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Characterisation of molecules regulating adult hippocampal neurogenesis

Final Report Summary - MOLREGNEUROGEN (Characterisation of molecules regulating adult hippocampal neurogenesis)


Executive Summary:

We have previously shown that depolarisation (used to mimic neural activity) stimulates a latent precursor population in the adult hippocampus (Walker et al., 2008). The major focus of this project was to characterise molecules that underpin this regulation. There is currently a need to identify more appropriate cell surface markers to isolate and characterise hippocampal precursor cells. In our search we have identified Prominin-1 and the lysophosphatidic acid receptor (LPAR) as novel hippocampal precursor cell markers. Prominin-1 (CD133) is commonly used to isolate stem and progenitor cells from the developing and adult nervous system and to identify cancer stem cells in brain tumors. However, no information about the expression of Prominin-1 by precursor cells of the adult hippocampus was available. We have recently shown that Prominin-1 is expressed by a significant number of cells in the subgranular zone of adult mice in vivo and ex vivo, including postmitotic astrocytes. A small subset of Prominin-1+ cells co-expressed the non-specific precursor cell marker Nestin as well as GFAP and Sox2. Upon fluorescence activated cell sorting (FACS), only Prominin-1/Nestin double-positive cells fulfilled the defining stem cell criteria of proliferation, self-renewal and multipotentiality as assessed by a neurosphere assay.

Using immunohistochemistry we have also found that LPAR-GFP is expressed in the precursor cells in the adult dentate gyrus of the transgenic reporter mice. Lysophosphatidic acid (LPA) is an extracellular signaling lysophospholipid that binds to the G protein-coupled receptors LPAR1-6 to regulate many important biological processes including neural cell development. FACS followed by neurosphere assays confirmed that the precursor activity was confined to the LPAR + cell population with >99% of total neurospheres being formed from the LPAR-GFP+ cells. Surprisingly, the more commonly used precursor cell reporter line Nestin-GFP was a less specific marker of these cells with only 80% of the total neurosphere formation from the Nestin+ population.

In summary, we show that the novel markers Prominin-1 and LPAR-GFP can be used to enrich precursor cells from the adult murine dentate gyrus. In addition, we describe novel marker combinations (Nestin/Prominin-1, GFAP-GFP/Prominin-1/CD24, GFAP-GFP/EGFR/PSA-NCAM and LPAR/Prominin-1/EGFR) for the simultaneous flow cytometric isolation of multiple cell types from the adult dentate gyrus. This approach will facilitate the isolation and characterization of homogeneous cell populations of astrocytes, neural precursor cells and immature neurons in order to gain a deeper understanding of the underlying molecular regulation of hippocampal neurogenesis in response to activity or injury in individual cell types rather than at the level of the entire dentate gyrus.

In addition, we have identified several promising candidate factors (Prominin-2 and LPA), which regulate hippocampal precursor proliferation. These two factors are potential therapeutic target molecules, which increase hippocampal neurogenesis may be useful for the treatment of neurodegenerative diseases.

Project Description and Objectives

Recent evidence has demonstrated that memory formation relies on continued neurogenesis i.e. the birth of new neurons in the adult brain. The primary brain structure involved in this process is the dentate gyrus of the hippocampus, where regulated neurogenesis throughout life underpins continuous learning and memory (Shors et al., 2001; Snyder et al., 2005). Various studies have shown that neurogenesis in the subgranular cell layer of the dentate gyrus is influenced by environmental factors (Kempermann et al., 1997), antidepressants (Malberg, 2000), disease states (Nakatomi et al., 2002) and behavioural stimuli (van Praag et al., 1999). In addition, with ageing, hippocampal neurogenesis declines, paralleled by the onset of diseases that affect memory, e.g. dementia (Kuhn et al., 1996). Physical activity as well as hormonal manipulations, counteract this decrease (Cameron et al., 1998; Kronenberg et al., 2006).

We have previously discovered a dormant or latent population of neural precursors in the adult hippocampus that can be triggered to generate new neurons (Walker et al., 2008). Identification of the molecules that trigger these latent neural precursors to generate new neurons should lead to the development of novel therapies to treat neurodegenerative conditions that affect the hippocampus or age-related decline. Thus, the major focus of this project was to characterise molecules that underpin this regulation. Before this can be achieved however, two key hurdles still need to be overcome. First, the stem cell in the hippocampus needs to be isolated and characterised, after which the factor/s capable of regulating the proliferation of the stem cell population need to be determined.

