Investigating Neural Stem and Progenitor Intracrine Signaling

Presented in Partial Fulfilment of the Requirements the Master of Arts in the Psychology

Graduate Program of The Ohio State University

By

Tyler Dause B.S.

Graduate Program in Psychology

Thesis Committee

Elizabeth Kirby Ph.D., Advisor

Kathryn Lenz Ph.D.

Jonathan Godbout Ph.D.

Copyrighted by

Tyler Dause

2019

ii Abstract

In the adult mammalian brain, there are two regions where neural stem and progenitor cells reside and proliferate throughout life: the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus. While much of the current research focuses on these cells’ ability to create new neurons, a process known as neurogenesis, new findings indicate that neural stem and progenitor cells (NSPCs) may influence their niches through the secretion of growth factors. Our previous work indicates that NSPCs express

1/3 of the vascular endothelial (VEGF) in the DG. While global VEGF has been shown to support the proliferation and maturation of adult-born DG neurons, the role of NSPC-derived VEGF is not entirely understood. Our data suggest that VEGF plays a role in regulating NSPC stemness in the DG. Currently, we aim to investigate the role of a VEGF/VEGFR2 intracellular autocrine (i.e. intracrine) signaling pathway in regulating

NSPC stemness and maintenance. This thesis contributes to our ongoing work by investigating the immediate effects of VEGF knockdown on NSPC stemness in vitro and modeling VEGF knockdown to determine the NSPC-derived VEGF signaling pathway in vivo. My results suggest NSPC-derived VEGF knockdown increases NSPC proliferation, which is indicative of impaired stemness, in vitro. To investigate VEGF intracrine signaling in vivo we utilized a transgenic mouse line of inducible NSPC-derived VEGF knockdown was accompanied by EYFP reporter expression. I investigated the possible limitations of this commonly used genetic model and discovered recombination induced expression of one fluorescent reported does not accurately predict recombination of another gene at a single cell level. These data indicate that we may not accurately identify NSPC-derived

VEGF intracrine signaling using our mouse model.

iii Acknowledgements

I would like to thank Dr. Kirby for her encouragement, professional guidance and support of my thesis work. I would also like to thank the members of the Kirby lab, especially Mark Fongheiser and Armaan Cheema, for assistance with data collection.

iv Vita

Personal Information

Graduate Education: The Ohio State University, Columbus, Ohio Field of Study: Psychology – Behavioral Neuroscience Advisor: Elizabeth Kirby

Bachelor of Science (Magna Cum Laude) The Ohio State University, Columbus, Ohio Major: Behavioral/Systems Neuroscience with Research Distinction in Neuroscience GPA: 3.703 | Major GPA: 3.918 | Spring 2017

Publications

Dause TJ, Kirby ED. (2019) Aging gracefully: social engagement joins exercise and enrichment as a key lifestyle factor in resistance to age-related cognitive decline. Neural Regeneration Research, 14, 39-42.

Rieskamp, JD, Denninger, JK, Dause, TJ. (2018). “Identifying the Unique Role of Notch3 in Adult Neural Stem Cell Maintenance.” Journal of Neuroscience, 38, 3157- 3159.

Fields of Study

Major Field: Psychology

v Table of Contents

Abstract ...... iii Acknowledgments ...... iv Vita ...... v List of Tables ...... vi List of Figures ...... vii Introduction ...... 1 Methods ………………………………………………………………………………… 6 Results …………………………………………………………………………………. 11 Discussion ………………………………………………………………………………22 Future Directions………………………………………………………………………..27 Summary…………………………………………………………………………….…..28 Bibliography……………………………………………………………………………. 30

vi List of Tables

Table 1: Primary and Secondary antibodies ………………………………………… 10

vii List of Figures

Figure 1: Paracrine vs Autocrine vs Intracrine ………………………………… 4 Figure 2: Intracellular vs Extracellular VEGF …………………………………... 5 Figure 3: NSPC lentiviral Infection in vitro ……………………………………… 12 Figure 4: Lentiviral Infection in vivo …………………………………………..… 14 Figure 5: NSPC derived VEGF knockdown in vivo ...…….…………………… 16 Figure 6: EYFP and tdTomato Recombination in SVZ NSPCs …..…………..18 Figure 7: EYFP and tdTomato Overlap in SVZ NSPCs ..………….…...... ….. 19 Figure 8: EYFP and tdTomato Recombination in DG NSPCs ………….……..20 Figure 9: EYFP and tdTomato Overlap in DG NSPCs ………………………… 20 Figure 10: Representative EYFP+ and tdTomato+ NSPC subpopulations .... 21

viii Introduction

Since the discovery of endogenous adult-born neurons in the 1960s suggested the possibility of adding functional neurons to the mature brain,1 the potential to use neural stem and progenitor cells (NSPCs) to produce new neurons in disease has garnered considerable attention. As our understanding of NSPC function in the brain develops, many scientists are investigating the use of stem cell therapies to combat a spectrum of neurological disorders. Usually, these therapies rely on the direct transplantation of exogenous stem cells or on targeting the endogenous population with different neurotrophic factors.1 Despite mixed success in rodents, clinical trials are pushing forward with stem cell-based therapy for a variety of neurodegenerative disorders. In many of these trials, the extracellular environment is manipulated to increase likelihood of success.1 For instance, in both endogenous1 and transplanted stem cell therapies1 exogenous extracellular growth factors are introduced to support stem cell engraftment, growth and survival. Therefore, it is critical to understand how, or even if, stem cells respond to these growth factors within the brain.

There are two neurogenic niches in the adult mammalian brain where NSPCs reside and proliferate throughout life: the subventricular zone (SVZ) and the dentate gyrus

(DG) of the hippocampus. For years, the primary focus of NSPC research was neurogenesis, or the production of new neurons. In rodent models, numerous studies support the importance of adult-born neurons for proper hippocampal function, including learning and memory.2 However, recent findings suggest that neuronal differentiation may not be the only function of NSPCs. For example, NSPCs may also regulate the DG

1 microenvironment through the expression of growth factors.3 Indeed, NSPCs have been shown to be a significant source of vascular endothelial growth factor (VEGF) in the DG,3 though the role of this VEGF is not entirely understood.

VEGF (also known as VEGFA) is expressed by neural precursors during development4 and NSPCs in the mature brain.3 In the developing embryo, VEGF expression from the neural tube is crucial for vasculogenesis, or the formation of the peri- neural vascular plexus.5 VEGF from neural precursors forms a chemotactic gradient, which attracts endothelial cells from the peri-neural vascular plexus and guides the ingression of sprouting vessels into the embryonic brain.6 Loss of just one VEGF allele in neural precursors is embryonic lethal due to improper cerebral vascularization as characterized by abnormal blood vessels7 and disrupted vessel patterning,4 underscoring the importance of neural precursor VEGF in development. Independent of brain vascularization, VEGF also supports the development of neurons and affects their structure,8 proliferation,9 and survival.10 In the adult brain, VEGF continues to play a role in angiogenesis, or the development of new blood vessels, particularly after injury.11

Similarly to development, VEGF also impacts the proliferation9 and survival of adult-born neurons.12 In most adult brain regions, astrocytes are the primary source of VEGF13 and in past studies of VEGF’s effects on NSPCs, this has been assumed to be the case in the neurogenic niches as well.9,14 However, my lab’s recently published data shows that

NSPCs synthesize their own VEGF and despite the presence of VEGF from astrocytic populations, this self-derived VEGF is necessary to maintain their stemness.3 The mechanism by which NSPC-derived VEGF supports stemness is unknown.

