TLX as a Novel Therapeutic Target

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).

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).

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).

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.

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-inflammatorycytokines 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).

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

Allen E. 1912. The cessation of mitosis in the central nervous system of the albino rat. J Comp Neurol.(19):547-568.

Altman J, Das G, D. 1965. Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. The Journal of Comparative Neurology

124(3):319-335.

Altman J, Das G, D. 1967. Postnatal neurogenesis in the guinea-pig. Nature 214:1098-1101.

Balu DT, Lucki I. 2009. Adult hippocampal neurogenesis: regulation, functional implications, and contribution to disease pathology. Neurosci Biobehav Rev 33(3):232-52.

Christie BR, Li AM, Redila VA, Booth H, Wong BK, Eadie BD, Ernst C, Simpson EM. 2006. Deletion of the nuclear receptor Nr2e1 impairs synaptic plasticity and dendritic structure in the mouse dentate gyrus. Neuroscience 137(3):1031-7.

Deng W, Aimone JB, Gage FH. 2010. New neurons and new memories: how does adult hippocampal neurogenesis affect learning and memory? Nat Rev Neurosci 11(5):339-50.

Duman RS, Nakagawa S, Malberg J. 2001. Regulation of adult neurogenesis by antidepressant treatment. Neuropsychopharmacology 25(6):836-44.

Emsley JG, Mitchell BD, Kempermann G, Macklis JD. 2005. Adult neurogenesis and repair of the adult CNS with neural progenitors, precursors, and stem cells. Prog Neurobiol 75(5):321-41.

Eriksson PS, Perfilieva E, Bjork-Eriksson T, Alborn AM, Nordborg C, Peterson DA, Gage FH. 1998. Neurogenesis in the adult human hippocampus. Nat Med 4(11):1313-7.

Gage FH. 2000. Mammalian neural stem cells. Science 287(5457):1433-8.

Green HF, Nolan YM. 2012. GSK-3 mediates the release of IL-1beta, TNF-alpha and IL-10 from cortical glia. Neurochem Int 61(5):666-71.

Green HF, Treacy E, Keohane AK, Sullivan AM, O’Keeffe GW, Nolan YM. 2012. A role for interleukin-1beta in determining the lineage fate of embryonic rat hippocampal neural precursor cells. Mol Cell Neurosci 49(3):311-21.

Hall PA, Watt FM. 1989. Stem cells: the generation and maintenance of cellular diversity. Development 106(4):619-33.

Keohane A, Ryan S, Maloney E, Sullivan AM, Nolan YM. 2010. Tumour necrosis factor-alpha impairs neuronal differentiation but not proliferation of hippocampal neural precursor cells: Role of Hes1. Mol Cell Neurosci 43(1):127-35.

Kozareva D, A.,, Hueston C, M.,, O’Leime C, S.,, Crotty S, Dockery P, Crayn J, F.,, Nolan Y, M. 2017. Absence of the neurogenesis-dependent nuclear receptor TLX induces inflammation in the hippocampus. Journal of Neuroimmunology:In Press.

Kreisel T, Frank MG, Licht T, Reshef R, Ben-Menachem-Zidon O, Baratta MV, Maier SF, Yirmiya R. 2014. Dynamic microglial alterations underlie stress-induced depressive-like behavior and suppressed neurogenesis. Mol Psychiatry 19(6):699-709.

Kuzumaki N, Ikegami D, Tamura R, Sasaki T, Niikura K, Narita M, Miyashita K, Imai S, Takeshima H, Ando T and others. 2010. Hippocampal epigenetic modification at the doublecortin gene is involved in the impairment of neurogenesis with aging. Synapse 64(8):611-6.

Li W, Sun G, Yang S, Qu Q, Nakashima K, Shi Y. 2008. Nuclear receptor TLX regulates cell cycle progression in neural stem cells of the developing brain. Mol Endocrinol 22(1):56-64.

Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P and others. 1995. The nuclear receptor superfamily: the second decade. Cell 83(6):835-9.

Ming GL, Song H. 2011. Adult neurogenesis in the mammalian brain: significant answers and significant questions. Neuron 70(4):687-702.

Monaghan AP, Bock D, Gass P, Schwager A, Wolfer DP, Lipp HP, Schutz G. 1997. Defective limbic system in mice lacking the tailless gene. Nature 390(6659):515-7.