1) Identification of a hippocampal stem cell marker

The development of new markers capable of identifying and isolating defined neural stem cell (NSC) populations are of utmost importance. A number of different antigens have been associated with the NSC cell phenotype such as Nestin (Lendahl et al., 1990), Musashi (Sakakibara et al., 2002), Sox2 (D’Amour and Gage, 2003) and Id1 (Nam and Benezra, 2009). However, their specificity as bona fide stem-cell markers remains questionable. Therefore, there is still a need to identify more appropriate cell surface markers to enable the use of flow cytometry to isolate and characterise the hippocampal precursor cells. Isolation of this population will then allow us to further define the stem and progenitor cell phenotype, with the longer-term goal of identifying key regulators of hippocampal neurogenesis using gene microarrays.

Based on our preliminary data, we have identified Prominin-1 as a candidate marker. Prominin-1 is a five-transmembrane protein that was originally identified in mouse neuroepithelial cells where it associates with microvilli and plasma membrane protrusions (Weigmann et al., 1997). In humans, Prominin-1 was originally identified as an antigenic marker expressed on hematopoietic stem cells (Miraglia et al., 1997) and has been used to enrich hematopoietic stem cells and human neural stem/progenitor cells for clinical purposes (Uchida et al., 2000). Prominin-1 (CD133) has also successfully been used as a marker for the isolation of neural stem and progenitor cells from the mouse embryonic forebrain (Corti et al., 2007), the adult SVZ (Corti et al., 2007) and the postnatal cerebellum (Lee et al., 2005). However, its potential as a marker of stem cells in the adult hippocampus remains to be confirmed.

2) Factors that stimulate hippocampal neurogenesis

Because of the importance of our discovery that neural activity stimulates a latent precursor population, including a bona fide hippocampal stem cell, to future therapeutic approaches to treat neurodegenerative diseases, much of the focus of this project will be on the characterisation of the molecules that underpin this regulation. Our preliminary in vitro evidence suggests that depolarisation of hippocampal cells leads to the release of an as yet unidentified factor, as we have shown that medium conditioned with KCl-depolarised primary hippocampal cells, but from which the KCl is subsequently removed, can activate precursor activity as effectively as direct application of KCl. Therefore, the second part of our proposal aims to characterise factors underpinning this activation, particularly molecules that can cross the blood-brain barrier to enhance brain repair in a non-invasive manner. In order to identify this unknown factor(s) we have performed microarray analysis comparing primary hippocampal cells cultured for 24 hours in either control or depolarising levels of KCl. In addition, these data have been compared to existing data available in the Kempermann lab comparing the expression profiles of control mice to those which were either allowed to run or were housed in an enriched environment, both of which have previously been shown to increase hippocampal neurogenesis.

The specific aims of the current proposal were:

Aim 1: To identify a cell surface marker for isolation and characterisation of adult hippocampal stem cells

Aim 2: To characterise novel target molecules capable of regulating adult hippocampal neu

Project Results/Outcomes

Aim 1: To identify a cell surface marker for isolation and characterisation of adult hippocampal stem cells

Based on our preliminary data, we identified Prominin-1 as a candidate marker. In humans, Prominin-1 was originally identified as an antigenic marker expressed on hematopoietic stem cells (Miraglia et al., 1997) and has been used to enrich hematopoietic stem cells and human neural stem/progenitor cells for clinical purposes (Uchida et al., 2000). Prominin-1 (CD133) has also successfully been used as a marker for the isolation of neural stem and progenitor cells from the mouse embryonic forebrain (Corti et al., 2007), the adult SVZ (Corti et al., 2007) and the postnatal cerebellum (Lee et al., 2005). However, its potential as a marker of stem cells in the adult hippocampus remains to be confirmed.

A. Confirmation of Prominin-1 expression in the adult mouse hippocampus

To determine whether the Prom1 gene was functional in the hippocampus we performed quantitative PCR using universal Prominin-1 primers designed to amplify all Prominin-1 splice variants identified to date (Fargeas et al., 2007). Interestingly, the comparison of the relative Prominin-1 expression at P2, P3, P5, P10, P15, P20 and adult revealed a stable expression of Prominin-1 transcripts in the maturing hippocampus from birth into adulthood.