2

While little is known about VEGF signaling in NSPCs, the intracellular signaling pathways of VEGF receptors are well-established in endothelial cells,15 hematopoietic stem cells16 and different forms of cancer cells.17–19 VEGF binds to two related

VEGF tyrosine kinases, VEGFR1 (Flt1) and VEGFR2 (KDR).20 NSPCs are known to express VEGFR2.3 Based on findings in other cell types, VEGF binding to

VEGFR2 leads to receptor dimerization,21 internalization,22 and the autophosphorylation of multiple tyrosine residues,23 specifically Y95124 and Y117525 in the intracellular domain.

Phosphorylation of Y951 promotes PI3K activation and translocation of Akt to the membrane.23,24 Once at the membrane, Akt is phosphorylated, which triggers the activation of downstream signaling cascades and promotes cell survival.28 Independently, phosphorylation of Y1175 activates PLCγ.29 PLCγ initiates the phosphorylation of the ras- raf-mek-erk pathway and promotes downstream cell proliferation pathways.25,30 These

VEGF/VEGFR2 signaling mechanisms were originally thought to be dependent upon paracrine or autocrine VEGF, but there is a third, less well-studied mechanism by which

VEGF can signal to VEGFR2 cell-internally (i.e. intracrine).

Intracellular , otherwise known as intracrine signaling, refers to in an internal, cell autonomous mechanism (Figure 1). VEGF intracrine signaling was originally described in hematopoietic stem cells16 and is suggested to play a role in endothelial VEGFR2 signaling.15 Though relatively little is known about VEGF intracrine signaling, previous studies have shown that VEGFR2 has a high turnover rate31 and is trafficked to and from the cell surface by endosomes. VEGFR2 signaling does not

3 end upon internalization, but continues intracellularly from the endosomes.32 It has also been shown that VEGFR2 can be phosphorylated within23 and signal from endosomes32 suggesting a potential target for internal VEGF. However, the role of intracrine VEGF signaling in NSPCs remains unclear.

Previous work from my graduate mentor establishes NSPCs as a significant source of VEGF in the DG, contributing up to 1/3 of the total VEGF.3 Dr. Kirby further showed that inducible NSPC- derived VEGF knockdown in adult mice leads to increased NSPC proliferation and exhaustion of the stem cell pool in the DG, indicating that NSPC-derived VEGF is necessary for maintenance of NSPC stemness in vivo.3 This phenotype of stem cell exhaustion preceded by a proliferate burst is commonly found when key genes related to stem cell quiescence are impaired, leading to over-activation of stem cells and their

4 eventual depletion.33–35 Dr. Kirby’s subsequent work with VEGF loss of function experiments in vitro suggests that NSPC-derived VEGF is necessary to maintain stemness via signaling within the NSPC population, not via feedback to/from endothelial or neuronal cells.3 Taken together, these findings suggest NSPCs do not depend on

VEGF from other niche cells to function. Currently, I aim to investigate the VEGF/VEGFR2 signaling pathway in NSPCs, specifically whether VEGF signaling is transmitted in an autocrine or intracrine manner. My lab has found that blocking intracellular VEGF signaling in vitro significantly reduces phosphorylation of the PI3K/Akt, but the use of an extracellular VEGF neutralizing antibody (nAb) does not (Figure 2A, C). Disruption of intracellular VEGF signaling also impairs NSPC maintenance in vitro (Figure 2B).

However, NSPC maintenance is not impaired after neutralizing extracellular VEGF

(Figure 2D). Taken together, these data suggest that cell-internal VEGF stimulates

VEGF/VEGFR2 signaling pathways and regulates cell stemness. Extracellular VEGF neutralization does not affect these processes indicating that NSPC-derived VEGF functions in a cell autonomous manner in vitro.

This thesis will first confirm the necessity of VEGF/VEGFR2 intracrine signaling for stemness maintenance in adult DG NSPCs at the single cell level and also investigate the validity of the most commonly used experimental paradigm to show intracrine signaling in vivo. First, the previous data from my lab focuses on how loss of self-derived

VEGF signaling across the entire population changes NSPC stemness over many cell passages. The aim of my first set of experiments is to understand the effect of VEGF knockdown in a more cell-specific manner. Second, previous in vivo work suggests that

5 knockdown of NSPC-derived VEGF disrupts NSPC stemness in the DG. However, these findings did not reveal whether knockdown affected stemness cell internally or via loss of extracellular VEGF that impacted all cells similarly. My in vivo experiments aim to determine the cell autonomous effects of NSPC-derived VEGF knockdown. However, in pursuing these experiments, I tested the core, underlying assumption of the dominant paradigm used to demonstrate intracrine signaling in vivo and found that assumption was severely flawed.

Methods:

Animals

All mice were housed in standard ventilated cages, with ad libitum access to food and water throughout all experiments and maintained on a 12h light cycle with lights on at 630h. For this thesis we utilize wild type C57BL/6J (WT) mice (Jackson #000664).

NestinCreERT2 (Jackson #016261) were crossed with VEGFlox/lox mice (Genentech,

Inc)36 to create NestinCreERT2;VEGFlox/lox mice. NestinCreERT2;Rosa-stop-floxed-

EYFP (Rosa EYFP Jackson #006148) mice were crossed with VEGFlox/lox mice to create NSPC-specific, inducible VEGF knockdown mice with a fluorescent reporter.

NestinCreERT2;Rosa-stop-floxed-EYFP mice were crossed with Rosa-stop-floxed- tdTomato mice (Jackson #007909) to create NSPC-specific fluorescent reporter mice. All animal use was in accordance with institutional guidelines approved by the Ohio State

University Institutional Animal Care and Use Committee.

NSPC Isolation and Maintenance

6 Adult hippocampal NSPCs were isolated from WT and VEGFlox/lox mice as described by Babu et al.37 NSPCs were maintained on poly-D-lysine (Sigma) and laminin

(Fisher) coated plates in Neurobasal A media (Invitrogen) with 1x B27 supplement without vitamin A (Fisher), 1x glutamax (Fisher) and 20 ng/ml each of EGF and FGF2

(Peprotech).37 No cells were used past passage 15. Two separate lines were used in this experiment, one from male WT mice and one from VEGFlox/lox mice.