Monaghan AP, Grau E, Bock D, Schutz G. 1995. The mouse homolog of the orphan nuclear receptor tailless is expressed in the developing forebrain. Development 121(3):839-53.

Mu Y, Gage FH. 2011. Adult hippocampal neurogenesis and its role in Alzheimer’s disease. Mol Neurodegener 6:85.

Niu W, Zou Y, Shen C, Zhang CL. 2011. Activation of postnatal neural stem cells requires nuclear receptor TLX. J Neurosci 31(39):13816-28.

Nolan Y, Maher FO, Martin DS, Clarke RM, Brady MT, Bolton AE, Mills KH, Lynch MA. 2005. Role of interleukin-4 in regulation of age-related inflammatory changes in the hippocampus. J Biol Chem 280(10):9354-62.

Nolan Y, Martin D, Campbell VA, Lynch MA. 2004. Evidence of a protective effect of phosphatidylserine-containing liposomes on lipopolysaccharide-induced impairment of long-term potentiation in the rat hippocampus. J Neuroimmunol 151(1-2):12-23.

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.

Olah M, Biber K, Vinet J, Boddeke HW. 2011. Microglia phenotype diversity. CNS Neurol Disord Drug Targets 10(1):108-18.

Paolicelli RC, Bolasco G, Pagani F, Maggi L, Scianni M, Panzanelli P, Giustetto M, Ferreira TA, Guiducci E, Dumas L and others. 2011. Synaptic pruning by microglia is necessary for normal brain development. Science 333(6048):1456-8.

Parent JM, Vexler ZS, Gong C, Derugin N, Ferriero DM. 2002. Rat forebrain neurogenesis and striatal neuron replacement after focal stroke. Ann Neurol 52(6):802-13.

Reif A, Fritzen S, Finger M, Strobel A, Lauer M, Schmitt A, Lesch KP. 2006. Neural stem cell proliferation is decreased in schizophrenia, but not in depression. Mol Psychiatry 11(5):514-22.

Ryan SM, O’Keeffe GW, O’Connor C, Keeshan K, Nolan YM. 2013. Negative regulation of TLX by IL-1beta correlates with an inhibition of adult hippocampal neural precursor cell proliferation. Brain Behav Immun 33:7-13.

Shi Y, Chichung Lie D, Taupin P, Nakashima K, Ray J, Yu RT, Gage FH, Evans RM. 2004. Expression and function of orphan nuclear receptor TLX in adult neural stem cells. Nature 427(6969):78-83.

Sierra A, Tremblay ME, Wake H. 2014. Never-resting microglia: physiological roles in the healthy brain and pathological implications. Front Cell Neurosci 8:240.

Sladek FM. 2003. Nuclear receptors as drug targets: new developments in coregulators, orphan receptors and major therapeutic areas. Expert Opin Ther Targets 7(5):679-84.

Sun G, Yu RT, Evans RM, Shi Y. 2007. Orphan nuclear receptor TLX recruits histone deacetylases to repress transcription and regulate neural stem cell proliferation. Proc Natl Acad Sci U S A 104(39):15282-7.

van den Berge SA, Middeldorp J, Zhang CE, Curtis MA, Leonard BW, Mastroeni D, Voorn P, van de Berg WD, Huitinga I, Hol EM. 2010. Longterm quiescent cells in the aged human subventricular neurogenic system specifically express GFAP-delta. Aging Cell 9(3):313-26.

Young KA, Berry ML, Mahaffey CL, Saionz JR, Hawes NL, Chang B, Zheng QY, Smith RS, Bronson RT, Nelson RJ and others. 2002. Fierce: a new mouse deletion of Nr2e1; violent behaviour and ocular abnormalities are background-dependent. Behavioural Brain Research 132(2):145-158.

Yu RT, McKeown M, Evans RM, Umesono K. 1994. Relationship between Drosophila gap gene tailless and a vertebrate nuclear receptor Tlx. Nature 370(6488):375-9.

Zhang CL, Zou Y, He W, Gage FH, Evans RM. 2008. A role for adult TLX-positive neural stem cells in learning and behaviour. Nature 451(7181):1004-7.