| Suppresion of neurogenesis in mouse dentate Gyrus |
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| Confocal image of immature neurons in mouse dentate gyrus stained green with Doublecortin (DCX) left image: abundant numbers of DCX+ cells, right image: absence of DCX+ cells [31] |
Adult neurogenesis has been recognized as a vital life-long process in the mammalian dentate gyrus (DG) within the hippocampus[1]. Newborn neurons exhibit enhanced synaptic plasticity, increased excitability and a lower threshold for long-term potentiation compared to mature neurons[2]. These unique properties allow young hippocampal neurons to play a key role in regulating the body’s stress response and learning and memory[1]. The 3 main stages of adult neurogenesis, cell proliferation, neuronal differentiation and cell survival can be negatively regulated by stress and certain neurodiseases[1]. The neurons in the DG are densely populated with glucocorticoid receptors, and acute and chronic stress has been shown to decrease hippocampal adult neurogenesis[1]. Increased circulating glucocorticoids and cytokines have been proposed as two principal mechanisms to explain the detrimental effects of stress on neurogenesis[1]. Also, neurogenesis is reduced in neurological disorders, such as Epilepsy, Alzheimer's disease(AD) and psychiatric illnesses, like Schizophrenia[3]. New neurons fail to migrate and integrate into the DG circuitry correctly in temporal lobe epilepsy animal models[3]. While, in AD and Schizophrenia, mutations in key genes that regulate neurogenesis can completely suppress neurogenesis and contribute to the progression of the disease[4]. Animal studies have demonstrated that the consequences of decreased neurogenesis include increases in anxiety-related behaviors and impairments in cognitive function[1]. By understanding the factors that negatively regulate neurogenesis, it is possible to create treatments to rescue the neurogenic potential in patients affected by different neurological diseases and brain injuries[3].
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Table of Contents
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1.0 Stress
| The process of neurogenesis in the DG |
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| Steps include neural progenitor cell proliferation, neuronal differentiation and cell survival. <http://www.abcam.com/index.html?> |
1.1 The effect of acute and chronic stressors
Acute stressors involve the application of a stressor (e.g. exposure to predator odor, restraint stress, social dominance) for short periods of times, from minutes to hours usually[1]. Acute stressful experiences decrease dentate gyrus cell proliferation in many different species (rat, mice, treeshrew, marmoset)[1]. Chronic stress involves the application of stressors over the course of days to several weeks[1]. While acute stressful experiences mainly decrease cell proliferation, exposure to chronic stressors negative regulates all three steps of adult neurogenesis: cell proliferation, neuronal differentiation and cell survival[1].
1.2 Mechanism underlying the effect of stress on Neurogenesis
1.2.1 Glucocorticoids
Stress activates the hypothalamo-pituitary-adrenal (HPA) axis to initiate the release of glucocorticoids from the adrenal cortex. Dentate gyrus granule cells are densely populated with the two types of corticosteroid receptors: mineralocorticoid receptor (MR) and glucocorticoid receptor (GR)[1]. Since the MRs have a higher affinity to bind GCs than GRs, they primarily regulate the hippocampal neuronal responses to the normal circadian rhythms of GCs[1]. GRs mediate the effect of stress-induced elevations of GCs on hippocampal neurons[1]. Using techniques to manipulate GC levels in animals, numerous studies have demonstrated that GCs strongly inhibit adult neurogenesis. By performing bilateral adrenalactomies (ADX) to remove all endogenous corticosteroids in adult rats, Gould and colleagues observed increased cell proliferation of neurons in the DG[5]. Also, exogenous administration of corticosterone to adult rats decreased cell proliferation and survival of newborn DG neurons[6]. Oomen et al (2007) showed that treatment of chronically stressed rats with GR antagonists prevented a reduction in neurogenesis, and this result confirmed that increased activation of GRs during stress reduces neurogenesis[7]. Though few human studies have explored the effect of high GC levels on neurogenesis, Cushing’s disease patients commonly have reduced hippocampal volumes[8].
