The Role of Gpr55 in Neural Stem Cell Proliferation, Differentiation, and Immune Responses to Chronic, Systemic Inflammation

THE ROLE OF GPR55 IN NEURAL STEM CELL PROLIFERATION, DIFFERENTIATION, AND IMMUNE RESPONSES TO CHRONIC, SYSTEMIC INFLAMMATION

A Dissertation

Submitted to

The Temple University Graduate Board

In partial fulfillment of the Requirement of the Degree

DOCTOR OF PHILOSOPHY

Jeremy D. Hill

December 2018

Examining Committee Members:

Yuri Persidsky, MD, PhD, Advisory Chair, Department of Pathology and Laboratory Medicine

Doina Ganea, PhD, Department of Microbiology and Immunology

Servio H. Ramirez, PhD, Department of Pathology and Laboratory Medicine

Seonhee Kim, PhD, Department of Anatomy and Cell Biology

Marcus Kaul, PhD, External Member, Division of Biomedical Sciences, University of California – Riverside School of Medicine

ii ABSTRACT

The cannabinoid system exerts functional regulation of neural stem cell (NSC) self- renewal, proliferation, and differentiation during both homeostatic and pathologic conditions. Recent evidence suggests that cannabinoid signaling is neuroprotective against reduction in NSC proliferation and neurogenesis caused by a multitude of conditions including injury due to HIV-1 associated neurotoxic proteins, neuroinflammation, and stroke. Yet not all effects of cannabinoids or cannabinoid-like compounds on neurogenesis can be attributed to signaling through either of the classical cannabinoid receptors CB1 or CB2. The recently de-orphaned GPR55 is targeted by numerous cannabinoid compounds suggesting GPR55 may be causing these aberrant effects. Activation of GPR55 has shown immune-modulatory effects outside the central nervous system (CNS) and anti-inflammatory actions on microglia, the resident immune cells within the CNS. New evidence has confirmed that both human and murine NSCs express functional levels of GPR55 yet the effects that GPR55 activation has on adult neurogenesis or NSC responses to inflammation has not been elucidated.

In the present study we sought to determine the role GPR55 signaling has on NSC proliferation and neurogenesis as well as possible neuroprotective mechanisms within the

NSC pool in response to inflammatory insult. Activation of GPR55 increased human

NSC proliferation in vitro as assessed by BrdU incorporation and flow cytometry.

Neuronal differentiation was also upregulated by signaling through GPR55 under homeostatic conditions in both human and murine NSC samples. Expression of NSC differentiation markers (nestin, sox2, GFAP, S100b, DCX, bIII-tubulin) in vitro was determined by immunohistochemistry, qPCR, and flow cytometry. In vivo, C57BL/6 and

iii GPR55-/- mice were administered the GPR55 agonist O-1602 (4 µg/kg/day) directly into the left hippocampus via stainless steel cannula connected to an osmotic mini-pump for a continuous 14 days. O-1602 treatment increased hippocampal NSC proliferation, survival, and immature neuron formation as compared to vehicle treated animals. These results were determined to be dependent on GPR55 activation as GPR55-/- animals did not show any response to agonist treatment. Interestingly, GPR55-/- mice displayed significantly reduced rates of hippocampal NSC proliferation and neuroblast formation as compared to C57BL/6 animals.

Chronic production of inflammatory mediators, such as IL-1b seen in neuroinflammation, to NSCs is known to reduce proliferation rates and attenuate neurogenesis both in vitro and in vivo. Addition of GPR55 agonists to IL-1b (10 ng/mL) treated human and murine NSC samples in vitro protected against reductions in neuron formation as assessed by immunohistochemistry and flow cytometry. Moreover, inflammatory cytokine receptor mRNA expression was down regulated by GPR55 activation in a neuroprotective manner. To determine inflammatory responses in vivo, we treated C57BL/6 and GPR55-/- mice with LPS (0.2 mg/kg/day) continuously for 14 days via osmotic mini-pump. Reductions in NSC survival (as determined by BrdU incorporation), immature neurons, and neuroblast formation due to LPS were attenuated by concurrent direct intrahippocampal administration of the GPR55 agonist, O-1602

(4µg/kg/day) in C57BL/6 mice but not in GPR55-/-mice. Neuroprotection by O-1602 treatment was not found to be microglia dependent as microglia activation was not altered by agonist administration. Molecular analysis of the hippocampal region showed a suppressed ability to regulate immune responses by GPR55-/- animals manifesting in a

iv prolonged inflammatory response (IL-1b, IL-6, TNFa) after chronic, systemic inflammation as compared to C57BL/6 animals.

Taken together, these results suggest a neuroprotective role of GPR55 activation on NSCs in vitro and in vivo and that GPR55 provides a novel therapeutic target against negative regulation of hippocampal neurogenesis by inflammatory insult.

v DEDICATIONS

To Nagehan, for whom I would not be in the position I am now. You have kept me on track to be the best scientist I can be and have shown me what a true partner in life is. It has not been the easiest time but I can think of no one I would rather go through this process with. I love you.

To TS, my beloved baby boy. Before you knew how to walk you knew what it meant to focus on a task and accomplish your goals. I’ll never have a better writing partner. You and your mother are what guide me to be my best.

To all my family. Your constant support, no matter what I chose to do, has meant the world to me. I am so happy that I am able to share this part of myself with you.

vi PERMISSIONS AND FUNDING

Figures 1-6, the corresponding results, and discussion were reproduced from the reference (Hill et al. 2018) with permission:

Hill JD, Zuluaga-Ramirez V, Gajghate S, Winfield M, and Persidsky Y.

Activation of GPR55 increases neural stem cell proliferation and promotes early adult hippocampal neurogenesis. Br J Pharmacol. 2018 August 175 (16) 3407-3421.

Jeremy Hill was partially supported by three National Institutes of Health grants: the

Center for Substance Abuse Research T32 training grant (T32DA007237-28 to Ellen

Unterwald) and through NIH-R37AA015913 and NIH-U01AA023552 to Yuri Persidsky.

vii ACKNOWLEDGEMENTS

First and foremost, I would like to thank my thesis advisor, Dr. Yuri Persidsky, for your guidance, trust, and insight during our time together. You have taught me more through your actions as the scientist you are than I ever expected. You challenged me personally, scientifically, and mentally in ways that I did not expect but greatly appreciate. I feel that working with you has prepared me to embark on the next chapter of my career with confidence and the knowledge that I have the capacity to, and will, make an impact.

I would like to thank my thesis committee, Drs. Doina Ganea, Servio H. Ramirez, and Seonhee Kim. Your insights and support during my time at Temple has been instrumental in preparing me for my career beyond the walls of MERB.

I would like to thank Dr. Marcus Kaul, my external examiner, for his time and effort on this project. You have always been a welcoming face at meetings and someone to bounce ideas off of whether they be scientific or career oriented.

I must thank, acknowledge, and bow to all the members, past and present, of the Persidksy lab. You have been the second family I have always wanted. A group of people who have each other’s backs both with work and personal matters. A shoulder to lean on, an ear to listen, or maybe just a chair to scoot down the aisle with. A beautiful mix of scientific prowess and simple hijinks to keep a person sane. You will all be missed.

I would also like to thank Drs. Slava Rom and Uma Sriram. It has been a pleasure to work with you and see your careers blossom the way they have.

I would like to thank all the members of graduate student island. Larisa, Steve, Lee Anne, and Nate. We may not have all been there at the same time but the support never diminished.

And lastly, Dr. Nicole Fernandes. You know who you are and the amazing force you’ve been in my, and my family’s, life. Here’s to a lifetime of bickering in the car.

viii TABLE OF CONTENTS

ABSTRACT……………………………………………………………………………..iii DEDICATION…………………………………………………………………………..vi PERMISSIONS AND FUNDING.…………………………………………………...... vii ACKNOWLEDGEMENTS……………………………………………………………viii LIST OF FIGURES………………………………………………………………….....xiii LIST OF TABLES………………………………………………………………………xv LIST OF ABBREVIATIONS…………………………………………………………..xvi

