'Neurogenesis' is the process of formation of new neurons and making functional connections known as synapses (Bruel-Jungerman, et al., 2005). Neurogenesis occurs throughout the brain during embryonic development and continuous throughout the postnatal life in a few restricted areas to form and maintain the central nervous system (CNS) (Aguado, et al., 2005). The CNS starts developing during embryogenesis primarily in two waves. First wave i.e. prenatal wave is when most of the neurons are generated and the second wave i.e. postnatal wave is when most of the astrocytes and oligodendrocytes are produced (Abrous,Â et al., 2005). The CNS was initially considered to become mitotically quiescent after completion of this second wave and therefore lacking the ability to repair damaged tissues upon injury to the brain such as neurodegenerative disorders (Abrous,Â et al., 2005). Since then, the researches on brain have focussed on promoting the survival of neurons, formation of new processes to establish functional cell connections in case of injury.
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The previous notion of a mitotically quiescent CNS was proven wrong when neurogenesis in the brains of adult vertebrates was clearly demonstrated to be occurring throughout post-natal life in some specific regions of the adult brain under normal conditions (Figure 1). Adult neurogenesis is restricted to the proliferation zones in the CNS of the mammals (Louis and Reynolds, 2008) (Figure 1). There are two most prominent and stable regions of the adult brain identified to have mitotic activity - the sub ventricular zone (SVZ) of the lateral ventricle in the frontal cortex (Figure 2) and the sub granular zone (SGZ) of the dentate gyrus in the hippocampus (Figure 3) (Zhao, et al., 2008). In the adult SVZ, the new neurons produced migrates through the rostral migratory stream (RMS) and become granule neurons and periglomerular neurons in the olfactory bulb (Zhao, et al., 2008). On the other hand, in adult SGZ new neurons migrate into the granule cell layer of the dentate gyrus and mature into dentate granule cells. The neuronal turnover in adult brain exhibits surprising plasticity. It can be modulated by sensory input and systemic signals generated from several environmental and behavioural responses (Kaslin, et al., 2008). However, this neuronal turnover is very low that only fewer neurons are produced that can survive for very long and can also functionally integrate into the neural circuitry (Malberg, 2004). The exact mechanism for regulating this process is still largely unknown (Kaslin, et al., 2008).
Figure 1. The different sites of neurogenesis in adult brain (Gould, 2007). The figure illustrates different site of neurogenesis in adult mouse brain. In early 1990, all the regions of brain were assumed to be 'non-neuogenic' (shown in grey). In late 1990, only dentate gyrus and olfactory bulb as well as the subventricular zone, which give rise to rostral migratory stream, were identified as 'neurogenic' region (shown in red). Today, many other neurogenic regions have been identified (shown in pink). However, the extent of neurogenesis in these areas is controversial and only very for low-level of neurogenesis may occur in them. (All the regions shown here are located approximately as they all are not present on same sagittal plane).
Adult neurogenesis is affected and largely regulated by physiological and pathological activities. Such activities include proliferation of adult neural stem cells (NSCs) and/ or neural precursors cells (NPCs), fate determination of progenitor cells, survival, maturation and integration of newborn neurons (Figure 4). Physiological activities like stress, calcium homeostasis, age, gender and lifestyle can also affect neurogenesis (Pardon and Rattray, 2008; Grote and Hannan, 2007). During exercise, hippocampal neurogenesis increases, leading to antidepressive effects and improvements in memory and learning occurs (Grote and Hannan, 2007). During stroke or other hypoxic insults, the proliferation of NSCs in SVG increases (Castrén, 2005). In response to physiological and pathological changes, the adult brain is capable of activating NSCs or NPCs within SGZ and SVZ, increasing their rate of self-renewal and neurogenesis (Castrén, 2005). Moreover, these cells may be necessary for certain forms of brain function involving learning and memory in the olfactory and the hippocampus (HP) (ref). Whether neurogenesis occurs in others areas of the adult mammalian brain besides SVG and SGZ still remains a topic of research (Gould, 2007; Rakic, 2002).