Using a combination of splice variant-specific primers we showed that a small facultative exon encoding 9 amino acid residues that could be either absent (e.g. s1 variant) or present (s2 variant) at the N-terminal domain is detected in the adult SVZ, dentate gyrus and kidney, suggesting that more than one splice variant can be simultaneously expressed. Interestingly, expression of the s6 variant, as defined by its C-terminal domain, appeared to be spatially restricted and could only be detected in the SVZ and kidney. In the dentate gyrus, the same primer pair could amplify only a smaller product corresponding to the C-terminal domain of either splice variant 3, 4, or 5. The s8 variant, which to date has only been detected in the retina, could not be detected in either of the brain regions tested. Thus, particular areas of the adult brain (e.g. the dentate gyrus versus the SVZ) seem to express distinct Prominin-1 splice variants indicating a region-specific regulation of the Prom1 gene.

Given that we could detect the Prominin-1 transcript in the adult hippocampus we next wanted to determine the tissue localization of Prominin-1 at a protein level using the 13A4 mAb. In the dentate gyrus, the intensity of the Prominin-1 immunoreaction was very low and only detectable upon close examination at high magnification. We also stained hippocampal sections from Nestin-GFP and GFAP-GFP transgenic mice with Prominin-1, which revealed Prominin-1 expression on both the Nestin+ precursor cells and on GFAP+ “vertical” type-1 astrocytes or Type-1 cells.

B. Isolation and characterisation of Prominin-1+ve cells from adult mouse hippocampus

Precursor cells can be prospectively isolated from the adult SVZ on the basis of co-expression of Prominin-1 and GFAP (Beckervordersandforth et al., 2010). This information, in combination with our detection of Prominin-1 expressing cells in the adult dentate gyrus, led us to next examine whether it would also be possible to prospectively isolate the hippocampal precursor cell population on the basis of Prominin-1. Fluorescence activated cell sorting (FACS) revealed that approximately 45% of cells were labeled with Prominin-1. Using the neurosphere-forming assay we showed that almost all of the precursor cells were in the Prominin-1+ population (94.1 ± 2.0% of the total neurospheres generated, n = 6 experiments), whereas the sorted cells negative for Prominin-1 generated only very few neurospheres (5.9 ± 2.0% of the total neurospheres generated).

Aim 2: To characterise novel target molecules capable of regulating adult hippocampal neurogenesis

Because of the importance of our discovery that neural activity stimulates a latent precursor population, including a bona fide hippocampal stem cell, to future therapeutic approaches to treat neurodegenerative diseases, much of the focus of this project was on the characterisation of the molecules that underpin this regulation.

A. Characterisation of Prominin-2 expression in adult mouse brain

Prominin-2 is the second pentaspan membrane glycoprotein that is structurally related to Prominin-1 (Fargeas et al., 2003). It has been suggested that there may be a redundancy in their role as organizers of plasma membrane protrusions (Florek et al., 2007). To test whether, assuming shared functionality, Prominin-2 might compensate for the loss of Prominin-1 in the hippocampal precursor cells of Prominin-1 knockout mice, we first confirmed the expression of Prominin-2 mRNA in the dentate gyrus. Reverse transcriptase PCR analysis with Prominin-2-specific primers, designed to span an intron to eliminate amplification from genomic DNA, amplified the expected product from the dentate gyrus cDNA (data not shown). Given that Prominin-2 was thus expressed in the proper location, we hypothesized that if Prominin-2 is compensating for the lack of Prominin-1 expression, then the relative expression of Prominin-2 should increase in the Prominin-1 null mice compared to their wild-type counterparts. Indeed, Prominin-2 expression was approximately 35% higher in the Prominin-1 null mice compared to their wild-type littermates (135.4 ± 10.7%, t-test p = 0.03 n = 6 mice per genotype).