CHAPTER 1 – INTRODUCTION……………………………………………………….1

Adult neurogenesis………………………………………………………………..1

Reduced neurogenesis is associated with numerous pathologic conditions………5

Neuroinflammation………………………………………………………………..9

Chronic neuroinflammation and adult neurogenesis…………………………….11

The cannabinoid system………………………………………………………….16

Cannabinoids and inflammation…………………………………………………18

Cannabinoid receptor function on neural stem cells……………………………..20

GPR55……………………………………………………………………………25

GPR55 and inflammation………………………………………………………..28

Scientific rationale and specific aims……………………………………………30

CHAPTER 2 – METHODS

Cell culture……………………………………………………………………….32

Human neural stem cells…………………………………………………32

Mouse hippocampal neural stem cell isolation and culture……………...33

HEK293 and hGPR55HEK cell culture………………………………….33

ix Reagents………………………………………………………………………….33

Pharmacological treatments……………………………………………………...35

In vitro inflammatory treatments………………………………………………...35

Flow cytometry…………………………………………………………………..36

Analysis of neural stem cell and neurogenesis markers…………………36

Analysis of proliferation (BrdU)…………………………………………36

RT-PCR………………………………………………………………………….37

qPCR……………………………………………………………………………..38

Animals…………………………………………………………………………..40

Genotyping……………………………………………………………………….41

Intrahippocampal injection of O-1602…………………………………………...42

Systemic LPS administration…………………………………………………….42

BrdU injections…………………………………………………………………..43

Immunofluorescent staining……………………………………………………...43

Cell counts and quantification…………………………………………………...44

Microglial morphology and quantification………………………………………45

RNAseq…………………………………………………………………………..45

Statistical analysis………………………………………………………………..46

CHAPTER 3 – RESULTS

Expression of GPR55 in human neural stem cells……………………………….48

Primary murine hippocampal NSCs express GPR55…………………………….49

x Activation of GPR55 increases proliferation of human neural stem cells in vitro……………………………………………………………………………....50

GPR55 increases neuronal differentiation and reduces astrogliosis of neural stem cells in vitro………………………………………………………………………52

Administration of O-1602 promotes hippocampal neural stem cell proliferation in vivo……………………………………………………………………………….55

Neural stem cell survival and neuroblast formation is increased due to O-1602 administration within the hippocampus in vivo………………………………….56

Activation of GPR55 is protective against reduced neuron formation due to IL-1b in vitro…………………………………………….……………………………..58

Human neural stem cell cytokine receptor expression is altered by GPR55 activation after inflammatory insult with IL-1b…………………………………61

Activation of GPR55 on murine neural stem cells increases neurogenesis and is protective against reduced neuron formation due to IL-1b in vitro……………..64 mNSC cytokine receptor expression is altered by GPR55 activation after inflammatory insult with IL-1b………………………………………………….66

Direct intrahippocampal infusion of GPR55 agonist O-1602 is neuroprotective during chronic inflammation in vivo……………………………………………..69

Microglial activation is not attenuated by intrahippocampal administration of

GPR55 agonist O-1602 during chronic inflammation in vivo…………………...72

Lack of GPR55 induces altered inflammatory responses after 14 days of chronic, low-level systemic inflammation in vivo………………………………………...74

xi CHAPTER 4 – GENERAL DISCUSSION……………………………………………...78

Summary of conclusions…………………………………………………………93

Future directions…………………………………………………………………98

REFERENCES…………………………………………………………………………102

xii LIST OF FIGURES

Figure ID Page

Figure 1: Expression of GPR55 in human neural stem cells…………………………….48

Figure 2: Expression of GPR55 in primary murine hippocampal neural stem cells…….50

Figure 3: Activation of GPR55 increases proliferation of human neural stem cells in vitro……………………………………………………………………………………...51

Figure 4: GPR55 increases neuronal differentiation and reduces astrogliosis of neural stem cells in vitro ……………………………………………………………………….53

Figure 5: O-1602, a GPR55 agonist, increases murine NSC proliferation in vivo………55

Figure 6: O-1602 promotes adult murine hippocampal neurogenesis in vivo…………...57

Figure 7: Activation of GPR55 is protective against reduced neuron formation due to IL-

1b in vitro………………………………………………………………………………..59

Figure 8: hNSC cytokine receptor expression is altered by GPR55 activation after inflammatory insult with IL-1b………………………………………………………….62

Figure 9: Activation of GPR55 on murine neural stem cells increases neurogenesis and is protective against reduced neuron formation due to IL-1b in vitro……………………...65

Figure 10: mNSC cytokine receptor expression is altered by GPR55 activation after inflammatory insult with IL-1b………………………………………………………….67

Figure 11: Direct intrahippocampal infusion of GPR55 agonist O-1602 is neuroprotective during chronic inflammation in vivo……………………………………………………..70

Figure 12: Microglial activation is not attenuated by intrahippocampal administration of

GPR55 agonist O-1602 during chronic inflammation in vivo…………………………...73

xiii Figure 13: Lack of GPR55 induces altered inflammatory responses after 14 days of chronic, low-level systemic inflammation in vivo………………………………………75

xiv

LIST OF TABLES

Table ID Page

Table 1: Human qPCR primer/probe sets………………………………………………..39

Table 2: Mouse qPCR primer/probe sets………………………………………………...40

Table 3: Statistics for human inflammatory cytokine receptor expression…………..…..63

Table 4: Statistics for murine inflammatory cytokine receptor expression…………...... 68

Table 5: Statistics for hippocampal expression of inflammatory mediators……………..76

Table 6: Effects of GPR55 activation on human NSCs………………………………….94

Table 7: Effects of GPR55 activation on mouse NSCs………………………………….96

xv LIST OF ABBREVIATIONS

2-AG – 2-arachidonoylglycerol

ACSF – artificial cerebral spinal fluid

AD – Alzheimer’s disease

AEA – N-arachidonoylethanolamine

APP – amyloid precursor protein

BBB – blood brain barrier

BDNF – brain-derived neurotrophic factor bFGF – basic fibroblast growth factor

BrdU - 5-bromo-2’-deoxyuridine

CB1 – cannabinoid receptor 1

CB2 – cannabinoid receptor 2

CBD – cannabidiol

CNR1 – cannabinoid receptor 1 gene

CNR2 – cannabinoid receptor 2 gene

CNS – central nervous system

COX2 – cyclooxygenase-2

CREB – cAMP response element-binding protein

CSF – cerebral spinal fluid

CUS – chronic unpredictable stress

DAGL – diacylglycerol lipase

DCX – doublecortin

xvi DG – dentate gyrus

DISC-1 – Disrupted-in-schizophrenia 1 eCB – endocannabinoid

EGF – epidermal growth factor

EGFR – epidermal growth factor receptor

ERK – extracellular signal-regulated kinase

FAAH – fatty acid amide hydrolase

FGFR1 – fibroblast growth factor receptor 1

GFAP – glial fibrillary acidic protein

GRM1 – glutamate receptor, metabotropic 1 (mGluR1)

GRM5 – glutamate receptor, metabotropic 5 (mGluR5)

HD – Huntington’s disease

IFN – interferon (a, b, g)

IL-10 – interleukin 10

IL-10Ra - interleukin 10 receptor a

IL-1b – interleukin 1b

IL-1R1 – interleukin 1 receptor 1

IL-1R2 – interleukin 1 receptor 2

IL-6 – interleukin 6

IL-6st – interleukin 6 signal transducer (gp130)

IRAK-1 – interleukin 1 receptor associated kinase 1

JAK – janus kinase

xvii LPI – L-α-lysophosphatidylinositol

LPS – lipopolysaccharide

LTP – long term potentiation

MAGL – monoacylglycerol lipase

MAP2 – microtubule associated protein 2

MDD – major depressive disorder

MyD88 – myeloid differentiation primary response 88

NAPE – N-arachidonoyl phosphatidyl ethanol

NSC – neural stem cell

NFkB – nuclear factor-kappa B

NFAT – nuclear factor of activated T-cells

NO – nitric oxide

PD – Parkinson’s disease

PKA – protein kinase A

PKC – protein kinase C

PLCb - phospholipase-C b

PSA-NCAM – polysialylated form of the neural cell adhesion molecule

ROS – reactive oxygen species

SGZ – subgranular zone

STAT – signal transducer and activator of transcription

SVZ – subventricular zone

TGFb - transforming growth factor-b

xviii THC - Δ9-tetrahydrocannabinol

TLR – toll-like receptor

TNFa - tumor necrosis factor-a

TNF-R1 – tumor necrosis factor a receptor 1

TNF-R2 – tumor necrosis factor a receptor 2

TRAF6 – TNF receptor associated factor 6

WT – wild type

xix

CHAPTER 1 – INTRODUCTION

Adult neurogenesis

Adult neurogenesis, or the generation of new neurons in the adult brain, was first reported by Joseph Altman in 1965(Altman and Das 1965). Since this discovery, it is now well established that neural stem cells (NSCs) reside within the adult brain and are restricted to two primary, major neurogenic niches: the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) within the dentate gyrus (DG) of the hippocampus. The defining characteristics of NSCs are the capacity for self-renewal and the ability to specifically differentiate into the major cell types of the CNS; neurons, astrocytes, and oligodendrocytes (Goncalves, Schafer, and Gage 2016). Within the hippocampus, NSCs make up only a fraction of the entire cellular population yet the

SGZ, the thin area between the granule cell layer and the hilus, provides a unique microenvironment which allows NSC proliferation and promotes the differentiation of adult granule neurons (Goncalves, Schafer, and Gage 2016).