Figure 2. Adult Neurogenesis in SGZ of mouse brain (Adapted from Alvarez-Buylla and Lim, 2004). A) Coronal section through mouse brain at level of the hippocampus (HP). The dentate gyrus (DG) is indicated by dotted line with arrow. B) The SGZ of the DG is enlarged to show the architecture of the SGZ. The SGZ stem cells (dark blue) give rise to progenitors (orange) which matures into new granule cells (red). These newly born granule cells integrate into the DG granule cell layer indicated by brown cells. BV, blood vessels; BL, basal lamina.
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Figure 3. Adult Neurogenesis in SVZ of mouse brain (Adapted from Alvarez-Buylla and Lim, 2004). A) Coronal section through the adult mouse brain. The lateral ventricles (LV) are indicated with light blue colour. B) The boxed region is enlarged to show the architecture of the SVZ. The SVZ stem cells (dark blue) give rise to progenitor cells (green) that rapidly divides to form neurobalst (red). This neuroblast migrates to the olfactory bulb where they become mature local interneurons. BV, blood vessels; BL, basal lamina; ciliated ependymal cells (grey).
Adult Neural stem cells:
The neural stem cells (NSCs) biology is of great interest to researchers, not only for their significant roles in CNS development but also their potential use in cell replacement therapy for neurodegenerative disorders (Ferretti, 2004). The NSCs exhibits extensive proliferation, capacity to self-renewal, production of transient amplifying progeny and differentiating into multiple lineages (Figure 4). These properties make adult NSCs an important element in adult brain tissue for maintaining cell number following injury and disease or natural turn over (Deng, et al., 2010). During injury, these cells migrate towards the site of injury and activate neurogenesis for which various signals are received from their "niches" (Imayoshi, et al., 2009). Recent findings suggested that memory may depend on the continuous production of new neural cells (Martino and Pluchino, 2006). However, more often they don't completely compensate for the damage.
Self-renewing and multipotent adult NSCs are found in the CNS of mammals throughout postnatal life and reside in discrete locations of adult brain. The traditional idea of adult NSCs only being present in those adult tissues that exhibit a great deal of cell turnover was rejected (Eriksson, et al., 1998; Ellis, et al., 2004) when proliferating stem cells were found in the SVZ and SGZ. However, NSCs have been recently identified in some non-neurogenic regions of adult brain explaining the theory that the cells migrate away from the original site of neurogenesis (ref). This regional localisation might be partly contributed by the SVZ or SGZ microenvironment or so called NSC niches (Pluchino, et al., 2005). In this experiment, we are mainly interested in the hippocampus region where new neurons are contributed by NSCs to dentate gyrus (DG) in SGZ.
Mature Dentate granule cell
Figure 4. The role of NSCs in DG of adult mouse brain (Adapted from Lie et al., 2004). The neural stem cells proliferate extensively to give rise to transit amplifying cells. The transit amplifying cells are determined to form neurons and differentiate into immature neurons. These immature neurons then migrate into the granule layer of the dentate gyrus where they completely gets integrated into the neural circuitry and give rise to mature dentate granule cells in DG.
The NSC niche serves as an "anatomical and functional compartment" for the NSCs (Williams andÂ Lavik, 2009). The neural stem cells functions and behaviour are controlled by integration of local signals from niche and distant signals (carried to the niche) from the vasculature and/or neural inputs (ref). The SVZ and SGZ serves as niches for NSCs in adult brain.
Figure 5. SVZ niche (Williams and Lavik, 2009). The architecture of SVZ niche consists of SVZ stem cells (NSCs, type B cells), astrocytes (supports NSCs), a layer of ependymal cells lining the lateral ventricles, transit amplifying cells derived from self-renewing NSCs (type C cells) and neuroblast (type A cells). Below the ependymal layer, bodies of type B cells are organised into tunnels through which type A cells can migrate. Extensive network of blood vessels run along with this layer. This vessel also produces laminin rich extravascular basal lamina which is organised into branched structures called fractions. The basal processes of NSCs are also contact this vasculature.