Recently, a study demonstrated that high GC levels could directly increase the rate of apoptosis in neural precursor cells (NPCs) in the subgranular zone (SGZ)[9]. Rat neonates injected with the synthetic glucocorticoid, dexamethasone (DEX), on PND 1 to 7 had significantly higher expression of pro-apoptotic caspases in NPCs and lower numbers of NPCs in the SGZ compared to control rats[9]. NPCs have a limited capacity for renewal and thus, GCs can permanently decrease the rate of adult neurogenesis by depleting the limited pool of NPCs in the neurogenic niches of the brain[9]. Though, GCs strongly inhibit adult neurogenesis in the DG, few neural progenitor cells and immature neurons, 4 weeks old and younger, express glucocorticoid receptors[10]. In the dentate gyrus, cells express GR along a gradient where the expression of GR increases from the SGZ to the outer layers of the granule cell layer (GCL), where mature hippocampal cells reside[9].
1.2.1.1 The Indirect Effects of Glucocorticoids on Neurogenesis
Since the main targets of adult neurogenesis do not express corticosteroid receptors ubiquitously, further studies are being conducted to determine the indirect effects of GCs on the inhibition of adult neurogenesis[10]. It is seen that GCs can modify multiple extrinsic factors that regulate adult neurogenesis. Firstly, GCs can inhibit growth of astrocytes and alter astrocytic release of growth factors involved in neurogenesis[11]. Secondly, GCs can interact with N-methyl-D-aspartate (NMDA) receptors on hippocampal cells to downregulate neurogenesis[12]. Excess GCs increase calcium influx into granule cells through NMDA receptors to make these neurons more susceptible to excitatotoxicity and oxidative stress[12][13]. Thirdly, GCs can alter the levels of neurotrophic factors in the brain[14]. Synthetic glucocorticoid treatment onto hippocampal slices decreases brain derived neurotrophic factor (BDNF) mRNA and protein expression in hippocampal cells[14]. Since BDNF is key for promoting cell birth, survival and maturation, this GC-induced decrease in BDNF levels can significantly inhibit adult neurogenesis[14]. Finally, mature granule cells, which have high levels of GRs in the hippocampus, can integrate signals from GCs and subsequently, alter the release of mitogens that regulate neurogenesis[15].
1.2.2 Cytokine: Interleukin-1β
Another mechanism that underlies the inhibitory effects of stress on neurogenesis involves the release of cytokines from immune cells[1]. Stressful experiences can trigger microglia in the central nervous system (CNS) to release the pro-inflammatory cytokine, Interleukin-1β (IL-1β)[16]. It binds to cell surface Interleukin-1 receptors (IL-1R1) to alter the expression of many genes[16]. The hippocampus is highly influenced by cytokines because it has the highest expression of IL-1R1 in the CNS[16]. Overall, IL-1β suppresses hippocampal adult neurogenesis. Activation of IL-1R1 on NPCs decreases NPC proliferation and survival[17]. Moreover, IL-1β has anti-neurogenic effects in the hippocampus because it induces undifferentiated, progenitor cells to develop into glial cells instead of neurons, which will eventually deplete the neuronal population in the hippocampus[18]. Also, IL-1β reduces the growth of dendrites from newly born neurons, which will hinder their incorporation into the hippocampal circuitry correctly and ability to regulate hippocampal functions later[18]. Finally, in vitro studies using rat hippocampal cultures have shown that IL-1β binding to its receptor increases IL-1R expression on cells to create a positive feed-forward mechanism that increases its detrimental effects on neurogenesis[18]. IL-1β is a key molecule that can downregulate adult neurogenesis, however, not all types of stress increase IL-1β levels and thus, this is likely a significant, but not universal mechanism[1].
2.0 Neurodiseases and Altered Neurogenesis
This section will summarize the abnormal forms of neurogenesis seen in different neurological disorders and psychiatric illnesses.