The capacity for NSCs to self-renew is necessary for the proper generation of new neurons in the hippocampus. In short, self-renewal of the NSC niche is the ability for mother cells to produce progeny (that can ultimately differentiate into either a neuron or glial cell) while maintaining a ‘stemness’ state and an uncommitted, undifferentiated phenotype (Bonaguidi et al. 2011). This is achieved through asymmetric division in which one cell will undergo the process of differentiation while the other retains the ability to divide and continues to produce daughter cells. It is important to note the difference between self-renewal and proliferation. Again, self-renewal is the ability to

1 continually produce offspring through asymmetric division while maintaining a

‘stemness’ state while proliferation only refers to the increase in the number of cells through either asymmetric or symmetric division. Progenies that begin to differentiate are then able to divide symmetrically thus producing two identical, cell-fate committed cells.

It is critical that a proper balance between self-renewal and differentiation is maintained to ensure stable hippocampal neurogenesis. NSC self-renewal is controlled intrinsically by stem cell specific ‘stemness’ genes including those controlled by Notch signaling

(Ahmed et al. 2009; Ables et al. 2011; Louvi and Artavanis-Tsakonas 2006). The differentiation of NSCs results from a gradual inactivation of self-renewal genes and the upregulation of pro-neural or pro-glial genes, a process which is tightly regulated by epigenetic variables (Ma et al. 2010).

The generation of adult granule neurons has tightly conserved developmental stages and Type 1 radial glia-like cells are thought to represent the true NSC population

(Goncalves, Schafer, and Gage 2016). These radial glia-like cells, characterized by combined expression of the molecular markers nestin, Sox2, and GFAP, divide asymmetrically to produce Type 2 transient amplifying cells, characterized by expression of nestin and Sox2 but not GFAP, which then continue to proliferate and give rise to doublecortin (DCX) expressing neuroblasts. Subsequently, neuroblasts further differentiate into fully mature glutamatergic granule neurons. During neuronal differentiation, cells migrate from the SGZ into the granule cell layer, project dendrites to form synapses with the entorhinal cortex, and extend mossy fibers toward the CA3 region of the hippocampus (van Praag et al. 2002). This process ensures integration of newly developed granule neurons into functioning neuronal circuits and promotes plasticity by

2 making synaptic connections with mature neurons (Ming and Song 2011). Proper integration of differentiating NSCs into the existing neuronal circuitry is critical for numerous processes such as learning, memory, and pattern separation (Christian, Song, and Ming 2014; Deng, Aimone, and Gage 2010; Sahay et al. 2011; Sahay, Wilson, and

Hen 2011). These processes are stringently regulated by numerous intrinsic factors including growth factor availability, the surrounding cellular environment, transcription factors, and endogenous signaling mechanisms.

Recent studies have suggested conflicting theories as to whether hippocampal neurogenesis exits in the adult human brain. The first evidence of hippocampal neurogenesis in the adult human brain was demonstrated by 5-bromo-2’-deoxyuridine

(BrdU) labeling of dividing cells with neuronal and astrocytic cell-type specific markers, such as NeuN and GFAP (Eriksson et al. 1998). BrdU-positive adult-born neurons and astrocytes were then detected using confocal microscopy. Since this seminal work, several independent laboratories have found evidence of adult hippocampal neurogenesis occurring in human tissue (Roy et al. 2000; Palmer et al. 2001; Boldrini et al. 2018). In addition, adult hippocampal neurogenesis has been found to be highly conserved across numerous mammalian species suggesting a significant role for hippocampal neurogenesis in brain function and behavioral outcomes (Patzke et al. 2015). Interestingly, across species, many studies have shown reductions of hippocampal neurogenesis during aging despite detection of molecular signals suggesting continuous NSC proliferation and neuron formation (Knoth et al. 2010; Spalding et al. 2013; Mathews et al. 2017).

Yet, even while accounting for decreases during aging, it may be difficult to determine adult hippocampal neurogenesis in the human hippocampus. The adult human

3 DG is ~500-150 mm3 and the number of new neurons identified per day by carbon dating is ~700 cells suggesting that adult neurogenesis in the hippocampus is sparse (Spalding et al. 2013; Dillon et al. 2017). A recent study by Boldrini et al. showed that healthy human brains maintained similar levels of neurogenesis from the ages of 14 to 79, suggesting higher neural plasticity in the human DG than was expected from previous studies

(Boldrini et al. 2018). In contrast to this, a study by Sorrells et al. came to a greatly different conclusion while using the same neurogenesis markers (DCX, PSA-NCAM) in human tissue (Sorrells et al. 2018). This study suggests that neurogenesis in the human

DG quickly decreases after birth and becomes undetectable before adulthood (Sorrells et al. 2018). So where do the discrepancies come from? One possible explanation would be differences in the duration of postmortem delay, fixation and sample preservation methodologies, and immunohistological staining protocols. The duration of postmortem delay is crucial for the detection of DCX and morphological signal (Boekhoorn, Joels, and Lucassen 2006). The study by Sorrells et al. used brain tissue which had longer delays in postmortem collection (up to 48 hours) as compared to the study by Boldrini et al. which could be a crucial factor in determining the number of adult-born neurons.

These two studies also had differences in the criteria for defining adult-born neurons.

Sorrells et al. defined only DCX+/PSA-NCAM+ cells as adult-born neurons claiming that DCX-/PSA-NCAM+ cells showed more mature morphological features and subsequently excluded this subset of cells from analysis. However, the developmental time course of adult-born neurons in the human brain has not been fully characterized and may take up to six months for the cells to fully mature into functional neurons (Kuhn,

Toda, and Gage 2018; Kohler et al. 2011). Furthermore, the current knowledge we have

4 of markers for adult neurogenesis is derived from rodent studies and we therefore do not know the exact expression time course of neuronal markers in the adult human hippocampus (Dayer et al. 2003; Jessberger et al. 2008; Andersen et al. 2014; Cahill et al.

2017). These discrepancies underscore a much needed criteria used to clearly determine the expression time course of neurogenesis markers in the human DG.

Altered neurogenesis is associated with pathological conditions

NSC differentiation has been confirmed to be altered under numerous pathological conditions such as Alzheimer’s disease (AD), Huntington’s disease (HD),

Parkinson’s disease (PD), stroke, schizophrenia, and mood disorders [major depressive disorder (MDD), bipolar disorder]. Studies of AD pathogenesis on NSC function have asserted that impaired neurogenesis is a common feature of transgenic mouse models of the disease and precedes neuronal loss. Most reported transgenic mouse models make use of overexpression of familial AD-associated mutant genes [amyloid precursor protein

(APP), presenilin ½ (PSEN1/2), Tau] which show both an increased age-dependent accumulation of amyloid-b peptides and an associated decrease in neurogenesis within the hippocampus (Rodriguez et al. 2008). This reduction in neurogenesis is thought primarily to be through down-regulation of NSC proliferation rather than reducing the rate in which NSCs are differentiating into mature neurons. Interestingly, analysis of human postmortem hippocampal samples of affected individuals in both early and late stages of AD has shown an increase in hippocampal NSC proliferation rather than a reduction as seen in murine studies (Boekhoorn, Joels, and Lucassen 2006). This observation was deemed to be due to a younger, presenile human population rather than

5 senile AD patients and that these brains had a more efficient response to deleterious events (Boekhoorn, Joels, and Lucassen 2006).

In the case of PD and HD, the specific alterations in neurogenic areas such as the dentate gyrus parallel the premotor symptoms (depression, anxiety) that are seen in the early stages of neurodegenerative disease (Simuni and Sethi 2008; Winner, Kohl, and

Gage 2011). Evidence suggests that dopamine depletion in PD patients and animal models of the disease reduces NSC proliferation both in the SVZ and SGZ, and that administration of dopamine receptor agonists can attenuate this effect (Van Kampen,

Hagg, and Robertson 2004; Hoglinger et al. 2004). As for HD, decreases in hippocampal

NSC proliferation and neuronal differentiation occur in numerous mouse and rat models of the disease (Gil et al. 2005; Simpson et al. 2011; Kandasamy et al. 2010). Of note, these reductions in proliferation and differentiation have been accompanied by an expansion of the quiescent NSC pool suggesting that the neurogenic process has stalled

(Kandasamy et al. 2010; Gil-Mohapel et al. 2011). The progressive declines in hippocampal neurogenesis in both PD and HD constitutes a common neuropathological feature that occurs early in the progression of these diseases and may play a role in hippocampal dysfunction and development of cognitive deficits characteristic for these diseases.

Ischemic stroke is characterized by an initial blood flow occlusion resulting in hypoxia and neuronal cell death followed by numerous secondary injury events including immune cell infiltration and inflammation. After stroke, NSCs within the SVZ and SGZ undergo extensive proliferation and functional neuronal differentiation in both rodents and humans (Arvidsson et al. 2002; Macas et al. 2006). These cells and neuroblasts

6 migrate toward the lesion site where they mature into neurons or die via apoptosis. It has been determined that this process occurs over the course of several months and is directed by the stromal derived factor 1a through its receptor CXCR4 (Thored et al. 2006).