The architecture of SVZ niche is arranged in such a way that NSCs stay protected from the external factors and retains its multipotency (Figure 5). In SVZ niche, NSCs exhibit typical morphology that appears like astrocyte in shape with multiple apical and basal processes which are deeply integrated into the walls of later ventricles (Figure 5). A layer of ependymal cells that lines the ventricle is penetrated by the apical process of NSCs with a short single cilium (Lledo, et al., 2006). The resident stem cell lineage in the SVG consists of the relatively quiescent NSCs, which can self-renew or give rise to rapidly dividing transit-amplyfying cells and ultimately generating migratory neuroblasts which enters the RMS (Lledo, et al., 2006; Martino and Pluchino, 2006).
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Figure 6. SGZ niche (Williams and Lavik, 2009). The architecture of SGZ niche contains NSCs (type1 and type 2), astrocytes, dividing immature cells, neuroblasts, endothelial cells and matured granule neurons. The type 1 NSCs has single process and type 2 lack such processes.
There are two groups of morphology of NSCs are found in SGZ (Figure 6). Type 1 NSCs are called as 'radial NSCs' which has a long process integrated into the granule layer of DG. The Type 2 NSCs are called as 'non-radial NSCs'. This non-radial NSCs will be referred as 'horizontal NSCs' throughout. The quiescent type 1 progenitors have a single radial process that spans the granule cell layer and their cell bodies are in close contact with both their lineage-related progenitors and with blood vessels. Radial NSCs are quiescent under normal condition. Type 2 or horizontal NSCs lacks such long processes and mostly appears in an irregular morphology but mostly circular. They are present towards the outside of granule layer and are rapidly diving under normal adult neurogenesis. Therefore they sometimes even appear flattened with cluster of 3-4 cells together. The lineage relationship between type 1 and 2 NSCs is unclear, but they ultimately give rise to migratory neuroblasts (Lugert, et al., 2010).
Alzheimer's disease (AD) was named after German scientist Alois Alzheimer, who first discovered dementia in a woman's brain in 1906. AD is most prevalent amongst the aged population of society as it is defined as an "age-related neurodegenerative disorder". 15 million people around the world suffer from AD (Wang, et al., 2010). About 15% of the population in developed countries are aged above 65 years and this percentage is predicted to increase by 5% in 2025 (Crews and Masliah, 2010). This common form of dementia associated with neurodegeneration is characterised by synaptic injury in initial stage followed by neuronal loss as the disease progresses (Crews and Masliah, 2010). The most common symptoms experienced by the patients suffering from AD are cognitive alteration, memory loss and behavioural changes (Kinsella and Velkoff, 2001).
1.5.01 Amyloid protein precursors and Presenilin 1
The early onset of AD is familial autosomal dominant disorder (FAD) caused by missense mutations in the amyloid precursor protein (APP) gene on chromosome 21 and in the presenilin 1(PS1) gene on chromosome 14 (Walder, et al., 2003). In 30% of the cases, FAD occurs with mutation in PS1 (Walder, et al., 2003).
PS1 and APP are primarily expressed in neurons. APP is thought to be critical for neuronal growth, survival and injury-repair (Umka, et al., 2010). PS1 helps in cleavage of APP to produce AÎ² peptides of various lengths. AÎ²42 and AÎ²40 (Amyloid-beta peptides) gets deposited to form extracellular plaques. All FAD-linked mutations in APP leads to increased production of AÎ²42. Therefore, AÎ²42 tends to aggregate at faster rate and lower concentration than AÎ²40 form (Waldau and Shetty, 2008). The exact of function of PS proteins is not yet fully elucidated. However, they are required for normal axial formation and survival of neurons in specific regions (Demars, et al., 2010). PS proteins have also been suggested to play role in neuronal apoptosis (Haughey, et al., 2002). Binding of PS proteins to APP may be associated with an increased production of the AÎ²42 peptide (Demars, et al., 2010).
Thus, FAD-linked mutations in APP increase the amount, length, or fibrillogenic properties of AÎ² species. FAD mutant PS1 alters APP processing to enhance the generation of AÎ²-42 peptides which is more fibrillogenic than AÎ²-40 (Waldau and Shetty, 2008).