In vitro lentivirus infection

NSPCs were passaged using accutase (Fisher) and counted with a hemocytometer to plate 5,000 cells/well in a 96 well plate coated with poly-D-lysine and laminin. Adherent NSPCs were infected with lentiviral vectors expressing mCherry-Cre or mCherry only (control) at low MOI designed to yield 1-2.5% infection. mCherry-Cre lentivirus plasmid was obtained from addgene (#27465) and mCherry only was created by restriction digest-mediated truncation of the Cre gene (Kirby et al). Viral plasmids were packaged by the Cincinnati Children’s Medical Center Viral Vector Core. NSPCs were incubated with 5-bromo-2'-deoxyuridine (BrdU) (Sigma) for 2 hours then fixed with 4% paraformaldehyde prior to immunohistochemical processing 2- and 4- days after viral infection.

Tamoxifen and bromodeoxyuridine administration in vivo

Tamoxifen (TAM) was dissolved in sterile sunflower oil, overnight with agitation at

37 deg C. TAM solution was stored at +4 deg C for up to 1 week. TAM (or oil vehicle) was injected (180 mg/kg/d, IP) for 5d.38 After 3, 21 or 60 days, mice were injected with 5- bromo-2'-deoxyuridine (BrdU) (Sigma) dissolved in physiological saline (Hospira) (150 mg/kg, IP) and sacrificed 2 hours later for immunofluorescent processing.

7 Stereotaxic Injections

Mice were anesthetized by inhalation of isoflurane (Akorn, 5% induction, 1-2% maintenance) in oxygen and mounted in the stereotaxic apparatus (Stoelting). Ocular lubricant (Puralube) was placed over the eyes to prevent evaporative dry eye. Following sterilization with alcohol (Fisher) and betadine swabs (Fisher) the skull was exposed and the lambda and bregma sutures were aligned in the same horizontal plane. A small bur hole was drilled in the skull and an automated injector (Stoelting) with a Hamilton syringe

(Hamilton) was lowered to the injection depth at a rate of -1.9 mm/min. Mice were injected with 0.5 µL of mCherry-Cre virus into the right hemisphere and 0.5 µL of mCherry only virus into the left hemisphere using at a rate of 0.1 µL/min. The injection coordinates from bregma were as follow: anterior/posterior -1.9 mm, medial/lateral ±1.6 mm, -1.9mm dorsal/ventral from dura. Post-surgery, the incision was sealed with tissue adhesive (3M) and the mouse was given saline (Hospira) and carprofen (Zoetis) injection ip. After 3 days or 1 month, mice were sacrificed for immunofluorescence processing.

IF staining/antibodies

In vitro

For immunofluorescence staining in vitro, adherent NSPCs were given 20µM BrdU

2h before 10-minute fixation with 4% paraformaldehyde. Fixed cells were rinsed with PBS

3 times then incubated in a blocking solution containing 1% normal donkey serum and

0.3% Triton X-100 (Acros) in PBS. Cells were then incubated in primary antibody (Table

1) diluted in blocking solution overnight at 4 deg C. The following day, after 3 rinses in

PBS, cells were incubated in secondary antibodies (Table 1) diluted in blocking solution

8 for 2 hours. After 3 PBS rinses, the cells were then fixed with 4% paraformaldehyde for

10 min, rinsed with PBS 3x and incubated with 2N HCl for 30 min at 37 deg C. After 3

PBS rinses and 30 min incubation in blocking, cells were incubated in BrdU primary antibody diluted in blocking solution overnight at 4 deg C. The next day, cells were rinsed

3 times with PBS and exposed to a secondary antibody diluted in blocking solution for 2h.

Cells were imaged at 10x magnification with a Zeiss apotome digital imaging system

(Zeiss) and were counted manually with ImageJ for quantification.

In vivo Brain Sections

All brains were harvested following perfusion with ice-cold PBS followed by fixation in 4% paraformaldehyde overnight at 4 deg C. After equilibration in 30% sucrose in PBS,

40 µm coronal brain sections were obtained in 1 in 12 series on a freezing microtome

(Leica), and stored in cryoprotectant at -20°C. Immunohistochemical staining was performed as above, in fixed adherent NSPCs, using primary and secondary antibodies from Table 1. The DG of the hippocampus was imaged in 15 µm Z-stacks at 20x magnification using a Zeiss apotome digital imaging system (Zeiss). NSPCs were identified and counted manually on ImageJ. Colocalization of immunofluorescent signal was analyzed with just another colocalization plugin (JACoP) software on ImageJ.39 First,

Z-stacks from each DG were separated into individual 1 µm thick images, 1 image per fluorescent channel. Images were thresholded then overlapped using anatomical features. The JACoP plugin then re-stacked the image files and analyzed overlap of

EYFP and tdTomato. These data were used to determine EYFP and tdTomato percent area, the proportion of EYFP overlapping tdTomato and the proportion of tdTomato overlapping EYFP.

9 Primary Antibody Vendor/ Product Dilution Secondary Vendor/ Product Dilution Use number number Rabbit mCherry Abcam ab167453 1:500 Donkey anti- ThermoFisher 1:500 In vitro/vivo Rabbit IgG (H+L) Scientific Highly Cross- A-31572 Adsorbed Secondary Antibody, Alexa Fluor 555 Mouse BD Biosciences 1:500 Donkey anti- Fisher 1:500 In vitro Anti-Brdu 347580 Mouse IgG (H+L) A-21202 Highly Cross- Adsorbed Secondary Antibody, Alexa Fluor 488 Hoescht Fisher 1:2000 N/A N/A N/A In vitro/vivo 33342 Goat Abcam 1:1000 Donkey anti-Goat Fisher 1:500 In vivo Anti-GFP Ab6673 IgG (H+L) Cross- A-11055 Antibody Adsorbed Secondary Antibody, Alexa Fluor 488 Mouse EMD Millipore 1:1000 Donkey anti- Fisher 1:500 In vivo Anti-Glial MAB360 Mouse IgG (H+L) A-31571 Fibrillary Acidic Highly Cross- , Clone Adsorbed GA5 (GFAP) Secondary Antibody, Alexa Fluor 647 Rabbit Cell Signaling 1:500 Donkey anti- Fisher 1:500 In vivo MCM2 Antibody Technology Rabbit IgG (H+L) A-31573 4007 Highly Cross- Adsorbed Secondary Antibody, Alexa Fluor 647 Rat Affymetrix 1:1000 Alexa Fluor® 647 Jackson 1:500 In vivo Anti- eBiosciences AffiniPure 102649-874 Human/Mouse 14-9811 Donkey Anti-Rat Sox2/Purifi ed IgG (H+L) Table 1: Table of primary and secondary antibodies used for immunofluorescent staining and imaging.

Statistical Analysis

NSPC proliferation after viral infection was compared by two-way ANOVA with

Sidak’s post-hoc comparison. Correlations between EYFP and tdTomato percent area were analyzed using Pearson’s correlation. Differences between EYFP and tdTomato expression were analyzed using paired t-tests. One-sample t-tests were used to determine the difference between fluorescent marker overlap and a theoretical value of

100%. All analyses were performed using Prism (v8.0; GraphPad Software) and p- values of <0.05 were considered significant.

10

Results

VEGF Knockdown Enhances NSPC Proliferation in an Intracrine Manner After 4 Days In

Vitro

Loss of a stem cell’s ability to self-renew leads to exhaustion of the stem cell pool.