2.1 Epilepsy and aberrant neural migration and integration
| Seizure induced aberrant neurogenesis |
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| Black = BrDU+ cells in Rat DG Seizure induced rats had (B)increased levels of neurogenesis at Day 9 & D increased numbers of hilar ectopic granule cells at Day 14 [20] |
Epilepsy is a devastating neurological disorder that affects 50 million people worldwide[3]. It is defined clinically as the occurrence of 2 or more unprovoked seizures[2]. Medial temporal lobe epilepsy (MTLE) is a common form of the disease that often involves disturbances in neurogenesis in the hippocampus[2]. Autopsies conducted in patients diagnosed with MTLE have revealed several structural abnormalities in the dentate gyrus, such as the appearance of ectopic granule cells in the hilus[3].Normally, newly generated neurons migrate a short distance into the GCL and integrate into it[19]. Parent et al (2006) showed that epileptic seizures increase newborn neuron migration into the hilus and the molecular cell layer (past the GCL)[20]. They looked at neurogenesis in the DG at different time points after inducing multiple spontaneous seizures in rats with pilocarpine (cholinergic agonist)[20]. Initially, they saw a significant increase in newborn neurons in the DG. Later, at the 2-week and 5-week time points, they saw increased hilar ectopic granule cells in the seizure induced group[20]. These hilar ectopic granule cells impair neurogenesis because they fail to integrate into the DG circuitry correctly[21]. The dendrites of the ectopic granule cells are abnormally innervated by axons from granule cells and hilar cells, which creates abnormal excitatory circuits in the DG[21]. Thus, these cells have been termed pro-epileptogenic because they promote further seizure activity in the brain[2].
2.2 Alzheimer's Disease: Mutations in PSEN1 and decreased neural progenitor cell population
| Structure of Presenilin |
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| <http://en.wikipedia.org/wiki/File:2KR6.pdb.png> |
Alzheimer's disease (AD) is a progressive neurodegenerative disease, characterized by neuronal damage, synaptic loss and accumulation of neurofibrillary tangles in many areas of the brain[22]. In the hippocampus, it is common to see pyramidal neuronal loss and disruptions in the perforant pathway during the early stages of AD, and impaired adult neurogenesis in later stages[22].
Mutations in Presenilin-1 (PSEN1) are seen in 50% of individuals diagnosed with early-onset Familial Alzheimer’s Disease (FAD)[23]. PSEN1 is a transmembrane protein that forms the key component of the ϒ-secretase complex, which cleaves the intracellular domains of membrane proteins to initiate various signaling cascades[23]. Though PSEN1 is essential for many neurodevelopmental processes, few studies have looked at how PSEN1 regulates the process of adult neurogenesis[23]. Handler and colleagues saw that fetal PSEN1 knockout mice had an increased number of differentiated post mitotic neurons, and a decreased neural precursor cell population in the DG[23]. The absence of PSEN1 in fetal mice promoted premature differentiation of NPCs, which depleted the NPC population[23]. This study showed that PSEN1 is required for appropriate cell differentiation of neural precursor cells into post mitotic neurons[23]. These results suggest that treatments that increase PSEN1 function in FAD to restore normal adult neurogenesis could be beneficial in delaying the cognitive and memory disturbances associated with the disease[4].
2.3 Schizophrenia
Schizophrenia is a well-known neuropsychiatric disorder with a genetic basis. Often, schizophrenics have abnormalities in granule cell neurogenesis in the hippocampus[24]. Studies have identified mutations in two key schizophrenia susceptibility genes that are associated with hippocampal structural and functional deficits[24].