B. Quantification of neurogenesis in AP2γ and Prominin-2 knock-out hippocampus

Gross morphological examination of the hippocampus from Prominin-2-/- and Prominin-2+/+ mice revealed no obvious differences. To determine the effect of Prominin-2 deletion on precursor proliferation in vivo, adult Prominin-2-/-, Prominin-2+/- and Prominin-2+/+ mice were given a single intraperitoneal injection of the thymidine analog bromodeoxyuridine (BrdU) and perfused 2 hours later. To determine the effect of Prominin-2 deletion on neuron survival (net neurogenesis) in vivo, adult Prominin-2-/-, Prominin-2+/- and Prominin-2+/+ mice were given a single ip injection of BrdU and perfused 4 weeks later. These brains are currently being processed for BrdU immunohistochemistry to determine the effect of Prominin-2 deletion on hippocampal precursor proliferation and net neurogenesis.

Given that it took some time into the project before the Prominin-2 -/- mice were available from our collaborators, we have additionally characterized the effect of Prominin-1 deletion on adult neurogenesis.

To determine the effect of Prominin-1 deletion on precursor proliferation in vivo, adult Prominin-1-/-, Prominin-1+/- and Prominin-1+/+ mice were given a single intraperitoneal injection of the thymidine analog bromodeoxyuridine (BrdU) and perfused 2 hours later. BrdU immunohistochemistry revealed no difference in the number of BrdU+ cells in the SGZ (Prominin-1-/-: 2380 ± 251.6 n = 4; Prominin-1+/-: 2431 ± 122.3 n = 16; Prominin-1+/+; 2097 ± 110.2 n = 4; F21 = 0.8 p = 0.4) or SVZ (Prominin-1-/-: 2746 ± 151.4 n = 4; Prominin-1+/-: 2573 ± 96.86 n = 16; Prominin-1+/+; 2835 ± 327.9 n = 4; F21 = 0.74 p = 0.5) between the three genotypes. Quantification of the number of DCX+ intermediate progenitor cells in the SGZ (Type-2b, Type-3 and early post-mitotic neurons) also revealed no significant difference between the genotypes (Prominin-1-/-: 31684 ± 1762, n = 6; Prominin-1+/-: 34123 ± 661, n = 6; Prominin-1+/+: 29540 ± 1731, n = 4; F13 = 2.3 p = 0.1). In addition, using flow cytometric analysis we compared the numbers of other major cell types in the Prominin-1-/- dentate gyrus to wild-type controls. These analyses revealed no change in the number of astrocytes (S100β+ or GFAP+), oligodendrocytes (Olig2+), neural precursor cells (Sox2+) or adult-born early post-mitotic neurons (Calretinin+) between the genotypes (data not shown).

In agreement with the corresponding in vivo proliferation data, there was no difference in the number of neurospheres derived from either the dentate gyrus (Prominin-1-/-: 101.2 ± 17.4 n = 5; Prominin-1+/+; 104.0 ± 10.1 n = 5; two-tailed t-test p = 0.12) or SVZ (Prominin-1-/-: 1271 ± 48.1 n = 5; Prominin-1+/+; 1481 ± 124.0 n = 5; two-tailed t-test p = 0.15) of Prominin-1-/- and Prominin-1+/+ mice.

Given that no effects on proliferation in either the SVZ or dentate gyrus could be detected, we were interested to determine whether Prominin-1 deletion affected net neurogenesis. To measure the consequences of net adult neurogenesis in vivo, adult Prominin-1-/- and Prominin-1+/+ mice were given a single intraperitoneal injection of BrdU and perfused 28 days later. Interestingly, BrdU immunohistochemistry revealed significantly fewer total BrdU+ cells in the SGZ of Prominin-1-/- compared to Prominin-1+/+ mice (BrdU+ cells: Prominin-1-/-: 858.0 ± 97.6 n = 7; Prominin-1+/+: 1244 ± 81.6 n = 8; two-tailed t-test p = 0.009). Although there was no significant difference in the percentage of newborn cells that became neurons, as determined by phenotyping 100 BrdU+ cells for expression of the mature neuron marker NeuN (Fox3; percentage BrdU+/Fox3+: Prominin-1-/-: 93.2 ± 1.6%, n = 7; Prominin-1+/+; 92.7 ± 1.5%, n = 8; two-tailed t-test p = 0.8) it did however result in a significant decrease in net neurogenesis in the Prominin-1-/- mice (total BrdU+/Fox3+: Prominin-1-/-: 804.4 ± 98.3 n = 7; Prominin-1+/+; 1157 ± 85.5 n = 8; two-tailed t-test p = 0.02). In contrast to the dentate gyrus, the deletion of Prominin-1 had no effect on net olfactory bulb neurogenesis (Prominin-1-/-: 5668 ± 802.0 n = 6; Prominin-1+/+: 5591 ± 353.2 n = 7; two-tailed t-test p = 0.9) or on the percentage of BrdU+ cells that expressed the mature neuron marker Fox3 (Prominin-1-/-: 99.3 ± 0.4 n = 7; Prominin-1+/+: 99.9 ± 0.1 n = 8; two-tailed t-test p = 0.2).