This is characterized by an increase in proliferating progenitors followed by a proliferative collapse because the stem cells cannot restore the progenitor pool. Our lab has previously demonstrated that NSPC-derived VEGF knockdown leads to a loss of stemness and exhaustion of the DG stem cell pool at the whole population-level.3 My current work explores whether NSPC-derived VEGF maintains stemness through cell autonomous signaling or via extracellular autocrine signaling. My lab previously found that disruption of intracellular VEGFR2 via a cell permeant small molecule inhibitor leads to an early over-proliferation of cultured NSPCs within 4 days, followed by a loss of proliferation in subsequent passages (Fig 2B). The lack of similar effect following neutralization of extracellular VEGF implies that VEGF maintains stemness cell-internally

(Fig 2D). However, a more direct proof of this hypothesis would demonstrate loss of stemness at the single cell level.

11 To determine the effects of VEGF knockdown on NSPC proliferation cell- specifically, I used cultured NSPCs from the DG of adult mice, isolated and maintained in vitro as per Babu et al.40 When maintained in these conditions adult NSPCs can self-renew and proliferate across dozens of passages.40 I infected both WT and VEGF floxed NSPCs with lentiviral vectors expressing mCherry or mCherry-P2A-Cre. Lentiviral infection was performed at a low MOI, and cell proliferation was investigated either 2- or 4-days after infection. Utilizing a low MOI allowed us to disrupt

NSPC-derived VEGF in individual cells without changing total extracellular VEGF for the whole in vitro population, creating a “neighbor rescue” experiment. After 2 days, ~6% of cells/well were mCherry+ across all cultures (Figure 3A). I then assessed proliferation in infected NSPCs by determining the percentage of mCherry+ cells that were labeled with

BrdU. After 2 days, there was no effect of NSPC-derived VEGF knockdown on cell proliferation in comparison to control virus or WT cells (Figure 3B). 4 days post-infection,

~10% of cells in each condition were mCherry+ independent of genotype or virus (Figure

3C). I then assessed NSPC proliferation 4 days post-infection. Analysis revealed a significant difference in NSPC proliferation 4 days post-infection (two-way ANOVA Cre- infection, F(1,8) = 7.581 p = 0.0249, Genotype, F(1,8) = 5.742 p = 0.2364, Interaction,

F(1,8) = 11.31 p = 0.0099). Sidak’s multiple comparisons test revealed VEGFlox/lox

12 treated with mCherry-P2A-Cre (M = 33.78, SE = 6.79) had a significant increase in their percentage of proliferating mCherry+ cells when compared to those treated with mCherry control virus (M = 30.94, SE = 0.67) (p = .0050) (Figure 3D).

These data suggest that NSPC-derived VEGF knockdown in vitro begins to functionally disrupt stemness in a cell autonomous manner (i.e. in mCherry-Cre+

VEGFlox/lox cells despite ample extracellular VEGF) in as little as 4 days. To further investigate autocrine vs intracrine VEGF signaling, I assessed proliferation in non- infected NSPCs. If NSPC-derived VEGF regulates stemness through intracrine signaling there should be no significand difference in mCherry- cell proliferation regardless of genotype and virus. If NSPC-derived VEGF is autocrine, we should see an increase in mCherry- NSPC proliferation. Indeed, we saw no change in mCherry- NSPC proliferation at 2 days (Figure 3E) and 4 days (Figure 3F), further confirming VEGF intracrine signaling in NSPCs. Replication of this experiment at least twice more is planned in the near future.

Modeling NSPC-derived VEGF Knockdown In Vivo

Our previous in vivo data indicate that NSPC-derived VEGF knockdown leads to a dysregulation of NSPC stemness and exhaustion of the DG stem cell pool.3 These experiments utilized NestinCreERT2;VEGFlox/lox mice where a TAM sensitive Cre is driven by the Nestin promoter, which is active selectively in NSPCs. While this is a useful tool for inducing recombination, it does not allow us to track the NSPCs where VEGF was knocked down. Without this ability, it is difficult to discern if NSPC-derived VEGF regulates stemness via paracrine, autocrine, or intracrine signaling in vivo. In order to

13 confirm the method of NSPC-derived VEGF signaling in vivo it is necessary to track recombination and VEGF knockdown cell-specifically.

To determine the effects of NSPC-derived VEGF knockdown cell-specifically in vivo, I used lentiviral vectors to express Cre recombinase in the brains of VEGFlox/lox and WT mice. Both WT and VEGFlox/lox mice received stereotaxic injections of mCherry only (control; left hemisphere) and mCherry-Cre lentivirus (right hemisphere) into the DG.

As lentiviruses infect all cell types, this would also allow us to analyze the impact of VEGF knockdown on NSPCs that had lost cell internal VEGF versus those that maintained internal VEGF while extracellular VEGF was reduced by infection of neighboring cells. Mice were sacrificed

3 days and 1 month after surgery. The 3-day timepoint was designed to assess transgene expression soon after infection but before significant changes to NSPC stemness occurred. After 21 days, if NSPC-derived

VEGF maintains cell stemness through intracrine

VEGFR2 signaling, mCherry-Cre infection is expected to increase mCherry+ progenitor proliferation but reduce mCherry+ radial glia-like stem cells (RGLs) in

VEGFlox/lox mice when compared to control virus and

WT mice. Additionally, if NSPC-derived VEGF signaling is cell autonomous, there would be no effect on non-infected progenitors and RGLs. mCherry-Cre

14 infection is not expected to impact cell survival of most other DG cells in VEGFlox/lox mice in comparison to controls based on.9,41 However, it may impact cell survival in endothelial cells, which is regulated VEGF/VEGFR2 intracrine signaling.15 If

VEGF/VEGFR2 signaling is extracellular autocrine, mCherry-Cre infection would be expected to increase proliferation of all progenitors and reduce the entire RGL population in the DG of VEGFlox/lox mice in comparison to controls. Unfortunately, I found lentiviral infection was exceedingly variable in the DG. In certain subjects, there was a track mark in the DG but a complete lack of mCherry+ cells (Figure 4A) while others were saturated with an unquantifiable number of mCherry+ cells (Figure 4B). Therefore, I elected to pursue a different method of tracking NSPCs after VEGF knockdown in vivo.

