesis is defined as the generation of new nerve cells in the adult central nervous system (CNS). Stimulating neurogenesis may potentially offer a therapeutic approach for neurodegenerative diseases such as Parkinson???s disease. However, it is not clear why neurogenesis does not normally replace neurons lost in these diseases. As a first step to address this problem it is necessary to identify mechanisms that regulate adult neurogenesis in the normal and diseased brain and further, determine if manipulating these mechanisms may offer therapeutic potential. In this thesis, we identify activin A, a member of the transforming growth factor ?? (TGF??) superfamily, as a significant regulator of neurogenesis. We demonstrate that mRNA encoding activin A is expressed after a KA injury, and that inhibition of this activin A profoundly impairs neurogenesis in the hippocampus. Further we demonstrate that activin A impairs gliosis and also has potent anti-inflammatory effects in the injured hippocampus. Finally, we provide evidence that the majority of activin A???s neurogenic effect results from its potent anti-inflammatory actions. Our study draws a clear link between neurogenesis and inflammation in the CNS and is the first to provide evidence that this process is regulated through activin signalling. Since inflammation is now believed to be an important component of many neurological diseases we suggest that therapeutic compounds that enhance activin A signalling may offer a therapeutic approach for treating these diseases by suppressing inflammation and stimulating neurogenesis.

Adult neurogenesis is defined as the generation of new nerve cells in the adult central nervous system (CNS). Stimulating neurogenesis may potentially offer a therapeutic approach for neurodegenerative diseases such as Parkinson???s disease. However, it is not clear why neurogenesis does not normally replace neurons lost in these diseases. As a first step to address this problem it is necessary to identify mechanisms that regulate adult neurogenesis in the normal and diseased brain and further, determine if manipulating these mechanisms may offer therapeutic potential. In this thesis, we identify activin A, a member of the transforming growth factor ?? (TGF??) superfamily, as a significant regulator of neurogenesis. We demonstrate that mRNA encoding activin A is expressed after a KA injury, and that inhibition of this activin A profoundly impairs neurogenesis in the hippocampus. Further we demonstrate that activin A impairs gliosis and also has potent anti-inflammatory effects in the injured hippocampus. Finally, we provide evidence that the majority of activin A???s neurogenic effect results from its potent anti-inflammatory actions. Our study draws a clear link between neurogenesis and inflammation in the CNS and is the first to provide evidence that this process is regulated through activin signalling. Since inflammation is now believed to be an important component of many neurological diseases we suggest that therapeutic compounds that enhance activin A signalling may offer a therapeutic approach for treating these diseases by suppressing inflammation and stimulating neurogenesis.

Adult neurogenesis is defined as the generation of new nerve cells in the adult central nervous system (CNS). Stimulating neurogenesis may potentially offer a therapeutic approach for neurodegenerative diseases such as Parkinson???s disease. However, it is not clear why neurogenesis does not normally replace neurons lost in these diseases. As a first step to address this problem it is necessary to identify mechanisms that regulate adult neurogenesis in the normal and diseased brain and further, determine if manipulating these mechanisms may offer therapeutic potential. In this thesis, we identify activin A, a member of the transforming growth factor ?? (TGF??) superfamily, as a significant regulator of neurogenesis. We demonstrate that mRNA encoding activin A is expressed after a KA injury, and that inhibition of this activin A profoundly impairs neurogenesis in the hippocampus. Further we demonstrate that activin A impairs gliosis and also has potent anti-inflammatory effects in the injured hippocampus. Finally, we provide evidence that the majority of activin A???s neurogenic effect results from its potent anti-inflammatory actions. Our study draws a clear link between neurogenesis and inflammation in the CNS and is the first to provide evidence that this process is regulated through activin signalling. Since inflammation is now believed to be an important component of many neurological diseases we suggest that therapeutic compounds that enhance activin A signalling may offer a therapeutic approach for treating these diseases by suppressing inflammation and stimulating neurogenesis.