Unfortunately, this increased number of proliferating NSCs quickly returns to pre- ischemia levels (Tanaka et al. 2004). Despite this increase in NSC proliferation, most of these cells prematurely die without repairing dead tissue and the underlying mechanisms for this phenomenon are as yet not fully determined, although there is a definite role of

CNS immune responses as well as infiltrating, peripheral immune cells.

Schizophrenia is a chronic, debilitating mental disorder characterized by a both positive (hallucinations, paranoia) and negative (cognitive deficits, decline in social interaction, lack of motivation) symptoms (Ross et al. 2006). Dysfunctional hippocampal neurogenesis and neuroanatomical and functional abnormalities within the hippocampus has been implicated in the symptomology of schizophrenia (Weinberger 1999).

Neuroimaging studies suggest that hippocampal volume and activity is significantly reduced in patients with schizophrenia as well as dysfunctional prefrontal-hippocampal connectivity (Heckers 2001; Vita et al. 2006; Sigurdsson et al. 2010). Studies of postmortem tissue from patients with schizophrenia have reported higher expression of the polysialylated form of the neural cell adhesion molecule (PSA-NCAM), a marker of immature neurons, in the hippocampus (Barbeau et al. 1995). Of note, Walton and colleagues identified an immature DG in patients with schizophrenia (Walton et al.

2012). This immature DG is characterized by greater NSC proliferation, an increase in the levels of markers for immature neurons, and a lack of markers for mature neurons

(calbidin). Mice with this type of immature DG displayed several behavioral traits that

7 reflect both positive and negative symptoms observed in human patients, including hyperactivity and deficits in social interaction and working memory (Walton et al. 2012).

Genetic models also support an important role for neurogenesis in the pathogenesis of schizophrenia. These studies focus on the gene Disrupted-in-schizophrenia 1 (DISC-1).

DISC-1 is mutated in patients with schizophrenia and is closely associated with other psychiatric disorders (Millar et al. 2000; Schumacher et al. 2009). DISC-1 is necessary for the development of hippocampal neurons and its mRNA is the highest expressed within the granule cell layer of the dentate gyrus in mice (Austin et al. 2004; Schurov et al. 2004). Dysfunction of DISC-1 was found to be associated with excessive acceleration of neuron formation followed by aberrant dendritic outgrowth, ectopic apical dendrites, and soma hypertrophy (Duan et al. 2007)

Dysfunction of hippocampal neurogenesis, and the necessity for therapeutic efficacy, has been studied at length in the context of mood disorders. Adult neurogenesis is required for some of the beneficial effects of antidepressants and in human subjects, hippocampal volume and adult hippocampal NSCs are reduced in depression while antidepressant treatment alleviate these changes (Santarelli et al. 2003; Campbell et al.

2004; Lucassen et al. 2010; Boldrini et al. 2012; Perera et al. 2007; Boldrini et al. 2013).

So, it is plausible that an increase in hippocampal neurogenesis mediates the effects of antidepressants although no consensus has been reached on the exact signaling mechanisms necessary for the observed alleviation of depressive-like symptoms.

Conversely, environmental challenges used to induce depressive-like phenotypes in animals (chronic mild stress, prenatal stress, chronic social defeat, early life stress) all impair adult hippocampal neurogenesis (Snyder et al. 2011; Mirescu, Peters, and Gould

8 2004; Santarelli et al. 2003; Surget et al. 2008; Coe et al. 2003; Gould et al. 1998). It may be that stressors create a vicious cycle in which stress impairs neurogenesis, low neurogenesis fails to mitigate stress, and further adult born neurons are lost (Toda et al.

2018). There has also been an observed decline in the action of neurotrophins such as brain-derived neurotrophic factor (BDNF), which aids in the regulation of hippocampal neurogenesis, in patients with mood disorders (Quiroz et al. 2010; Chiu et al. 2013). The risk of bipolar disorder has been linked to single nucleotide polymorphisms in the gene that codes for BDNF while patients with depression have low serum levels of BDNF (Liu et al. 2008).

Neuroinflammation

In biological systems, inflammation is a non-homeostatic response of vascular tissues to numerous stimuli such as pathogens, injury, or xenobiotics. Throughout evolution, inflammation was developed as a mechanism of self-defense and survival pertaining to both beneficial and detrimental outcomes depending on timing, cell types involved, and the severity of the insult (Kizil, Kyritsis, and Brand 2015). It is now widely accepted that neuroinflammation (the inflammatory process within the brain) plays a key role in the pathogenesis of various neurological disorders including AD, PD, and major depressive disorder (Aid and Bosetti 2011; Vivekanantham et al. 2015; Zunszain,

Hepgul, and Pariante 2013). Neuroinflammation was once thought to be a local tissue response without any, or at least very little, involvement of the peripheral immune system. This was due to the CNS being an immune privileged site and being separated from the peripheral immune system by the blood brain barrier (BBB). The concept of neuroinflammation has widened over the past number or years to include not only the

9 CNS response to altered homeostasis but also infiltration of cells of the innate and adaptive immune responses through the BBB into the CNS. Neuroinflammation can be triggered by various biological mechanisms including glial reactions, oxidative stress, and infiltration of peripheral leukocytes. All neuroinflammatory reactions start by the activation of the innate branch of the immune system. Within the CNS, the innate immune system is comprised primarily of myeloid-derived microglia which have the ability to recognize a spectrum of molecules, both endogenous as in the case of dying cells or foreign molecules from microorganisms (Konat, Kielian, and Marriott 2006).

Upon activation, microglia retract their processes, change from a stellate to ameboid shape, and release nitric oxide, reactive oxygen species (ROS), and chemokines and cytokines such as interleukin-1b (IL-1b), IL-6, IL-12, tumor necrosis factor-a (TNFa), and Type II Interferons (IFNs). These secreted molecules are used to attract peripheral immune cells (neutrophils, macrophages, T cells) to the site of injury and as the later stages of inflammation is prolonged, there is an extended prevalence of infiltrating cells and consequent resolution of inflammation over long periods of time (Kyritsis, Kizil, and

Brand 2014; Das and Basu 2008). This pro-inflammatory response by microglia is often termed the M1 response and it has been recognized that once triggered, this response may not cease and instead will maintain a chronic inflammatory state with possible long-term consequences. In addition to the M1 response, M2 microglia secrete anti-inflammatory molecules [transforming growth factor-b (TGFb), IL-10] which aid in suppression of inflammation until resolution of the inflammatory insult (Iadecola and Anrather 2011). In conjuncture with the increase in inflammatory state, the BBB becomes permeable enabling peripheral immune-cell infiltration. Although microglia are the predominant

10 immune cell in the CNS, astrocytes are also involved in the inflammatory process.

Astrocytres express innate immune pattern recognition receptors (TLRs, complement receptors) and contribute to the inflammatory response by producing pro-inflammatory cytokines (IL-1b, IL-6, TNFa, IFNg) chemokines, and ROS upon stimulation with the bacterial endotoxin LPS (Trapp et al. 1999; van Neerven et al. 2010; Farina, Aloisi, and

Meinl 2007; Lieberman et al. 1989). Any inflammatory insult to the CNS is associated with reactive astrogliosis which, in short, is a change in the morphology and hypertrophy of astrocytes followed by cytokine and complement C1q secretion leading to scar formation (Sofroniew 2009). Reactive astrogliosis is also associated with a number of neuropathologic diseases including AD, PD, and autism (Johnstone, Gearing, and Miller

1999; Sofroniew and Vinters 2010).

Chronic neuroinflammation and adult neurogenesis

Neuroinflammatory processes can either effect NSCs directly or indirectly through changes in the neurogenic niche with the end result being altered NSC proliferation, differentiation, or migration. The first reports of detrimental effects of neuroinflammation on NSCs were through studies utilizing the bacterial endotoxin LPS and demonstrating that LPS administration activated the resident microglia which in turn reduced neurogenesis rates in the SGZ (Monje et al. 2002; Ekdahl et al. 2003). It was determined that this effect was mediated through upregulated production of IL-6 by activated microglia (Monje, Toda, and Palmer 2003). Exposure of NSCs in vitro to recombinant IL-6 and TNFa dramatically decreased neurogenesis rates and it was shown that blockade of IL-6 was enough to attenuate the reductions seen (Monje, Toda, and

Palmer 2003). Chronic overexpression of IL-6 in astrocytes of young mice significantly

11 reduces the number of new neurons formed in the SGZ even though NSC distribution and glia formation rates remained normal (Vallieres et al. 2002). IL-6 has since been postulated as the pivotal cytokine responsible for suppression of neurogenesis. Further study into the role of IL-6 signaling in inhibition of neuron formation confirmed that the

Janus Kinase and Signal Transducer and Activator of Transcription (JAK/STAT) 3 pathway is essential for astrocytic differentiation from the NSC population (Namihira and

Nakashima 2013). Furthermore, signaling through the RAF/MEK/ERK pathway modulates the JAK/ STAT 3 pathway by regulating gp130 expression (the membrane bound co-activator of IL-6 signaling; IL-6st) in NSCs and consequently contributes to a switch from neurogenesis to astrocytogenesis(Li et al. 2012).