C. Determine the in vivo effects of exogenous AP2γ on hippocampal neurogenesis

Due to some initial problems with AP2γ as a candidate factor we decided to focus our work on another promising candidate, lysophosphatidic acid receptor (LPAR).

LPAR was identified by searching publically available Gene Expression Nervous System Atlas (GENSAT; (Heintz, 2004)) database for transgenic mice with a GFP expression pattern corresponding to the type-1 radial-glial like stem cells of the adult dentate gyrus. Lysophosphatidic acid (LPA, 1-acyl-2-sn-glycerol-3-phosphate) is a membrane-synthesized phospholipid that acts as an intercellular signaling molecule through six G-protein receptor sub-types (LPA1-6; (Choi et al., 2010)). The first of these receptors to be described, LPA1, mediates proliferation, migration and survival of neural progenitor cells during development (Estivill-Torrus et al., 2008). In addition, LPA1 is involved in the regulation of adult hippocampal neurogenesis, with LPA1 deletion affecting neurogenesis, differentiation and survival (Matas-Rico et al., 2008) as well as causing spatial memory deficits (Santin et al., 2009). Given the expression pattern of LPA1-GFP within dentate gyrus, as well as its known involvement in hippocampal neurogenesis, we thus decided to follow this candidate to determine whether LPA1-GFP transgenic mice could be used as a novel tool to identify and prospectively isolate the stem cells of the hippocampal subgranular zone (SGZ) and whether exogenous LPA could promote precursor proliferation and/or neuronal survival in vitro and in vivo.

Using immunohistochemistry we showed that LPA1-GFP is expressed in the precursor cells in the developing and adult dentate gyrus but not SVZ of transgenic reporter mice. Given that we could detect LPA1-GFP expression in the precursor cells of the adult dentate gyrus by immunofluorescence, we also examined whether it would also be possible to prospectively isolate the hippocampal precursor cell population on the basis of LPA1-GFP. The dentate gyri from 8 week-old male LPA1-GFP mice or WT littermates were dissected and dissociated into a single-cell suspension. When sorting cells on the basis of LPA1-GFP almost all of the neurospheres were formed from the LPA1-GFP+ population (99.8 ± 0.2% of the total neurospheres generated, n = 4 experiments), whereas the sorted cells negative for LPA1 generated only very few neurospheres (0.2 ± 0.2% of the total neurospheres generated). Plating the LPA1-GFP+ population in the presence of depolarizing levels of KCl increased the number (LPA1-GFP+, no KCl: 19.9 ± 5.5 vs +KCl: 24.4 ± 6.4 neurospheres per 1000 cells) and size of the neurospheres, but had no effect on the LPA1-GFP- cells (LPA1-GFP- no KCl: 0.03 ± 0.03 vs +KCl: 0.02 ± 0.02 neurospheres per 1000 cells). Remarkably, the neurosphere forming frequency of the LPA1+ population was over 600-fold enriched compared to the negative population (LPA1+: 19.9 ± 5.5 vs. LPA1-: 0.03 ± 0.03 neurospheres per 1000 cells; n = 4 experiments). Given that Nestin is a commonly used marker of neural stem cells we next compared the efficacy of precursor isolation using the LPA1-GFP transgenic mice with that of Nestin-GFP mice. Similar to LPA1-GFP+, almost all of the neurospheres were formed from the Nestin-GFP+ population (Nestin-GFP+: 33.8 ± 6.1 vs. Nestin-GFP-: 2.0 ± 0.4 neurospheres per 1000 cells; n = 6 experiments; p = 0.0022). In comparison to the LPA1-GFP, we have previously shown that the Nestin-GFP sort is less specific for the precursor cells with approximately 20% of the total neurosphere-forming frequency coming from the Nestin-GFP- population (Nestin-GFP+: 80.6 ± 8.2 vs. Nestin-GFP-: 19.4 ± 8.2; (Walker et al., 2013)).

