Danka Kozareva PhD Candidate, University College Cork
The generation of neurons from stem cells
The process of generating functional neurons from stem cells in the central nervous system (CNS) was believed to occur strictly during embryonic development and the early postnatal days in mammals (Ming and Song, 2011). A century ago this idea was challenged with the discovery of adult neurogenesis. Ezra Allen was the first to demonstrate that mitosis persisted in the lateral walls of adult albino rats (Allen, 1912). Several decades later Altman and Das (1965) followed up this research and determined that neurogenesis occurred in the adult rat and guinea pig hippocampus (Altman and Das, 1965; Altman and Das, 1967). Not long after, evidence began to accumulate in favour of the existence of this process in the human brain as well. It was further illustrated that the rate of proliferation and the process of functional integration of adult born neurons into exiting circuitry was remarkably similar across species (Eriksson et al., 1998). Neural stem cells (NSCs) are of great therapeutic interest because they have the potential to regenerate and replace neurons lost in brain injury and neurodegenerative diseases such as Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, and multiple sclerosis (Parent et al., 2002; Emsley et al., 2005; van den Berge et al., 2010).
Properties of CNS Stem Cells
By definition CNS stem cells possess the following three properties: they have an infinite capacity to self-renew; they continuously undergo mitosis and they can differentiate in multiple cell types of the CNS (Hall and Watt, 1989). Stem cells of the adult CNS are heterogeneous in their origin, mechanism of operation and marker expression. The precursor cells, progeny of the stem cells, are committed to a particular cell lineage (neuronal, glial or astroglial) (Emsley et al., 2005). To date, multiple neurogenic niches have been identified in the adult mammalian brain. One such region, the focus of my research, is the subgranular zone (SGZ) – a layer of the hippocampus located between the dentate gyrus and the hilus. The putative hippocampal precursors are multipotent and divide infrequently. Morphologically they are similar to radial glia with a triangular cell body and a large apical process extending and forming multiple arborizations into the dentate granule cell layer. The end processes of these stem cells terminate onto the vasculature. The progeny of SGZ stem cells migrate to the granule cell layer of the dentate gyrus where they integrate into hippocampal circuitry as mature excitatory neurons (Gage, 2000).
Adult hippocampal neurogenesis is a key regulator of neural plasticity, learning, as well as processing of different types of memory (Deng et al., 2010). Adult neurogenesis has also been shown to play a role in mood regulation and disorders related to it, such as depression (Balu and Lucki, 2009). Interestingly, the effectiveness of antidepressant treatment matches the time that is needed for newborn cells to be integrated into hippocampal circuitry. Electroconvulsive therapy, lithium and the antidepressant fluoxetine have been shown to increase the number of newborn granule cells in mice models of depression (Duman et al., 2001). Lower levels of proliferation in the hippocampus were also demonstrated in patients suffering schizophrenia. What is more, genetic studies suggest that the decrease in newborn cells might be an important contributing factor to the development of the disease. The impaired cognitive function in schizophrenic patients resembles the impairments observed when hippocampal neurogenesis is inhibited (Reif et al., 2006). Moreover, decreased hippocampal neurogenesis is recognized as an important mechanism underlying cognitive deficits associated with aging itself as well as neurodegenerative disorders such as Alzheimer’s disease and various types of dementia (Kuzumaki et al., 2010; Mu and Gage, 2011). The processes contributing to deregulation of hippocampal neurogenesis under pathological conditions are not fully understood, but it is proposed that extracellular signals provided by the hippocampal microenvironment may interact with cell-intrinsic factors (Nolan et al., 2004; Keohane et al., 2010).
TLX and Neurogenesis
Recent attention has focused on the role of nuclear receptors in neurogenesis. Nuclear receptors are a superfamily of transcription factors that regulate genes involved in physiological and developmental processes and have proven to be important drug targets for a host of diseases (Sladek, 2003). The orphan nuclear receptor subfamily 2 group E member 1 (Nr2e1), commonly known as TLX, is an evolutionary conserved member of the nuclear receptor superfamily found in both vertebrates and invertebrates (Mangelsdorf et al., 1995). An alignment of drosophila, murine and human TLX proteins reveals remarkable interspecies conservation with 70% – 99% homology between the three species (Yu et al., 1994). Expression of TLX is specific to the developing forebrain and retina. In the mouse embryo, TLX expression is detectable at embryonic day 8 (E8), peaks at E14 and declines thereafter. Postnatal TLX expression increases with high levels present in the neurogenic niches of the adult brain and more specifically in the neural precursor cells in the SGZ and subventricular zone (Monaghan et al., 1995; Li et al., 2008).