The most common method for determining cell-autonomous versus paracrine/neighbor-induced effects of gene knockdown is to ablate gene function in a subset of the cell of interest, then track changes in that population in comparison to neighboring cells of the same type.16 In the Cre-lox system for knockdown, the knockdown versus non-knockdown cells are identified based on presence or absence of a fluorescent reporter from a stop-floxed construct driven by a universal promoter. To determine the cell-specific effects of VEGF inducible knockdown in NSPCs, I crossed

NestinCreERT2;Rosa-stop-floxed-EYFP mice with VEGFlox/lox mice to create NSPC- specific, inducible VEGF knockdown mice that were accompanied by EYFP reporter expression. VEGF KD in this model has been previously verified in Kirby et al., 2015.3 To induce knockdown of VEGF and expression of EYFP in NSPCs, NestinCreERT2;Rosa- stop-floxed-EYFP;VEGFlox/lox (EiKD) and their NestinCreERT2;Rosa-stop-floxed-

15 EYFP;VEGFwt/wt (EWT) littermates were submitted to 5 days of TAM injections, which is a standard regimen that leads to highly efficient and specific recombination of floxed genetic sequences in our hands3 and others’.42,43 After 21- and 60- days, mice were injected with BrdU and sacrificed 2 hours later for immunofluorescent processing. This experiment is ongoing. In planned tissue analysis, EYFP+ cells will be colabeled with markers to identify them as RGLs (Figure 5A) or progenitor cells (Figure 5B) in order to track their fate. If VEGF/VEGFR2 signaling is intracrine, there would be an increase in

EYFP+ progenitors and a concurrent loss of EYFP+ RGLs at 21 days in EiKD mice when compared to EWT controls. However, there would be no effect on EYFP- NSPCs. After

60 days there would be exhaustion of the EYFP+ stem cell pool with very few EYFP+ progenitors and RGLs in the DG of EiKD mice when compared to EYFP- NSPCs in EiKD mice and WT controls. These results would suggest that NSPCs rely upon cell autonomous VEGF signaling to regulate stemness in the DG. If VEGF/VEGFR2 signaling is autocrine, there would be an increase in progenitor proliferation and loss of RGLs at 21 days followed by exhaustion of the whole stem cell pool after 60 days, regardless of EYFP expression in comparison to WT controls. These results would suggest that NSPCs rely on VEGF from the environment to regulate stemness in the DG.

Recombination of One Gene Does Not Predict Recombination of Another at a Single

Cell Level

16 Our current model for tracking VEGF recombination in DG NSPCs relies on the assumption that recombination-induced knockdown of VEGF and expression of EYFP under the Rosa locus occur at similar efficiencies and within the same cells with a high probability. While this is a common methodology used to track recombination, it may not be an accurate assumption. To my knowledge, this assumption--that recombination in one gene accurately predicts recombination in another at the single cell level--has not been tested. Because gene recombination in two loci are separate events, it is possible they may not correlate well cell by cell. If this were the case, it would imply that using fluorescent reporters to indicate that a single cell also has recombination in a second gene is not a reliable methodology. In our study, for example, if

EYFP and VEGF recombine in different cells there may be many false positives where we identify NSPCs as EYFP+ but they have not lost their VEGF expression and false negatives where NSPC-derived VEGF is lost but not labeled with EYFP. In order to assess the validity of this common method for tracking recombination, I developed a mouse line to study the coexpression of two conditionally activated fluorescent reporters in NSPCs.

17 NestinCreERT2;Rosa-stop-floxed-EYFP mice were crossed with Rosa-stop-floxed- tdTomato mice to create NestinCreERT2(het);Rosa(EYFP/tdTom) mice. TAM administration was used to induce recombination and expression of EYFP and tdTomato in NSPCs. In these mice, both fluorescent reporters are driven by the same Rosa promoter in NSPCs, one on each allele. I began by comparing the SVZ and DG of oil injected controls to TAM injected mice and found that oil injection did not lead to recombination and expression of EYFP or tdTomato in NSPCs. These data indicate that recombination of both reporters is tightly dependent upon TAM administration, as expected.

Next, I analyzed NSPC expression of EYFP and tdTomato in the SVZ of TAM injected mice (Figure 6). A paired samples t-test revealed a significant difference between tdTomato (M = 0.8024, SE =

0.09484) and EYFP (M = 0.4988, SE = 0.06704) percent area in the SVZ; t(9)=5.089, p

= 0.0007 (Figure 7A). These data suggest that, despite their expression under identical promoters, EYFP and tdTomato constructs experienced recombination at different rates in the SVZ. However, results of the Pearson’s correlation indicated a strong and significant correlation between the total amount of tdTomato and EYFP expression in the

SVZ of TAM injected mice; r(9) = 0.7809, p = 0.0077 (Figure 7B). These findings suggest

18 that mice with high recombination of one reporter gene also have high recombination of the other and support the possibility that recombination of one allele might still be somewhat predictive of recombination in the second allele.

I next analyzed the percentage of immunofluorescent NSPCs that were positive for both fluorescent reporters. If recombination in one gene strongly predicts recombination of the other in the same cell, then the percentage of EYFP+ cells that coexpress tdTomato will be near 100%. As tdTomato percent area is significantly higher than EYFP percent area, it is not possible for 100% of tdTomato+ cells to coexpress

EYFP. However, if our assumption that dual recombination occurs with high probability is true; a high percentage of tdTomato+ cells will still coexpress EYFP. In contrast, if recombination of one gene is not a strong predictor of recombination of the other there will be a lower percentage of cells that express both markers. In my qualitative analysis of TAM injected mice, many SVZ NSPCs were yellow (EYFP+, tdTomato+), but there was also a substantial number of green (EYFP+, tdTomato-) and red (EYFP-, tdTomato+), indicating that EYFP and tdTomato were not always coexpressed in the same cell. When quantified, 57% of EYFP area was overlapped by tdTomato, which was significantly less than 100% co-expression (one sample t-test vs 100%, t(9)= 13.66, p < 0.0001) (Figure 7C). EYFP was not expected to

19 fully overlap on tdTomato+ area because tdTomato area was greater than EYFP+ area.

Quantification revealed that 40% of tdTomato area was overlapped by EYFP (M = 0.4063, SE = 0.04348), (Figure 7D). These results suggest that the fluorescent markers are not always coexpressed in the same NSPCs. Therefore, tdTomato expression is a poor indicator of EYFP expression, and vice versa, in SVZ

NSPCs.

My ongoing in vivo experiments investigate VEGF intracrine signaling in

DG NSPCs. Thus, it is necessary to examine recombination overlap at the single cell level in this population. 3 mice were analyzed for recombination and coexpression of the two fluorescent reporters in the DG (Figure 8). TAM injections resulted in less NSPC recombination in the DG overall, an expected result because there are fewer

20 NSPCs in the DG than SVZ of mice.38 In contrast to SVZ NSPCs, EYFP (M = 0.2736,

SE = 0.05423) and tdTomato (M= 0.2948, SD = 0.03554) percent area in the DG was similar; t(2) = .9603, p = 0.4382. (Figure 9A). These results suggest that either EYFP and tdTomato had similar recombination efficiencies in DG NSPCs, or that a lower percent area of each gene in the DG, when compared to the SVZ, coupled with a low sample size, made it difficult to detect any potential differences. Results of the Pearson’s correlation indicated a strong but non- significant correlation between tdTomato and

EYFP expression in the DG of TAM injected mice; r(2) =

0.9644, p = 0.1703 (Figure 9B). These results are likely not significant due to low sample size. These data suggest that high recombination in one marker is correlated with high recombination in the other, similar to findings in the SVZ. However, also like the SVZ, assessment at the single cell level revealed divergence in reporter expression. EYFP overlapped tdTomato 36.7% (M = 0.3671, SE = 0.02654), significantly less than 100% of the time in the DG; t(2)= 23.85, p = 0.0018 (Figure 9C). Similarly, tdTomato overlapped

EYFP 36.3% (M = 0.3630, SE = 0.05654), which was also significantly less than 100% of the time in the DG; t(2)= 11.27, p = 0.0078 (Figure 9D). These findings indicate that the fluorescent markers are not coexpressed in the same NSPCs, suggesting that tdTomato expression is a poor indicator of EYFP expression, and vice versa, in DG NSPCs.