D. Examine the effects of AP2γ and Prominin-2 deletion on hippocampal-based learning

Due to the longer than expected period of time that was required to access the colony of transgenic Prominin-2 knock-out mice, this last objective has not yet been completed. To assess the effect of Prominin-2 deletion on adult hippocampal proliferation and net neurogenesis, we have injected 8-week-old Prominin-2 knock-out mice with CldU and 4 weeks later with IdU. These mice were perfused and the brains are ready for analysis. These experiments are still ongoing and will be completed and analysed within the next 1-2 months. If from this analysis we detect an effect of Prominin-2 deletion on adult hippocampal neurogenesis, we will then assess whether this deficit translates to a deficit in hippocampal -based learning and memory using the Morris Water Maze task.

Project Impact

The burden of neurodegenerative disease affecting the hippocampus inflicts an immense social and economic impact on the EU economy. To date there are no effective pharmaceutical interventions available to prevent or delay the progression of these debilitating conditions. Thus, the primary aim of this research was to further understand the fundamental mechanisms underlying hippocampal neurogenesis in order to tap into the potential of adult generated neurons for therapeutic or preventive purposes. Solving this problem may in the future lead to effective ways to prevent cognitive decline in a variety of neurodegenerative diseases that affect the hippocampus.

Within this project we have successfully identified two novel hippocampal stem cell markers (Prominin-1 and LPAR), both of which can now be used for the isolation and downstream characterization of the stem cells. This is of wider importance, as detailed transcription profiling of the stem cell population will potentially lead to the discovery of a molecular pathway, which could either prevent the decline in neurogenesis or increase recruitment from the neural stem cell pool would have far-reaching implications in the treatment of neurodegenerative and age-related cognitive disease. In addition, we have identified several promising candidate factors (Prominin-2 and LPA), which regulate hippocampal precursor proliferation. These two factors are potential therapeutic target molecules, which increase hippocampal neurogenesis may be useful for the treatment of neurodegenerative diseases.

This project has generated data that has resulted in one first author published open-access manuscript

Walker TL, Wierick A, Sykes AM, Waldau B, Corbeil D, Carmeliet P and Kempermann G (2013) Prominin-1 allows prospective isolation of neural stem cells from the adult murine hippocampus. Journal of Neuroscience 33, 3010-3024.

In addition, a second manuscript is currently in advanced stages of preparation

Walker TL, Vogler S, Lasse D, Kempermann G and Fabel K. Selective and sensitive prospective isolation of adult hippocampus-specific neural precursor cells based on lysophosphatidic acid receptor expression. Manuscript in Preparation

This data has also been presented at several national and international conferences including:

1. Regulation of Adult Neurogenesis: from Epigenetics to Behaviour (Barcelona, Spain 2012) – poster presentation
2. Dresden Stem Cell Congress (Dresden, Germany 2012) – Poster presentation
3. First International Meeting of the Consortium Neuro-Rhine - Neuroprotection and Neurogenesis: Focus on Alzheimer’s disease and Chemotherapy-Induced Peripheral Neuropathy (Strasborg, France March 2013) – Invited speaker
4. Baltic Stem Cell Meeting (Szczecin May 2013) – Invited speaker

Training, transfer of knowledge and integration activities

During the period of this fellowship, I have been given the opportunity to participate in many training courses including animal handling and flow cytometry, as well as the opportunity to attend numerous national and international conferences. In addition, as part of my training/integration I have been attending German language classes organised by the Max Planck Institute and the CRTD for the past 2 years. During this time I have also been able to transfer my knowledge, being responsible for the supervision/mentoring of four students, one, Ms Ann Wierick, a medical PhD student who was directly involved in the described project. I have also been involved in integration activities in the form of presenting our research and institute to the wider audience, at such public events as those organised by the Sächsisches Staatsministerium für Wissenschaft und Kunst and the Lange Nacht Wissenschaft, as well being involved in the organisation of the 2012 Route28 Summits in Neurobiology meeting.