Yet, IL-6 is not the only cytokine with negative effects on adult neurogenesis. IL-

1b has been implicated by numerous groups as a key regulator of hippocampal neurogenesis. Within the SGZ, IL-1b acts directly on the NSC pool through interaction with IL-1R1 and reduced NSC proliferation both in vitro and in vivo (Arguello et al.

2009; Koo and Duman 2008). This effect was attributed to the activation of the nuclear factor-kappa B (NFkB) signaling pathway (Koo and Duman 2008). IL-1b is also implicated as having anti-neurogenic properties in association with neuropathologic conditions. Elevated levels of IL-1b has been demonstrated in patients suffering from

AD, in blood and CSF samples from depressed patients, and high levels of IL-1b are known to affect synaptic transmission causing learning impairments and memory dysfunction (Valero et al. 2014; Zonis et al. 2015; Yirmiya and Goshen 2011; Mossner et al. 2007; Raison, Capuron, and Miller 2006). Of note, memory impairment following

West Nile virus neuroinvasive disease is associated with reductions in hippocampal

12 neurogenesis and is due to chronic upregulation of IL-1b by activated astrocytes (Garber et al. 2018).

TNFa is upregulated in most immune responses as well as a myriad of neuropathological conditions. TNFa has anti-proliferative and anti-neurogenic actions within the NSC population as evidenced by reduced BrdU uptake and decreased microtubule-associated protein (MAP)-2 after treatment with recombinant TNFa in vitro

(Ben-Hur et al. 2003; Liu, Lin, and Tzeng 2005). These data suggested a probable inhibitory effect of neuronal survival and differentiation. Interestingly, the effect TNFa has on hippocampal neurogenesis is dependent up which receptor, TNF-R1 or TNF-R2, is activated demonstrating that the effects seen by administration of TNFa is not solely dependent on the ligand but also which specific receptor subtype is being targeted and which signaling pathways are triggered. Studies of TNFR1-/- and TNFR1/2-/- mice showed a significant increase in hippocampal NSC proliferation and neuroblast formation while TNFR2-/- mice showed the opposite effect, a significant reduction of neuron formation within the SGZ (Iosif et al. 2006). These data indicate TNFR1 signaling acts as a suppressor of NSC proliferation and differentiation while, conversely, activation through TNFR2 increases proliferation rates and survival of newly formed neurons

(Wang and Jin 2015). Further study utilizing irradiation injury showed TNFR2 activation to be more neuroprotective compared to the pro-inflammatory effects seen by TNFR1 signaling (Chen and Palmer 2013).

Type I (IFNa, IFNb) and Type II interferons (IFNg) are all potent pro- inflammatory cytokines produced after inflammatory insult or viral infection. Type I

13 interferons are primarily produced in the periphery by activated immune cells while the

Type II interferons can be produced by microglia within the CNS. Yet, microglia and astrocytes can both be targeted by either Type I or Type II interferons. Interestingly, even though the signaling pathways activated by interferons are conserved between microglia and astrocytes, the level to which these cells are activated is different (Li et al. 2018).

Type I interferons, specifically IFNa, has been shown to have a number of effects on the brain ranging from neurotoxicity to the development of behavioral phenotypes (Alboni et al. 2013; Felger et al. 2007; Hepgul et al. 2016). In particular, when IFNa has been used as a therapeutic treatment for chronic viral hepatitis, patients have increased depressive symptoms as well as other neuropsychiatric side effects such as anxiety, sleep disorders, and memory impairment (Raison, Capuron, and Miller 2006). Research has demonstrated in animal models that peripheral administration of IFNa negatively regulates NSC proliferation and neurogenesis within the hippocampus (Zheng et al. 2014). These findings indicate that the hippocampus is affected by IFNa and that the behavioral phenotypes arising from IFNa therapy may be the consequence of altered neurogenesis or neuronal function within the hippocampus. As for the Type II interferons, or IFNg, the evidence of the role IFNg plays on NSC function is controversial. In vitro studies of neurospheres showed that IFNg increased apoptosis and inhibited NSC proliferation

(Ben-Hur et al.). Interestingly, is was also reported that IFNg treatment increased the percentage of bIII-tubulin positive cells and that IFNg stimulation of microglia can achieve the same increase (Kim et al. 2007; Butovsky et al. 2006). Yet, not all data have supported a pro-neurogenic action of IFNg treatment. Recent data show that IFNg exerts a cytotoxic influence on NSCs by increasing caspase 3/7 activity and is anti-proliferative

14 (Walter et al. 2011). Further study by qPCR found that IFNg treatment could upregulate not only bIII-tubulin and MAP2a-c transcripts but also GFAP. Immunocytochemical analysis found that IFNg could drive this differentiation into an abnormal marker profile:

GFAP+/bIII-tubulin+, where these cells are functionally distinct from neurons and mature astrocytes (Walter et al. 2011). More recently, IFNg was shown to inhibit neurogenesis both in vitro and in vivo and these effects were dependent on the

JAK/STAT1 pathway and negative regulation of Neurog2, a critical initiator of neuronal differentiation (Ahn, Lee, and Kim 2015).

As mentioned previously, systemic inflammation that can be modeled by lipopolysaccharide (LPS) administration has been found to initiate numerous molecular signaling and cellular processes leading to pathological neuroinflammation (Valero et al.

2014; Qin et al. 2007; Godbout et al. 2005). Systemic LPS administration initiates an acute peripheral immune response which leads to BBB dysfunction through tight junction disruption (Persidsky et al. 2006; Qin et al. 2015). Once administered, LPS binds to a specific toll-like receptor, TLR4, which is expressed by monocytes, macrophages, and dendritic cells as well as resident microglia within the brain (Hornung et al. 2002;

Visintin et al. 2001; Lehnardt et al. 2003). Activation of TLR4 results in phosphorylation of numerous intracellular kinases culminating in the phosphorylation of IkB and release of NFkB which translocated to the nucleus and initiates the transcription of inflammatory genes cyclooxygenase-2 (COX-2), TNFa, IL-1b, and IL-6 (Dev et al. 2011; Brasier

2010; Chew, Takanohashi, and Bell 2006; Krakauer 2004). Through TLR4 signaling, microglia become activated and take on an M1 pro-inflammatory state and through prolonged production of inflammatory molecules can create a hostile environment for

15 neuronal survival and neurogenesis (Cunningham et al. 2005; Ekdahl et al. 2003; Monje,

Toda, and Palmer 2003). The increases in inflammatory mediators due to LPS administration within the CNS, and particularly within the hippocampus, lead to inhibition of NSC proliferation, decreases newborn neuron survival, and induces depressive-like behaviors in rodents (Tang et al. 2016). Interestingly, the results of decreased neurogenesis were correlated with induction of depressive-like behaviors suggesting that the depressive phenotype seen was due to negative regulation of hippocampal neurogenesis due to LPS insult (Tang et al. 2016). Unfortunately, even after inflammatory resolution evidence suggest a lasting impact of reduced neurogenesis. A single LPS-mediated inflammatory insult resulted in impaired spatial memory retention as well as cognition deficits demonstrating the serious impact of a single inflammatory insult within the brain (Valero et al. 2014).

The cannabinoid system

The cannabinoid system has recently been recognized for its role in NSC biology and adult neurogenesis. The cannabinoid system is made up of endocannabinoids (eCBs), plant-derived cannabinoids (phytocannabinoids), and their main target receptors, type-1

(CB1) and type-2 (CB2) G-protein coupled cannabinoid receptors. CB1 and CB2 receptors couple to either Gi or G0 classes of G proteins and their activation inhibits adenylyl cyclases and activates several mitogen-activated protein kinases inwardly rectifying potassium channels (Howlett et al. 2002). Activation of these receptors exerts diverse effects on cellular physiology, including synaptic function, gene transcription, and cell motility (Lu and Mackie 2016; Howlett et al. 2002). CB1 receptors are abundant in the

CNS, especially in cortex, cerebellum, and hippocampus, and are present on axon

16 terminals and preterminal axon segments (Mackie 2005; Nyiri et al. 2005). CB2 receptors on the other hand, are expressed at much lower levels in the CNS as compared to CB1.

These receptors are primarily present in microglia and vascular elements yet there is expression of CB2 by NSCs and some neurons under certain pathological conditions

(Molina-Holgado et al. 2007; Ramirez et al. 2012; Walter et al. 2003; Van Sickle et al.

2005; Viscomi et al. 2009).