21 Ongoing experiments are quantifying recombination via cell counts (rather than % area) and quantifying recombination in specific subclasses of NSPCs (i.e. RGL, TAP etc). I also plan to determine if there is a difference in EYFP and tdTomato coexpression between

RGLs (Figure 10A) and progenitors (Figure 10B) in this region.

Discussion

Though most VEGF signaling occurs through binding of extracellular ligand to cell surface receptors, a cell autonomous VEGF/VEGFR2 signaling pathway has been described in several select cell types.15–19 I aim to investigate the role of VEGF/VEGFR2 intracrine signaling in NSPCs of the DG, which, to the best of my knowledge, remains unestablished. This thesis expands upon previous work in my lab, which identifies the importance of intracellular VEGF/VEGFR2 signaling for maintaining NSPC stemness in vitro. Here, I demonstrate that VEGF knockdown functionally impairs NSPC stemness selectively in cells experiencing knockdown. Attempts to investigate whether VEGF maintains stemness via intracrine signaling in vivo led to my experiments examining the most common methodology for determining intracrine necessity of specific genes in vivo.

This model relies on the assumption that recombination-induced VEGF knockdown and fluorescent reporter expression occur at similar efficiencies and within the same cells. I tested if the assumption was correct with the use of a transgenic mouse model where I induced recombination of 2 separate fluorescent reporters in NSPCs. I measured the expression of these reporters in both SVZ and DG NSPCs and found that recombination of one gene did not accurately predict recombination of the other on a single cell level.

22 NSPC-derived VEGF Knockdown Impairs NSPC Stemness In Vitro

Cell autonomous VEGF signaling in stem cells was first discovered in hematopoietic stem cells.16 In this population, the VEGF/VEGFR2 intracrine loop is necessary for cell survival both in vitro and in vivo.16 Previous work from my lab established that NSPCs are a significant source of VEGF in the DG and that this VEGF is necessary to maintain stemness via signaling to NSPC-expressed VEGFR2.3 However, these findings not reveal whether NSPC-derived VEGF signals in an autocrine or intracrine manner to regulate stemness. My lab’s current data suggests that extracellular

VEGF does not affect VEGFR2 signaling or NSPC stemness in vitro while intracellular

VEGFR2 inhibition significantly disrupts the AKT pathway and NSPC maintenance. These data suggest that VEGF signaling is cell autonomous and regulates NSPC stemness.

My thesis investigates the effects of NSPC-derived VEGF knockdown in individual cells based on their recombination status. I infected NSPCs from the DG of WT and

VEGFlox/lox mice with lentiviral vectors expressing Cre recombinase fused to mCherry by P2A linker. I found that after 4 days NSPC-derived VEGF knockdown with mCherry-

Cre, but not control, virus increased proliferation in mCherry+ VEGFlox/lox cells, with no effect mCherry+ WT cells. Viral infection had no effect on mCherry- NSPC proliferation, regardless of genotype. Because the percent of mCherry+ cells was kept low, extracellular VEGF should remain high in the extracellular space of these cultures while the mCherry-Cre+ cells will experience intracellular loss of VEGF. These data therefore indicate that VEGF knockdown impairs stemness only in cells that have lost cell internal

VEGF despite high extracellular VEGF. In the future, this experiment will be repeated at least 2 more times. To build on this data, I am also currently analyzing the effect of internal

23 VEGFR2 inhibition on expression of stemness related genes. The results of these experiments will inform us of the effects of disrupting VEGF/VEGFR2 signaling on stemness gene expression. Taken together, these data will further our understanding of

NSPC-derived VEGF intracrine signaling in vitro.

NSPC-derived VEGF Knockdown in the DG

While our in vitro experiments suggest NSPC-derived VEGF signals in an intracrine manner, it is important that we test the mechanism of VEGF signaling in vivo as well, to better our understanding of NSPC function within the DG. To determine if

NSPC-derived VEGF signals cell autonomously, I chose the approach of knocking down

VEGF in a subpopulation of NSPCs and then tracking them over time to determine whether knockdown affected just the knockdown subset or also affected neighboring

NSPCs. In my first attempt, I injected lentiviral vectors expressing mCherry-Cre recombinase into the DG of VEGFlox/lox and WT mice. If NSPC-derived VEGF signals in an intracrine-manner, the number of mCherry+ progenitors would increase after 1 month in iKD mice, in comparison to control virus and WT mice. Additionally, we would expect to see a loss of mCherry+ RGLs after 1 month in these mice as well, and no effect on mCherry-negative NSPCs in VEGFlox/lox mice. If VEGF signals via extracellular autocrine mechanisms, mCherry-negative cells would experience the same increase in progenitor proliferation, and RGL loss, as seen in mCherry+ cells. Unfortunately, lentiviral expression varied tremendously, and the results of the experiment were unquantifiable.

Current ongoing experiments use a transgenic mouse model whereby inducible

NSPC-derived VEGF knockdown is coupled with Cre-dependent EYFP reporter

24 coexpression. Similar to above, I expect to see an increase in EYFP+ progenitors and a reduction of EYFP+ RGLs after 21 days in EiKD mice when compared to EWT mice if

EYFP is a reliable indicator of VEGF recombination. This would further confirm VEGF’s role in maintaining stemness in NSPCs. If there is no change in EYFP- progenitors and

RGLs at 21 days in EiKD mice, this would suggest that NSPC-derived VEGF signaling is intracrine. However, if EYFP- progenitors and RGLs follow the same trends as their

EYFP+ counterparts in EiKD mice this would suggest that NSPC-derived VEGF is autocrine (or that the assumption that EYFP+ = VEGF KD is not accurate). After 60 days, we expect to see complete exhaustion of the EYFP+ NSPC pool in EiKD mice when compared to EWT controls. Similar to the 21-day timepoint, no effect on the EYFP- NSPC pool, in EiKD mice, would indicate intracrine signaling, while comparable trends to the

EYFP+ NSPC pool would indicate NSPC-derived VEGF signaling is autocrine.

Importantly, this model relies on the assumption that NSPC-derived VEGF knockdown and EYFP expression occur at similar efficiencies and within the same cells. This assumption would be particularly important for interpreting data showing that EYFP+ and

EYFP- cells exhaust at similar rates—one possibility in this case is that VEGF signals in an extracellular autocrine fashion, but the other is that EYFP signal does not reliably indicate VEGF recombination within a single cell.