2.3.1 Mutations in DISC1 and abnormal neurogenesis
| Layers within the Granule Cell Layer of the Dentate Gyrus [25] |
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| DISC1 controls neuronal migration in dentate gyrus |
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| New neurons were stained green with Green Fluorescent protein (GFP) Image A, B & C show normal neuronal migration of newborn neuron Image D, E & F show abnormal neuronal migration into molecular cell layer due to absence of DISC1[25] |
Disrupted in Schizophrenia 1 (DISC1) is an important susceptibility gene for schizophrenia4]. The link between schizophrenia and DISC1 was established after DISC1 mutations were discovered in a Scottish family that had several members diagnosed with schizophrenia and other mood-affective disorders[4]. The DISC1 gene is expressed widely throughout the brain during embryonic development[24]. However, in adulthood, its expression is limited to a few brain regions, including the hippocampus[24]. The hippocampus has the highest expression of DISC1 in rats, mice and primates[24][25]. DISC1 is a scaffolding protein that links the neuronal cytoskeleton to various signaling molecules (eg. GABA, glutamate), to regulate many important processes in the brain, especially adult neurogenesis[24]. It plays a major role in controlling the integration of newborn neurons into the existing hippocampal circuitry[24][25].
Duan et al (2007) showed that inhibiting DISC1 expression in newborn DG granule cells leads to the migration of immature neurons from the SGZ, past the GCL and into the molecular cell layer[25]. This result showed that DISC1 normally sends inhibitory signals to the active cytoskeleton within cells to correctly position new neurons within the GCL[25]. This study also showed that DISC1 defines the synchronized and sustained firing patterns of immature neurons that are essential for proper neuronal integration into adult hippocampal circuitry[25]. Finally, DISC1 knockouts exhibited abnormal neurite outgrowth and cell morphology as well[25]. Overall, DISC1 acts as a negative regulator of the key steps involved in neuronal integration into the hippocampal circuitry[25]. Presently, studies are being conducted to determine how these defects in adult neurogenesis contribute to the etiology and progression of schizophrenia.
2.3.2 Mutations in NPAS3 and premature neuronal cell death
Neuronal PAS domain-containing protein 3 (NPAS3) is a transcription factor in the brain that regulates the expression of proteins involved a wide array of functions including adult neurogenesis[4]. Most mutations occur in the intron region of the NPAS3 gene, which deletes the DNA binding domain, and subsequently the mutated protein cannot control the transcription of genes[4]. NPAS3 knockout mice display the key symptoms of schizophrenia, and consequently, NPSA3 null mutants have become the main animal model of Schizophrenia[4]. These NPSA3 knockout mice exhibit a major loss in hippocampal adult neurogenesis as well[26]. Pieper and colleagues discovered that NPSA3 controls cell survival of newborn neurons in the DG by acting as a “survival checkpoint”, where it promotes the survival of neurons that have integrated into the hippocampal circuitry correctly[27]. NPAS3 deficiency increases apoptosis in newborn neurons and this will impair adult neurogenesis[27].
3.0 Consequences of decreased Adult Neurogenesis
The functional role of hippocampal newborn neurons is a highly debated topic in the field of Neuroscience[28]. Newborn neurons have unique properties, which allow them to control important physiological functions[2].
3.1 Increase in Anxiety-related behaviours
| The HPA Axis |
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| The Hypothalamo-Pituitary-Adrenal (HPA) axis regulates the levels of corticosteroids in the body. <http://www.nature.com/neuro/journal/v12/n3/fig_tab/nn0309-241_F2.html > |
Hippocampal neurons normally inhibit HPA axis activity[28]. Thus, it was seen that adult rats with suppressed hippocampal neurogenesis had significantly higher levels of plasma corticosterone after exposure to a mild stressor[28]. Also, researchers have found that neurogenesis in the hippocampus can regulate affective states and behaviours, such as fear and anxiety through the HPA axis[29].Interestingly, Revest et al (2009) showed that the loss of neurogenesis increases anxiety-like behaviours in mice[30]. The neurogenesis deficient mice were transgenic mice that overexpressed the pro-apoptotic protein, Bax, in neuronal precursor cells of the dentate gyrus[30]. These mice underwent four behavioural tests (elevated plus maze, the light/dark emergence test, novel object test and predator exposure test) to assess their levels of anxiety[30]. It was found that mice with impaired neurogenesis avoided new and potentially threatening situations, such as brightly lit and open spaces in each test more often than control mice[30]. This study confirmed that the loss of neurogenesis could produce persistent changes in behaviour, and a later breakthrough study by Snyder and colleagues highlighted a key mechanism underlying these anxiety-related behavioural changes[28].