Peroxynitrite (ONOO-), produced from the reaction of superoxide anion and nitric oxide (NO), plays important roles in many physiological and pathological processes as it is far more active than its precursors. However, the lack of specific and direct detection methods slow down the pace of its related research. To this end, five new peroxynitrite probes have been tested with different properties, including HKGreen-4A, 9-32, HKYellow-AM, 9-40 and MitoPN-1. Overwhelming evidence shows peroxynitrite is responsible for neurotoxicity in cerebral ischemia-reperfusion injury. Development of drugs for scavenging ONOO- becomes an important therapeutic strategy for ischemic stroke. Danshen, the dried root of salvia miltiorrhiza Bunge, is a traditional Chinese Medicine commonly used for cerebrovascular diseases. With the screening platform based on the new probe HKYellow-AM, several hydrophilic compounds from Danshen were identified to be peroxynitrite scavengers. Among them, Salvianolic acid B (SAB) possessed the highest peroxynitrite scavenging activity. Another compound, Sodium Danshensu (SDSS), the sodium salt of Danshensu, is not only a representative active compound of Danshen but also the degradation product of these hydrophilic compounds in vivo. Therefore, the potential neuroprotective effects of SAB and SDSS were studied. Both SAB and SDSS possessed strong ONOO- scavenging activity and consequently protected neuronal cell line SH-SY5Y and cortical neurons from both ONOO- and oxygen-glucose deprivation–reoxygenation induced cell death. SDSS also inhibited ONOO- formation partly by scavenging NO and superoxide. In vivo focal cerebral ischemia-reperfusion experiments revealed that both SAB and SDSS remarkably reduced protein nitration level, cell death and infarct volume in ischemia-reperfused rat brains. However, as “coin has its two sides”, whether peroxynitrite could also act as a signaling molecule has not been well known yet. The discovery of adult neurogenesis brings hope for brain repair after ischemic stroke. Recent studies indicate low concentration of peroxynitrite promotes endothelial cell growth for angiogenesis and contributes to hypoxia-induced muscle cell proliferation. To investigate the role of peroxynitrite in neurogenesis, several experiments on neural stem/progenitor cells (NSCs) were performed. The results indicated low concentrations of peroxynitrite promoted NSCs proliferation, self-renewal and neuronal differentiation. The increased peroxynitrite during hypoxia has been visualized by MitoPN-1 staining. Meanwhile, peroxynitrite decomposition catalysts (PDCs, FeTMPyP and FeTPPS) treatment reduced hypoxia-induced peroxynitrite formation, NSCs proliferation, self-renewal and neuronal differentiation. Moreover, effects of peroxynitrite on neurogenesis were partly through activating HIF-1α correlated with enhanced Wnt/β-catenin signaling pathway. In addition, the different effects of peroxynitrite on neurogenesis and neurotoxicity depended on its concentration. In summary, five new probes with different properties have been tested to be sensitive and specific peroxynitrite probes. With these probes, I found low concentration of peroxynitrite promoted neurogenesis, and endogenous peroxynitrite generation contributed to hypoxia-induced neurogenesis. On the other hand, high concentrations of peroxynitrite-induced neurotoxicity could be attenuated by SAB and SDSS, which partly contribute to their protective effects against ischemia-reperfusion injury. These findings extend the understanding on the biological function of peroxynitrite, drug discovery for targeting peroxynitrite, in both technological and experimental contexts. / published_or_final_version / Chinese Medicine / Doctoral / Doctor of Philosophy

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No description available.

To investigate the lineage relationships that are postulated to underlie the origins of phenotypically different neurons, Thy-1.1 $ leftrightarrow$ Thy-1.2 and hNF-L $ leftrightarrow$ +/+ mouse chimeras were examined for the distribution of the two neuron genotypes. Throughout the nervous system, a finely variegated pattern of mosaicism was always observed and, in each chimera, similar genotype proportions were found in all analysed neuronal populations of the peripheral and central nervous system. These findings require that the chimera neuroectoderm was a homogeneous mix of the two genotypes and that different neuronal phenotypes do not arise clonally from a small number of prespecified progenitors. Rather it would seem that all progenitors contribute daughter cells to all of the neuronal subpopulations at each level of the neuroaxis.