The two best- characterized eCBs are N-arachidonoylethanolamine (anandamide,

AEA) and 2-arachidonoylglycerol (2-AG) while the most prominent phytocannabinoids are Δ9-tetrahydrocannabinol (THC, the main psychoactive component of Cannabis sativa) and cannabidiol (CBD) (Luchicchi and Pistis 2012; Pertwee 2012; Elsohly and

Slade 2005). Although both AEA and 2-AG contain arachidonic acid, their routes of synthesis and degradation are almost completely distinct (Pacher, Batkai, and Kunos

2006). AEA appears to be produced from N-arachidonoyl phosphatidyl ethanol (NAPE), whereas 2-AG is primarily produced by arachidonoyl-containing phosphatidyl inositol bisphosphate through sequential hydrolysis by phospholipase-C b (PLCb) followed by diacylglycerol lipase (DAGL) (Mackie 2005; Shonesy et al. 2015; Jung et al. 2007;

Murataeva, Straiker, and Mackie 2014). It is important to consider that in addition to serving as an endogenous cannabinoid ligand, 2-AG is an important metabolic mediator intermediate in lipid synthesis and serves as a major source of arachidonic acid in prostaglandin synthesis (Nomura et al. 2011). Also, synthesis of AEA has been proposed to occur through multiple pathways and this varies in distinct brain regions suggesting that different pathways may be favored for distinct physiological and pathophysiological processes (Mackie 2005; Lu and Mackie 2016). Degradation of AEA within the CNS is

17 primarily by the enzyme fatty acid amide hydrolase (FAAH) yet degradation of AEA has also been shown via oxidation by COX-2 to produce prostamides (Cravatt et al. 1996;

Woodward, Liang, and Krauss 2008). Primarily, 2-AG degradation is accomplished through three hydrolytic enzymes; monoacylglycerol lipase (MGL), alpha/beta domain- containing hydrolase 6 (ABHD6), and alpha/beta domain-containing hydrolase 12

(ABHD12) (Blankman, Simon, and Cravatt 2007). Similar to AEA, 2-AG can also be degraded through oxidation by COX-2 or hydrolyzed by FAAH (Hermanson et al. 2014).

The presynaptic location of CB1 receptors and their ability to inhibit synaptic transmission in conjuncture with the postsynaptic localization of eCB synthesizing enzymes suggest that eCBs act as retrograde messengers (Kano et al. 2009). Considerable evidence supports this idea and three basic forms of eCB-mediated synaptic plasticity involving eCBs acting as retrograde messengers have been described: 1) depolarization- inducing suppression of inhibition and depolarization-induced suppression of excitation;

2) metabotropic-induced suppression of inhibition or excitation, also known as eCB- mediated short-term depression; and 3) eCB-mediated long-term depression (Kano et al.

2009; Lu and Mackie 2016; Safo, Cravatt, and Regehr 2006).

Cannabinoids and inflammation

Cannabinoid ligands have been shown to downregulate inflammatory conditions in numerous experimental models as well as in ex vivo experiments. Ex vivo studies show that activation of the cannabinoid system is linked to reductions in inflammatory cell recruitment and enhanced anti-inflammatory cytokine production as well as downregulation of leukocyte migration, production of ROS, and release of proinflammatory cytokines (Kerr et al. 2013; Costola-de-Souza et al. 2013; Zoppi et al.

18 2014; Ortega-Gutierrez, Molina-Holgado, and Guaza 2005; Rockwell et al. 2008).

Synthetic cannabinoid agonists are able to mimic most of the anti-inflammatory effects seen by eCBs suggesting possible therapeutic potential in targeting cannabinoid receptors as well as receptors that are activated by cannabinoid and cannabinoid-like compounds.

Recent studies have demonstrated that cannabinoid receptors, especially CB2 receptors, are markedly increased in reactive microglia in response to inflammatory conditions within the CNS (Viscomi et al. 2009). The unexpected presence of CB2 receptors rather than CB1 in microglia is actually of no surprise since microglia are distinctly part of the immune system and myeloid in derivation. In line with evidence of anti-inflammatory consequences of cannabinoid system activation during times of inflammatory insult, activation of CB2 receptors in microglia has been reported to potentiate microglial proliferation and migration, while attenuating the release of pro- inflammatory mediators like IL-1b, IL-6, TNFa, and ROS (Walter et al. 2003; Ramirez et al. 2005; Kim et al. 2006; Dirikoc et al. 2007). Similar to microglia, astrocytes also play a pivotal role in central immunity through direct reaction to pathogens as well as secretion of cytokines and chemokines to influence other immune cells (Pekny and Pekna

2014). Few studies have addressed the expression profile of cannabinoid receptors on astrocytes yet, it is of interest that cultured astrocytes from different rodent species or brain regions show great variability of cannabinoid receptor expression. For instance, astrocytes cultured from rat brain express CB1 receptors while mouse astrocytes do not

(Blazquez et al. 1999; Sanchez et al. 1998). While focusing on the immunomodulatory role of astrocytes, activation of CB1 receptors on these cells restricts the production of inflammatory mediators NO and IL-1b (Molina-Holgado et al. 2002; Sheng et al. 2005).

19 Indeed, synthetic cannabinoid agonists were found to inhibit the expression of NO,

TNFa, and certain chemokines (CXCL10, CCL2, CCL5) in astrocytes that were stimulated by IL-1b and these effects were partially antagonized by both CB1 or CB2 receptor specific synthetic antagonists (Sheng et al. 2005).

Cannabinoid function and adult neural stem cells

Recent studies suggest a crucial role of eCBs on adult neurogenesis and NSC maintenance. NSCs express both CB1 and CB2 receptors, and as NSCs begin to differentiate CB1 receptor expression increases while CB2 expression is lost as these cells become mature neurons (Begbie, Doherty, and Graham 2004; Palazuelos et al. 2006).

Signaling through the CB2 receptor is implicated in increased NSC proliferation rates and the ability of these cells to self-renew through the mTORC-1 pathway (Molina-Holgado et al. 2007; Palazuelos et al. 2012) while NSCs deficient in CB2 receptors have impaired proliferation both in vitro and within the mouse hippocampus (Palazuelos et al. 2006).

The CB1 receptor has been associated with NSC differentiation and neurogenesis in that chronic treatment with the cannabinoid compound HU-210 increased rat hippocampal

-/- neurogenesis while CB1 mice display reduced rates of hippocampal neurogenesis

(Xapelli et al. 2013; Jiang et al. 2005; Jin et al. 2004). Cannabinoid receptor signaling also induces the production of neurotrophins, such as BDNF, and other molecules that control the proliferation and survival of newborn neurons (Harkany et al. 2008).

Voluntary exercise, a positive regulator of adult neurogenesis, increases AEA levels and promotes NSC proliferation within the hippocampus while blockade of the CB1 receptor with AM251 prevented increases in running-induced adult hippocampal neurogenesis

(Hill et al. 2010). Manipulation of the enzymes responsible for AEA and 2-AG synthesis

20 and degradation has also been shown to regulate neurogenesis. Genetic inhibition of

FAAH increases hippocampal neurogenesis while ablation of DAGLa/b decreases cell proliferation, survival, and the number of cells committed along a neuronal lineage within the DG (Diaz-Alonso et al. 2015; Gao et al. 2010; Jenniches et al. 2016; de Oliveira et al.

2018). Evidence from studies on phytocannabinoids, such as THC and cannabidiol, have shown conflicting results. Neither acute or chronic (3 week) treatment with THC elicited a change in NSC proliferation within the DG of adult animals (Campos et al. 2017).

However, extending the treatment paradigm of THC of 6 weeks did reduce proliferation within the hippocampus and animals also showed a simultaneous impairment in spatial memory (Wolf et al. 2010).

Interestingly, it has recently been reported that there is an interaction between

CB1 and CB2 receptor regulation of rat NSC proliferation and neurogenesis (Rodrigues et al. 2017). Of note, data presented by Rodrigues et al. demonstrate that SVZ and SGZ neurogenic niches respond differently to the same cannabinoid pharmacological treatment. Results obtained from studies using CB1 receptor antagonists or inverse agonists also demonstrate contradictory evidence. For example, Rimonabant® was shown to decrease DCX expression in the DG, yet other studies show CB1 receptor antagonism facilitates the proliferation and survival of hippocampal neuroblasts

(Jenniches et al. 2016; Campos et al. 2017; Lee et al. 2009). These data reveal that activation of either CB1 or CB2 receptors does not account for all effects seen after cannabinoid treatment of NSC populations, which suggests that cannabinoid-like ligands may activate multiple receptors within these niches (Prenderville, Kelly, and Downer

2015).