1.5.02 Alzheimer Disease Pathology
AD is also called as a protein mis-folding disease caused by accumulation of abnormally folded AÎ² and tau proteins in the brain (Baizabal, et al, 2003). Plaques are developed from small peptides of AÎ² (Figure 7 A). These fragments give rise to fibrils of beta-amyloid which form clumps that deposit outside neurons (Figure 7 B). Another protein called tau stabilizes the micrcotubules when phosphorylated. In AD, tau becomes hyperphosphorylated creating neurofibrillary tangles and disintegrating the neuron's transport system (Figure 7 C).
This abnormal processing of the amyloid precursor protein results increases production of a self-aggregating AÎ² is thought to be initiating event in the pathogenesis of AD (Figure 7) (Ellis, et al., 2004). Recent studies have shown that AÎ² can adversely affect mitotic cells like inhibiting the proliferation of astrocytes and inducing apoptosis in endothelial cells (Chuang, 2010). AÎ² promotes synaptic dysfunction and death of mature cholinergic neurons in AD (Haughey, et al., 2002; Chuang, 2010). Studies have reported the adverse effect of AÎ² on the NPC and contribute to the depletion of neurons by inducing cellular oxidative stress and dysregulating the cellular calcium homeostasis (Imayoshi, et al., 2009) (Figure 7). However, expression of which ligand- and voltage-gated calcium channels are involved in NPCs that is associated with changes in their proliferation and differentiation has not been established (Louis and Reynolds, 2008).
Therefore, the exact disease mechanism of how the disruption of production and aggregation of the beta amyloid peptide gives rise to the pathology is still not clearly understood.
Figure 7. Alzheimer Disease neuropathology (Adapted from Frank et al., 2007 and Keheren, 2007). A) Formation and accumulation of Amyloid beta protein in Alzheimer brain. AÎ² form the clumps within the brain cells and give rise to AÎ² plaque and tangles (the inner support of brain comprised of protein tau). B) Neurodegeneration due to Amyloid beta protein in Alzheimer brain. The abnormal level of plaques and tangles kills surrounding neurons. C) Amyloid-Î² (AÎ²) is produced intracellular or taken up from extracellular sources. Intracellular AÎ² exist as a monomer which further aggregates into oligomers. The intracellular AÎ² contribute to pathology events by facilitating tau hyperphophorylation, disrupting proteosome, mitochondria function, induction of oxidative stress and triggering calcium and synaptic dysfunction. ROS, reactive oxygen species.
Experimental Mouse Model:
Previously, the single transgenic mouse model of APP mutation was demonstrated to show many features of AD including the elevated levels of beta-amyloid peptide 1-42 by age of 5 months (Borchelt et al., 1997). Another mouse model is with the double mutation APPswe/PS1dE9 was demonstrated to over-expresses APP swe (Swedish) as well as PS1 and shows elevated AÎ² levels at 2 months of age (Arendash et al., 2001) which is much early as compared to single transgenic mouse model.
For this experiment, a mouse model which represents pathological conditions and neurodegeneration of AD as well as enables the detection of NSC within their niches is required. Hence, a novel experimental mouse model APPswe/PS1dE9/Sox1-GFP is used for this experiment that can recapitulate some of the molecular and cellular mechanism, neurodegeneration in AD as well as help identification of NSCs.
1.6.02 Sox1-GFP mouse line
The major limitation in studying the stem cells from brain tissues is due the lack of acceptable markers for the unambiguous identification of neural stem cells in the CNS as well as to produce more relevant information. However, in spite of the difficulty there are some markers for NSCs recognised and currently being used. Markers such as Sox1, Sox2, Sox9 (intranuclear) and Nestin (intracellular) are recognised for NSCs and neural progenitors (Barraud, et al., 2005; Alcock andÂ Sottile, 2009).
The transcription factor Sox1 is one of the known markers for NSCs. Previous studies have demonstrated that Sox1 meet the requirements of a neural stem cell marker to identify the cells that fulfil the criteria of self-renewal and multipotency (Sottile et al., 2006, Alcock and Sottile, 2009). Sox1 get expressed in the neuroectoderm and widely expressed in the ventricular zone and granular zone of adult brain throughout the development of CNS (ref). The HMG box transcription factor gets expressed in NSCs at all stages of mouse CNS development ((D'Amour and Gage, 2003). To address the current issue, a transgenic mice that express green fluorescent protein (GFP) under the control of the endogenous locus-regulatory region of Sox1 gene was developed by GFP knock-in (Aubert et al.,2003). This Sox1-GFP transgenic line has been demonstrated to successfully label Sox1 with GFP (Ying et al., 2003; Zhao et al., 2004). The Sox1-GFP is an asymptomatic mutation and hence can be used as controls for this experiment (Figure 8). Cells producing green fluorescent i.e. co-express Sox1 protein and GFP are identified and quantified in the specific region of NSCs niches using this transgenic mouse.