TLX & Neural Precursor Cells
In 2002, Simpson and colleagues discovered a novel mouse mutation (Nr2e1-null) named “fierce” and have shown that these mice have altered neurogenesis, cortical and limbic abnormalities, aggression and cognitive impairment (Young et al., 2002; Christie et al., 2006). The functional importance of TLX is now apparent from studies showing that TLX maintains adult hippocampal neural precursor cells in a proliferative, undifferentiated state (Shi et al., 2004; Sun et al., 2007). It has recently been reported that TLX also controls the activation status and the proliferative ability of hippocampal neural precursors by repressing cell cycle-related genes such as pten (Niu et al., 2011). Experiments have confirmed that TLX has a role to play in spatial learning, memory and synaptic plasticity (Christie et al., 2006; Zhang et al., 2008; O’Leary et al., 2016a; O’Leary et al., 2016b). Furthermore, it has been reported that TLX knockout mice have a reduction in the size of the dentate gyrus and amygdala (Monaghan et al., 1997). These mice also demonstrate a trend for higher circulating levels of the stress hormone corticosterone and show deficits in fear conditioning (Young et al., 2002). The TLX knockout mice can thus be a useful model for investigating the cognitive impairment associated with deregulation of hippocampal neurogenesis.
Inflammation and TLX
Next to impaired hippocampal neurogenesis, inflammation has also been implicated in the pathology of many neurodegenerative and psychiatric disorders including Alzheimer’s disease and stress-induced depression. Furthermore, it has been shown that inflammation influences hippocampal neurogenesis and negatively impacts upon cognitive function (Nolan et al., 2005; Green et al., 2012). Additionally, extracellular stimulation of both embryonic and adult hippocampal neural stem cells with pro-inflammatory cytokine IL-1β has recently been shown to detrimentally affect TLX expression (Green and Nolan, 2012; Ryan et al., 2013; O’Leime et al., 2017). Microglia are the resident innate immune cells in the brain and mediate the effects of inflammation, stress and injury. In their ramified state, microglia inspect their microenvironment for potential threats. Any type of brain injury or increase in pro-inflammatory cytokines would trigger an activated or neuroprotective state of the microglia (Olah et al., 2011). Moreover, microglia play a crucial role in the regulation of adult neurogenesis. On the one hand, they phagocyte the newborn neurons, whose synapses fail to prune (Paolicelli et al., 2011). On the other hand, microglia can facilitate or inhibit synaptic transmission and synaptic morphology in response to anti- and pro- inflammatory cytokines, respectively. Last but not least, microglia have been shown to influence the proliferation of neural precursor cells and the survival of newborn neurons, depending on the signals from the microenvironment of the neurogenic niche (Sierra et al., 2014). It has also been proposed that dynamic microglia alterations underlie stress-induced depressive-like behavior and suppressed neurogenesis (Kreisel et al., 2014). Thus, the interaction between TLX and inflammation could be a useful model for exploring drug therapy developments for hippocampal-dependent disorders where impaired neurogenesis is implicated (Kozareva et al., 2017).
My PhD Project
The aim of my PhD project is to position TLX as a novel therapeutic target for hippocampal-dependent neurodegenerative and psychiatric disorders that have a strong inflammatory component. To achieve this, the following specific objectives are investigated:
The involvement of TLX in microglia-stem cell interactions and the regulatory role of TLX in known and novel genes controlling neurogenesis and hippocampal-dependant cognitive behaviors in mice and rats during adulthood and adolescence
The role of exercise as rescue strategy for TLX-deficiency-caused impairments
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O’Leary JD, Kozareva DA, Hueston CM, O’Leary OF, Cryan JF, Nolan YM. 2016a. The nuclear receptor Tlx regulates motor, cognitive and anxiety-related behaviours during adolescence and adulthood. Behavioural Brain Research 306:36-47.
O’Leary JD, O’Leary OF, Cryan JF, Nolan YM. 2016b. Regulation of behaviour by the nuclear receptor TLX. Genes, brain, and behavior.
O’Leime CS, Cryan JF, Nolan YM. 2017. Nuclear deterrents: Intrinsic regulators of IL-1beta-induced effects on hippocampal neurogenesis. Brain Behav Immun.
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