21 Recently, the cannabinoid system has been proposed to regulate the NSC niche within the hippocampus under numerous pathologic conditions. To begin, evidence suggests that NSCs have bi-directional cross-talk between the cannabinoid system (CB1 and CB2) and inflammatory cytokines (IL-1b, TNFa) that is necessary for NSC proliferation (Rubio-Araiz et al. 2008; Garcia-Ovejero et al. 2013). In the case of AD, postmortem brain samples show an increase in the levels of FAAH on plaque-associated astrocytes and an increase in CB2 receptor expression in the hippocampus and prefrontal cortex without changes to CB1 expression suggesting the involvement of CB2 in the pathogenesis of AD (Benito et al. 2003; Bedse et al. 2014; Solas et al. 2013). In an attempt to find therapeutics for AD, 15 days of chronic treatment with cannabidiol was found to counteract the Amyloid b-induced depletion of DCX in the hippocampus and stimulate basal neurogenesis in rats (Esposito et al. 2011). However, chronic treatment with the synthetic cannabinoid agonist HU-210 failed to improve cognitive deficits, impairments in contextual fear conditioning, and did not affect adult hippocampal neurogenesis in a double transgenic mouse model of AD (Chen et al. 2010).

During pregnancy, the recreational or medical use of cannabis has been associated with attention deficits, impaired learning and memory, and behavioral changes related to schizophrenia in offspring (Wu, Jew, and Lu 2011; Richardson, Hester, and McLemore

2016). However, the extent of these associations is still highly controversial and the effects of THC or synthetic cannabinoids on embryonic development are highly variable

(Fried 2002; Hall and Degenhardt 2009; El Marroun et al. 2011; Tortoriello et al. 2014).

According to several studies, CB1 and CB2 receptor function, as well as AEA and 2-AG level, are involved in the pathophysiology of schizophrenia (Fakhoury 2017). Cannabis

22 abuse during the critical period of adolescence is also associated with both positive and negative manifestations of psychosis and changes in eCB-related genes have been reported in the brains of patients with schizophrenia (Stefanis et al. 2004; Ujike et al.

2002; De Marchi et al. 2003; Martinez-Gras et al. 2006). Yet, very few studies have explored the link between schizophrenia, neurogenesis, and cannabinoids. One study demonstrated that a 2-week administration of the non-selective cannabinoid agonist WIN

55,212-2 to juvenile rats increased the survival of new-born cells and that these changes were accompanied by behavioral alterations that are potentially related to attention deficits, such as slow reaction time and increased novelty-seeking behaviors (Bortolato et al. 2014).

As mentioned above, the attenuation of hippocampal neurogenesis may facilitate anxiety and despair-related behaviors in rodents (Hollands, Bartolotti, and Lazarov 2016;

Revest et al. 2009). Yet, despite extensive pre-clinical evidence suggesting that cannabinoid signaling can regulate adult hippocampal neurogenesis, the exact mechanisms that connect cannabinoids, alterations in neurogenesis, and changes in behavioral phenotypes are still not fully understood. Recent studies have determined that circulating eCBs in human depressed patients are dysregulated. Serum concentrations of

2-AG and AEA have both been found to be significantly decreased as compared to matched controls, and that 2-AG levels were correlated with the length of the depressive episode; the longer the duration, the lower the 2-AG concentrations (Hill et al. 2009; Hill et al. 2008; Hillard and Liu 2014). In rodent studies, chronic unpredictable stress (CUS) has been implemented to induce depressive-like behaviors and subsequently study the core cellular and molecular mechanisms of depression (Willner 2017). In mimicking

23 depression, CUS impairs LTP and decreases the number of proliferating NSCs within the

DG and reduces neuronal differentiation (Li et al. 2006; Li et al. 2008; Zhang et al.

2015). Interestingly, blocking the degradation of 2-AG by MAGL produced antidepressant-like effects in rodents while also increasing hippocampal neurogenesis and

LTP within the DG (Zhang et al. 2015). Repeated administration of cannabidiol has also been shown to exert anxiolytic-like effects and reduce anhedonia (Campos et al. 2013).

These effects were suggested to be dependent on hippocampal neurogenesis since genetic ablation of proliferating progenitor cells in the hippocampus attenuated the anxiolytic- like effects of cannabidiol treatment (Campos et al. 2013). To further confirm an antidepressant-like action of cannabinoid-like compounds, chronic administration of the

CB1/CB2 receptor agonist HU-210 showed increased NSC proliferation within the DG and produced antidepressant-like effects in rats (Hill and Gorzalka 2005). Furthermore, transgenic mice with overexpression of CB2 receptors showed a decrease in depressive- like behaviors after being exposed to CUS with a concurrent increase in the expression of

BDNF within the hippocampus suggesting an increase in neuroplasticity (Garcia-

Gutierrez et al. 2010).

It has also been postulated that NSCs are protected from neuroinflammatory conditions as evidenced from results showing neuroprotection from inflammatory conditions arising due to Amyloid b plaques (Esposito et al. 2011). Activation of CB2 receptors rescued impaired hippocampal neurogenesis caused by chronic insult by HIV-1 neurotoxic protein gp120 further suggesting a neuroprotective role of cannabinoid-like ligands and cannabinoid receptor activation in the NSC niche (Avraham et al. 2014). In contrast with these results, it was found that pharmacological blockade of either CB1 or

24 CB2 receptors attenuated negative regulation of NSC proliferation within the hippocampus by repeated cocaine administration indicating that the cannabinoid system has numerous roles in regulating hippocampal neurogenesis under pathologic and inflammatory conditions (Blanco-Calvo et al. 2014).

GPR55

GPR55 has been considered a possible third cannabinoid receptor since the discovery that certain cannabinoid-like compounds activate this receptor even though the binding pocket of GPR55 does not share a “functional fingerprint” with CB1 or CB2 receptors (Petitet, Donlan, and Michel 2006) and the receptor itself shares a relatively low amino acid sequence homology with CB1 receptors (13.5%) and CB2 receptors

(14.4%) (Elbegdorj, Westkaemper, and Zhang 2013; Ryberg et al. 2007). The human

GPR55 gene was mapped to chromosome 2q37 and encodes a protein of 319 amino acids in length (Sawzdargo et al. 1999). GPR55 is a member of the d group of rhodopsin-like

(Class A) GPCRs and shares the closest homology with a selection of lysophospholipid- sensitive receptors in the purinergic subfamily (GPR23 30%, GPR92 30%, GPR35 27%) and the purinoreceptor P2Y5 (29%) (Fredriksson et al. 2003; Baker et al. 2006;

Sawzdargo et al. 1999). The human GPR55 protein has a relatively short N-terminus of only 24 amino acids, which contains a putative site for N-glycosylation (N5). Thorough examination of the receptor shows two protein kinase C (PKC) phosphorylation sites

(T315 and S317), three protein kinase A (PKA) phosphorylation sites (S134, T215,

S305), and another putative N-glycosylation site (N171) throughout the sequence. The C- terminus contains 27 amino acids and three of the potential PKA/PKC phosphorylation

25 sites are within this region (S305, T315, S317) (Sawzdargo et al. 1999; Balenga et al.

2011).

Recent evidence has demonstrated that GPR55’s endogenous ligand is L-α- lysophosphatidylinositol (LPI), a non-cannabinoid lipid signaling molecule (Oka et al.

2007). GPR55 is also thought to be activated by a number of endo-, phyto- and synthetic cannabinoid ligands including AEA, 2-AG, THC, CB1 receptor antagonists AM251 and

SR141716A (Rimonabant®), and the synthetic compounds, abnormal cannabidiol (Abn-

CBD), O-1602, and ML184 (Zhang et al. 2010; Ryberg et al. 2007; Johns et al. 2007;

Henstridge et al. 2010; Heynen-Genel et al. 2010). Activation of GPR55 by LPI induces a

2+ sustained, oscillatory Ca release pathway which is dependent on Gα13 and requires

RhoA activation (Henstridge et al. 2009; Sharir and Abood 2010). Further study has suggested that ligand binding to GPR55 also phosphorylates ERK1/2, CREB, activates the NFκB pathway, and translocates NFAT to the nucleus, yet these responses are ligand dependent which further complicates the pharmacology of this receptor (Henstridge et al.

2010; Henstridge et al. 2009). Differences in signaling mechanisms may be due to a wide distribution of GPR55 in the body.

Despite its widespread distribution, the physiological function of GPR55 remains to be fully elucidated. GPR55 shows high expression in certain regions of the CNS including the hippocampus, caudate, putamen, hypothalamus, cerebellum, thalamus, and pons. Within the CNS, blockade of GPR55 has been shown to increase the performance of mice in the T-maze paradigm suggesting a role for GPR55 in procedural memory

(Marichal-Cancino et al. 2016). GPR55 is also implicated as being a necessary aspect of axonal target innervation and nose-cone growth (Cherif et al. 2015). Targeting GPR55

26 reduces anxiety-like behaviors in rodents in numerous paradigms. In mice that went through acute stress, the GPR55 agonist O-1602 mediated anxiety-like effects in a medial orbital cortex dependent fashion (Shi et al. 2017). Two other groups found similar findings in that activating GPR55 reduces anxiety-like behaviors in both rats and mice and that antagonism increased these behaviors (Romero-Zerbo et al. 2017; Rahimi,

Hajizadeh Moghaddam, and Roohbakhsh 2015). Studies elucidating the role of GPR55 in the hippocampus have demonstrated microglial-dependent neuroprotective effects and

LPI-mediated increases in hippocampal CA1 and CA3 LTP (Kallendrusch et al. 2013;

Hurst et al. 2017).