Figure 8. Sox1-GFP mouse line. The Spatial alternation of Sox1-GFP mice is same as Wild type (WT) mice indicating good memory and hence, Sox1-GFP mutation is an asymptomatic. Significantly different compared to 50% alternation is considered to have good memory (Unpublished, Pardon Marie-Christine).
1.6.01 APPswe/PS1dE9/Sox1-GFP mouse model
A novel triple transgenic mouse model of AD was developed by Dr. Marie-Christine Pardon and Dr. Virginie Sottile which has the major hallmarks of AD i.e. mutation in APP and PS1. This model is made by combining transgenic mouse line which over-expresses amyloid (APPswe and PS1dE9 transgenes inducing AD phenotype) with expression of fluorescent label to stem cells (Sox1-GFP knock-in). Which means heterozygous male APPswe/PS1dE9 is crossed with heterozygous Sox1-GFP female mice to produce this novel APPswe/PS1dE9/Sox1-GFP mouse model (Figure 9). The presence of this fluorescent marker enables the direct detection and quantification of NSCs, providing with the unique opportunity to visualise NSCs present within SGZ and SVZ. However this model was shown to develop memory deficit at age of 6 months (Appendix 6.2, Figure 20).
Figure 9. APPswe/PS1dE9/Sox1-GFP mouse model. The heterozygous male APP/PS1 transgenic mouse crossed with heterozygous female Sox1-GFP transgenic mouse to give APP/PS1/Sox1-GFP triple transgenic mouse. (APPswe/PS1dE9/Sox1-GFP mouse Model developed by Dr. Marie Pardon and Dr. Virginie Sottile).
Aims and Objectives:
Effect of AD on NSCs
Neurogenesis in CNS plays important role in learning and memory processes under normal and diseased condition (Abrous,Â et al., 2005). This neurogenesis is impaired in age-related AD suggesting the main element of neurogenesis i.e. NSCs may contribute to the pathogenesis of disorders. The role of NSCs in AD pathology is not well understood. Using AD (APPswe/PS1dE9/sox1-GFP) mice and control (Sox1-GFP) mice it is investigated that whether NSCs properties are impaired when the cognitive symptoms develop. Hence, the aim of this experiment is to determine whether NSC number, distribution, morphology and proliferation in dentate gyrus of the hippocampus are impaired in novel triple transgenic mouse model of Alzheimer's disease. This particular experiment focuses on the behaviour of NSC in the SGZ region of the mouse adult brain.
Effect of AD morphology of NSC
There are two major morphology of NSC in SGZ are associated with the quiescent (radial NSCs) or active (horizontal NSCs) state. With use of proliferation marker, the number of radial NSCs and horizontal NSCs and their proliferative state will be determined. The second aim of this experiment is to determine the effect of AD-like pathology on number of these two sub-populations of NSCs in SGZ.
Effect of age on NSCs
Age is associated with memory impairment in AD. Some papers have suggested heavy impairment in hippocampal neurogenesis due aging (Clelland et al., 2009; Garthe et al., 2009). Hence, the third aim is to determine the effect of age number NSCs, radial NSCs and horizontal NSCs in SGZ of 4 months and 6 months old AD mice.
Effect of gender on NSCs
Epidemiology studies have revealed that there is high prevalence of AD in females (Fratiglinoni et al., 2000). Some studies have shown that the neurogenesis gets affected at faster rate in female under neurodegenerative disorder compared to males due to decrease in the estrogens level after menopause (Lee et al., 2002; Fillit et al., 2002). Hence, the fourth aim is to investigate the effect of gender on the number of NSCs in SGZ of AD mice.