Recombination of a Fluorescent Reporter Does Not Accurately Predict Recombination of

Another Gene

It is common practice to use a transgenic mouse model where recombination of one gene is tracked via the recombination of a fluorescent reporter on another gene.44–46

25 However, it may be inaccurate to assume that recombination of both genes is occurring simultaneously in the same cells. To test this assumption, I bred a transgenic mouse line where I expression of two different fluorescent reporters in NSPCs was inducible by TAM treatment. If recombination of one fluorescent reporter accurately predicts recombination in the other, we would see coexpression of the reporters on a single cell level with high frequency. If not, the assumption that tracking one gene with the recombination of a fluorescent reporter on a different gene would be incorrect. I analyzed fluorescent reporter recombination in NSPCs of the SVZ and found that the fluorescent reporters recombined at different rates, despite their location in identical genetic loci. However, there was a correlation between fluorescent reporter expression, meaning mice that expressed one reporter highly were likely to have high expression of the other reporter. Despite these findings, fluorescent reporter overlap at the single cell level was significantly less than

100%. These findings indicate that the recombination of one fluorescent reporter did not accurately predict recombination in the other.

Finally, I examined fluorescent reporter recombination in NSPCs of the DG, the region of interest for my in vivo NSPC-derived VEGF knockdown experiments. Similar to the SVZ, I found that while overall recombination efficiencies of the fluorescent reporters appeared to correlate well, fluorescent reporter recombination overlap at the single cell level was significantly less than 100% in the DG, once again indicating that recombination of one fluorescent reporter does not accurately predict recombination in the other. In the future, I plan to analyze fluorescent reporter overlap in subpopulations of NSPCs, including RGLs and progenitors, to determine if there is a difference in recombination of the two fluorescent reporters between these cell types.

26 Taken together, these data imply that using fluorescent reporters to track recombination in a second gene at the single cell level is not a reliable methodology.

While it is possible for a strong intracrine effect to permeate through the inaccurate labeling by recombination, it is not likely that smaller effects would have enough power to do so. Additionally, it may be difficult to discern if NSPC-derived VEGF signaling is intracrine in my in vivo experiments if EYFP does not correctly label NSPCs with VEGF knocked down. This could lead to both false-negatives, where NSPC-derived VEGF is knocked down with no fluorescent labeling, and false-positives, where VEGF is not knocked down but the NSPC is EYFP+. Either occurrence would skew data analysis and interpretation towards showing no cell-specific effect of VEGF KD (i.e. appearance of autocrine signaling even if that were not the case). It is important to weigh these limitations when considering the use of a transgenic mouse model where recombination of one gene is tracked via the recombination of a fluorescent reporter on another gene.

Future Directions

This thesis first provides support for an intracrine role of VEGF in maintaining stemness in NSPCs and second provides considerable evidence to support that transgenic mouse models where recombination of a target gene is tracked via recombination-induced expression of an immunoflourescent reporter are inaccurate. This discovery has substantial implications for experimental design where gene recombination is tracked on a single cell level. While tracking gene recombination in this matter is common in the literature,44–46 it would be inappropriate to use this methodology when investigating NSPC-derived VEGF signaling in vivo. Without single cell resonance, it is

27 unlikely that I could accurately discern if NSPC-derived VEGF signals in an autocrine or intracrine manner. Therefore, we must adopt a different methodology to study this function. My original design utilized mCherry-Cre lentiviral injections into the DG of WT or

VEGFlox/lox mice. Had this been successful, we would have known the effects of VEGF knockdown in both infected and non-infected NSPCs as well as other cells of the neurogenic niche. This method failed in my hands due to inconsistent lentiviral infection.

In the future, I will revisit this methodology as a more exact measure for tracking NSPC- derived VEGF knockdown in vivo. In order to achieve more consistent lentiviral infection

I will reevaluate an appropriate virus titer. Additionally, I will address inconsistencies with the stereotaxic surgeries and reconfirm injection coordinates.

Summary

As our understanding of neural stem cell function continues to progress, the potential use of stem-cell based therapies to replace lost neurons becomes a reality.

While some therapies seek to target the endogenous NSPC population, others rely on the direct transplantation of exogenous stem cells. In both cases, the extracellular environment can be manipulated by treatment with growth factors in attempt to improve stem cell growth and survival to increase the therapy’s viability. However, without proper knowledge of how stem cells respond to extracellular growth factors, this may prove unsuccessful. In the scope of this thesis, our discovery that VEGF regulates NSPC survival in a cell autonomous manner – independent of extracellular VEGF – suggests that supplementing a stem cell therapy with extracellular VEGF would not impact stem

28 cell survival and be ineffective for the therapy in that regard. These findings highlight a need for detailed understanding of how NSPCs respond to local growth factors.

29 References

1. TAKAGI, Y. History of Neural Stem Cell Research and Its Clinical Application.

Neurol. Med. Chir. (Tokyo) 56, 110–124 (2016).

2. Clelland, C. D. et al. A functional role for adult hippocampal neurogenesis in spatial

pattern separation. Science 325, 210–213 (2009).

3. Kirby, E. D., Kuwahara, A. A., Messer, R. L. & Wyss-Coray, T. Adult hippocampal

neural stem and progenitor cells regulate the neurogenic niche by secreting VEGF.

Proc. Natl. Acad. Sci. U. S. A. 112, 4128–4133 (2015).

4. James, J. M., Gewolb, C. & Bautch, V. L. Neurovascular development uses VEGF-A

signaling to regulate blood vessel ingression into the neural tube. Dev. Camb. Engl.

136, 833–841 (2009).

5. Hogan, K. A., Ambler, C. A., Chapman, D. L. & Bautch, V. L. The neural tube

patterns vessels developmentally using the VEGF signaling pathway. Dev. Camb.

Engl. 131, 1503–1513 (2004).

6. Ruhrberg, C. et al. Spatially restricted patterning cues provided by heparin-binding

VEGF-A control blood vessel branching morphogenesis. Genes Dev. 16, 2684–

2698 (2002).

7. Carmeliet, P. et al. Abnormal blood vessel development and lethality in embryos

lacking a single VEGF allele. Nature 380, 435 (1996).

8. Rosenstein, J. M., Krum, J. M. & Ruhrberg, C. VEGF in the nervous system.

Organogenesis 6, 107–114 (2010).

9. Licht, T. et al. Reversible modulations of neuronal plasticity by VEGF. Proc. Natl.

Acad. Sci. U. S. A. 108, 5081–5086 (2011).

30 10. Raab, S. et al. Impaired brain angiogenesis and neuronal induced by

conditional homozygous inactivation of vascular endothelial growth factor. Thromb.

Haemost. 91, 595–605 (2004).

11. Merrill, M. J. & Oldfield, E. H. A reassessment of vascular endothelial growth factor

in central nervous system pathology. J. Neurosurg. 103, 853–868 (2005).

12. Jin, K. et al. Vascular endothelial growth factor (VEGF) stimulates neurogenesis in

vitro and in vivo. Proc. Natl. Acad. Sci. U. S. A. 99, 11946–11950 (2002).

13. Acker, T., Beck, H. & Plate, K. H. Cell type specific expression of vascular

endothelial growth factor and angiopoietin-1 and -2 suggests an important role of

astrocytes in cerebellar vascularization. Mech. Dev. 108, 45–57 (2001).