Snyder et al (2011) demonstrated that newborn neurons in the hippocampus are critical for shutting off the HPA axis to allow circulating glucocorticoid levels to return to baseline[31]. Firstly, they created transgenic mice that expressed the Herpes Simplex Virus Thymidine Kinase (TK)[31]. Then, they injected the drug, valganciclovir (v), into these mice to eliminate neurogenesis in the brain[31]. The v-TK mice, which had impaired adult neurogenesis, had significantly higher corticosterone levels after 30 minute exposure to restraint stress compared to the v-WT (control) mice[31]. Also, local irradiation of the dentate gyrus in mice, which did not stop neurogenesis in the subventricular zone, produced the same result[31]. Thus, this study showed that hippocampal neurogenesis is required for the efficient recovery of the HPA axis[31]. Also, using the forced swim test and saccharin preference test, the researchers showed that the v-TK mice displayed an increase in behaviours associated with depression, such as helplessness and anhedonia[31]. The impaired neurogenesis in the v-TK group was associated with decreased responsiveness of the HPA axis to the dexamethasone suppression test, where v-TK mice had significantly elevated corticosterone levels after dexamethasone injection compared to v-WT mice[31]. Impaired responsiveness of the HPA axis is seen in approximately 50% of patients diagnosed with clinical depression[31].
This was a groundbreaking paper because it was the first study to show a direct link between neurogenesis, HPA axis function and depression. It showed that new neurons in the hippocampus are critical for resetting the stress-regulatory system, the HPA axis, to prevent the emergence of stress-related mood disorders. Interestingly, they showed that chronic stress may produce a maladaptive cycle, where stress activates the HPA axis, decreases adult neurogenesis, decreases stress resilience, and this would continuously activate the HPA axis and later, lead to the development of different stress-related pathologies[31]. Though the etiology of clinical depression involves complex changes in stress-regulatory systems and brain processes (synaptogenesis, dendritic plasticity), this paper demonstrated that adult neurogenesis is also a key process in the pathology of depression[28]. This suggests that decreased neurogenesis, seen in various neurodiseases, may increase an individuals’ risk of developing anxiety-related disorders and depression[28].
3.2 Impairment of Cognitive functions
| Performance in DNMS task with short and long delay |
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| Winocur et al (2005) showed that irradiated rats had impaired performance in DNMS task with 120s delay[33] |
It is well accepted that newborn neurons in the hippocampus are involved in regulating different cognitive functions, especially learning and memory[28]. Young hippocampal neurons are necessary for spatial long-term memory formation in rats[32]. Snyder and colleagues showed that irradiated rats learn the morris water maze (MWM) at a normal rate, but they perform significantly worse than control rats at remembering the hidden location of the platform during the 2-week and 4-week probe trials[32]. The neurogenesis deficient rats could store and retrieve information in long-term memory normally as well[32]. So, the unique properties of new neurons in the hippocampus, especially their lower LTP induction threshold, allow for the encoding of highly specific details of events, which are required for the formation of spatial long-term memories[32].
Another role of new neurons is to connect events that are part of the same context across time[33]. Rats that had impaired hippocampal neurogenesis performed poorly on the delayed non-match to sample task (DNMS) with long delays[33]. There was no difference in performance between the irradiated and control rats in the DNMS task with short delays because it is a hippocampal independent task[33]. However, when the interval between the sample and test trial was increased to 120s, this task relied on the hippocampus and subsequently, the irradiated rats performed significantly worse than control rats[33]. Therefore, adult hippocampal neurogenesis is required to recall events after long-delays[33]. These results suggest that reductions in adult neurogenesis can produce significant cognitive deficits.
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