14. Licht, T. et al. VEGF is required for dendritogenesis of newly born olfactory bulb

interneurons. Dev. Camb. Engl. 137, 261–271 (2010).

15. Lee, S. et al. Autocrine VEGF signaling is required for vascular homeostasis. Cell

130, 691–703 (2007).

16. Gerber, H.-P. et al. VEGF regulates haematopoietic stem cell survival by an internal

autocrine loop mechanism. Nature 417, 954–958 (2002).

17. Matsumoto, T. et al. VEGF receptor-2 Y951 signaling and a role for the adapter

molecule TSAd in tumor angiogenesis. EMBO J. 24, 2342–2353 (2005).

18. Chatterjee, S. et al. Tumor VEGF:VEGFR2 autocrine feed-forward loop triggers

angiogenesis in lung cancer. J. Clin. Invest. 123, 1732–1740 (2013).

19. Dias, S. et al. Autocrine stimulation of VEGFR-2 activates human leukemic cell

growth and migration. J. Clin. Invest. 106, 511–521 (2000).

31 20. Koch, S. & Claesson-Welsh, L. by vascular endothelial growth

factor receptors. Cold Spring Harb. Perspect. Med. 2, a006502 (2012).

21. Ruch, C., Skiniotis, G., Steinmetz, M. O., Walz, T. & Ballmer-Hofer, K. Structure of a

VEGF–VEGF receptor complex determined by electron microscopy. Nat. Struct.

Mol. Biol. 14, 249–250 (2007).

22. Ewan, L. C. et al. Intrinsic tyrosine kinase activity is required for vascular endothelial

growth factor receptor 2 ubiquitination, sorting and degradation in endothelial cells.

Traffic Cph. Den. 7, 1270–1282 (2006).

23. Clegg, L. W. & Mac Gabhann, F. Site-Specific Phosphorylation of VEGFR2 Is

Mediated by Receptor Trafficking: Insights from a Computational Model. PLoS

Comput. Biol. 11, e1004158 (2015).

24. Dougher, M. & Terman, B. I. Autophosphorylation of KDR in the kinase domain is

required for maximal VEGF-stimulated kinase activity and receptor internalization.

Oncogene 18, 1619–1627 (1999).

25. Takahashi, T., Yamaguchi, S., Chida, K. & Shibuya, M. A single

autophosphorylation site on KDR/Flk-1 is essential for VEGF-A-dependent activation

of PLC-γ and DNA synthesis in vascular endothelial cells. EMBO J. 20, 2768–2778

(2001).

26. Eming, S. A. & Hubbell, J. A. Extracellular matrix in angiogenesis: dynamic

structures with translational potential. Exp. Dermatol. 20, 605–613 (2011).

27. Fujio, Y. & Walsh, K. Akt mediates cytoprotection of endothelial cells by vascular

endothelial growth factor in an anchorage-dependent manner. J. Biol. Chem. 274,

16349–16354 (1999).

32 28. Song, G., Ouyang, G. & Bao, S. The activation of Akt/PKB signaling pathway and

cell survival. J. Cell. Mol. Med. 9, 59–71 (2005).

29. Cunningham, S. A., Arrate, M. P., Brock, T. A. & Waxham, M. N. Interactions of

FLT-1 and KDR with phospholipase C gamma: identification of the phosphotyrosine

binding sites. Biochem. Biophys. Res. Commun. 240, 635–639 (1997).

30. Sun, Y. et al. Signaling pathway of MAPK/ERK in cell proliferation, differentiation,

migration, senescence and apoptosis. J. Recept. Signal Transduct. Res. 35, 600–

604 (2015).

31. Jopling, H. M., Howell, G. J., Gamper, N. & Ponnambalam, S. The VEGFR2

undergoes constitutive endosome-to-plasma membrane

recycling. Biochem. Biophys. Res. Commun. 410, 170–176 (2011).

32. Lampugnani, M. G., Orsenigo, F., Gagliani, M. C., Tacchetti, C. & Dejana, E.

Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from

intracellular compartments. J. Cell Biol. 174, 593–604 (2006).

33. Ehm, O. et al. RBPJkappa-dependent signaling is essential for long-term

maintenance of neural stem cells in the adult hippocampus. J. Neurosci. Off. J. Soc.

Neurosci. 30, 13794–13807 (2010).

34. Kawaguchi, D., Furutachi, S., Kawai, H., Hozumi, K. & Gotoh, Y. Dll1 maintains

quiescence of adult neural stem cells and segregates asymmetrically during mitosis.

Nat. Commun. 4, 1880 (2013).

35. Marqués-Torrejón, M. Á. et al. Cyclin-dependent kinase inhibitor p21 controls adult

neural stem cell expansion by regulating Sox2 gene expression. Cell Stem Cell 12,

88–100 (2013).

33 36. Gerber, H. P. et al. VEGF is required for growth and survival in neonatal mice. Dev.

Camb. Engl. 126, 1149–1159 (1999).

37. Babu, H. et al. A protocol for isolation and enriched monolayer cultivation of neural

precursor cells from mouse dentate gyrus. Front. Neurosci. 5, 89 (2011).

38. Lagace, D. C. et al. Dynamic Contribution of Nestin-Expressing Stem Cells to Adult

Neurogenesis. J. Neurosci. 27, 12623–12629 (2007).

39. Bolte, S. & Cordelières, F. P. A guided tour into subcellular colocalization analysis in

light microscopy. J. Microsc. 224, 213–232 (2006).

40. Babu, H., Cheung, G., Kettenmann, H., Palmer, T. D. & Kempermann, G. Enriched

monolayer precursor cell cultures from micro-dissected adult mouse dentate gyrus

yield functional granule cell-like neurons. PloS One 2, e388 (2007).

41. Cao, L. et al. VEGF links hippocampal activity with neurogenesis, learning and

memory. Nat. Genet. 36, 827–835 (2004).

42. Sun, M.-Y., Yetman, M. J., Lee, T.-C., Chen, Y. & Jankowsky, J. L. Specificity and

efficiency of reporter expression in adult neural progenitors vary substantially among

nestin-CreER(T2) lines. J. Comp. Neurol. 522, 1191–1208 (2014).

43. Witcher, K. G. et al. Traumatic brain injury-induced neuronal damage in the

somatosensory cortex causes formation of rod-shaped microglia that promote

astrogliosis and persistent neuroinflammation. Glia (2018). doi:10.1002/glia.23523

44. Dhaliwal, J. et al. Adult hippocampal neurogenesis occurs in the absence of

Presenilin 1 and Presenilin 2. Sci. Rep. 8, 17931 (2018).

34 45. Neckles, V. N. et al. A transgenic inducible GFP extracellular-vesicle reporter

(TIGER) mouse illuminates neonatal cortical astrocytes as a source of

immunomodulatory extracellular vesicles. Sci. Rep. 9, 3094 (2019).

46. Carrica, L. et al. Genetic inactivation of hypoxia inducible factor 1-alpha (HIF-1α) in

adult hippocampal progenitors impairs neurogenesis and pattern discrimination

learning. Neurobiol. Learn. Mem. 157, 79–85 (2019).

35