Outside of the CNS, GPR55 has been implicated in a wide array of physiological functions. Activation of GPR55 inhibited mouse osteoclast formation and significantly reduced bone resorption in vivo (Whyte et al. 2009). In human patients, the GPR55/LPI system has been found to be positively associated with obesity in that obese patients with type 2 diabetes show significantly increased levels of GPR55 mRNA in adipose and hepatic tissue as well as increased circulating levels of LPI in blood as compared to lean subjects (Moreno-Navarrete et al. 2012). There also is evidence of localization of GPR55 on myenteric neurons within mouse and human colon tissue. Activation of GPR55 with

O-1602 reduced evoked contractions in muscle strips from the colon and injection of O-

1602 either i.p. or directly into the CNS slowed whole gut transit times further suggesting a role of GPR55 in GI physiology (Li et al. 2013).

27 GPR55 and inflammation

Since GPR55 has a wide dispersion throughout the body and has been suggested to be implicated in many physiological roles it is also likely that GPR55 is involved in pathophysiological processes as well. Signaling through GPR55 in peripheral tissues has demonstrated pro- and anti-inflammatory effects depending on cell type and cause of inflammation. Studies using gastrointestinal disease models in rodents discovered increased expression of GPR55 in the intestine of LPS treated animals (Lin et al. 2011).

GPR55-/- mice showed less severe colitis induced by dextran sulfate sodium as compared

-/- -/- to CB1 or CB2 mice suggesting a pro-inflammatory role of GPR55 (Schicho et al.

2011). Furthermore, GPR55-/- animals display reduced neuropathic hypersensitivity and inflammation in a mechanical hyperalgesia model using Freud’s complete adjuvant

(Staton et al. 2008). GPR55 has also been suggested to be responsible for neutrophil chemotaxis and recruitment via crosstalk with CB2 receptors (Balenga et al. 2011). In support of a possible role of GPR55 regulation of immune cells, it has also been reported that GPR55 is highly expressed on monocytes and natural killer (NK) cells and that activation of GPR55 in LPS treated monocytes and NK cells increases pro-inflammatory cytokine release and cytolytic activity (Chiurchiu et al. 2015). In contrast to these pro- inflammatory data, it was determined that GPR55 reduces the release of nerve growth factor on inflammation-activated mast cells and attenuates neuroinflammation and chronic pain from colitis induced by intracolonic administration of dinitrobenzenesulfonic acid (Cantarella et al. 2011; Borrelli et al. 2015). In the CNS, the endogenous agonist for GPR55 (LPI) also shows neuroprotective effects after excitotoxic lesion in a microglial and GPR55-dependent manner (Kallendrusch et al. 2013).

28 Furthermore, GPR55 mRNA expression in microglia is down-regulated after treatment with LPS or IFNg, suggesting that GPR55 is involved in neuroinflammation (Pietr et al.

2009). These data further suggest that GPR55 is a novel therapeutic candidate for treatment of inflammatory based diseases.

29 Scientific rationale and specific aims

Collectively, the literature shows that adult hippocampal neurogenesis is a highly regulated and necessary mechanism for proper memory integration, pattern separation, and mood regulation. These processes are interrupted and impaired during bacterial or viral infection, leading to increases in inflammatory mediators (cytokines, chemokines,

ROS) within the CNS which exert differential effects on NSC function at the molecular, cellular, and behavioral level. Recent evidence suggests a functional regulation of hippocampal neurogenesis by the cannabinoid system in terms of both NSC proliferation and neuronal differentiation. Additionally, activation of this system has shown anti- inflammatory effects both within the CNS as well as the periphery and specifically, a neuroprotective role on NSCs. These effects are mediated through both of the known cannabinoid receptors (CB1 and CB2). Yet, cannabinoid-like ligands exert effects that cannot be attributed to either CB1 or CB2. The recently de-orphaned GPCR GPR55 has been deemed a candidate cannabinoid receptor due to activity of numerous cannabinoid- like compounds and its expression throughout the body. Studies of GPR55 during inflammatory conditions have shown conflicting results suggesting that the immune- regulatory role GPR55 plays is cell type specific. However, GPR55 has never been studied in the context of hippocampal neurogenesis or in the possibility of acting as a therapeutic target during neuroinflammatory conditions. Therefore, studies detailing the role that GPR55 activation plays during both homeostatic and pathologic conditions are urgently needed. As such, we hypothesize that activation of GPR55 exerts functional regulation of neural stem cell proliferation and differentiation and offers protection against neuroinflammatory conditions. The specific aims of this study are as follows:

30 • Specific Aim 1: To evaluate the effects of GPR55 activation on NSC proliferation

and differentiation under homeostatic conditions. First, we determined that

GPR55 activation increases human NSC proliferation rates in vitro. Second, we

evaluated the ability of GPR55 activation to increase rates of human and murine

NSC neuronal differentiation in vitro. Third, we analyzed the effects of O-1602, a

selective GPR55 agonist, on murine hippocampal neuroblast formation and

proliferation rates within the SGZ in vivo.

• Specific Aim 2: To evaluate the effects of GPR55 activation on decreased NSC

neurogenesis and immune responses during neuroinflammatory insult in vitro.

First, we evaluated the ability of GPR55 activation to alleviate reduced

neurogenesis produced by inflammatory insult with IL-1β in vitro. Second, we

determined that signaling through GPR55 alters NSC immune receptor expression

(IL-1R1, IL-6st, TNFR2, IL-10Ra) after inflammatory insult with IL-1β in vitro.

• Specific Aim 3: To determine whether activation of GPR55 can protect against

reduced hippocampal NSC proliferation and neuroblast formation caused by

neuroinflammation in vivo. First, we assessed the neuroprotective ability of O-

1602 within the SCZ of the hippocampus after systemic inflammatory insult in

vivo. Second, we determined differences in immune and inflammatory responses

within the hippocampus between C57BL/6 and GPR55-/- mice using RNAseq due

to systemic LPS administration.

31 CHAPTER 2 – METHODS

Cell culture

Human neural stem cells

In vitro human NSC experiments utilized primary human NSCs (ReNcell VM) commercially obtained from Millipore Inc. (Billerica, MA, USA; Cat# SCC008,

RRID:CVCL_E921). The ReNcell VM cell line is an immortalized human NSC line derived from the ventral mesencephalon region of human fetal brain. These cells can differentiate into dopaminergic neurons, astrocytes, and oligodendrocytes after growth factor withdrawal (Donato et al. 2007). The hNSCs were regularly characterized by flow cytometry for the molecular markers nestin and Sox2. Undifferentiated cells were maintained and cultured in ReNcell NSC maintenance medium (Millipore) supplemented with epidermal growth factor (EGF; 20 ng/ml; Millipore) and basic fibroblast growth factor (bFGF; 20 ng/ml; Millipore). The cells were grown as an adherent monolayer on laminin-coated (mouse laminin, Millipore, 10 µg/ml in DMEM/F12 w/o HEPES, w/ L- glutamine) 75 cm2 cell culture flasks, 6-well plates, or cover slips at 37° C in a humidified atmosphere of 95% air and 5% CO2. Cells were passaged when flasks were

~80-90% confluent. Media was changed every 2-3 days. Differentiation of hNSCs was induced by removal of growth factors (bFGF, EGF) from the culture medium. Cells were passaged when ~80-90% confluent. Media was changed every 2-3 days.

32 Murine neural stem cell isolation and culture

Primary hippocampal NSCs were obtained from C57BL/6 and GPR55-/- mice at 4-6 weeks of age. Briefly, whole brains were removed and placed in ice-cold wash buffer

(30% glucose, 1M HEPES, penicillin/streptomycin in HBSS) on ice. Brains were washed in wash buffer and transferred to a coronal brain matrix where brains were cut into 1mm sections. The hippocampus was carefully sectioned out and cut into small pieces. Pieces were then digested in a collagenase solution (Collagenase type IV, Worthington

Biochemical, Lakewood, NJ, USA) for 30 minutes, further digested with trypsin for 10 minutes, and triturated. Dissociated cells were cultured at a density of 2x105 cells/mL in proliferation medium (DMEM/F12 (1:1) supplemented with 0.2% heparin, 1xB27 supplement (Gibco), 20 ng/mL mouse EGF, 10 ng/mL mouse bFGF, Pen/Strep, and L- glutamine). Neurospheres were allowed to grow for seven days with half medium changed every other day. For expansion as an adherent monolayer, neurospheres were dissociated with Accutase and plated on Matrigel (Corning, Corning, NY, USA) coated 6 well plates. Proliferation medium was changed every other day. To differentiate murine

NSCs, cells were passaged and plated on Matrigel coated 8-well chamber slides

(Millipore). Differentiation media consisted of DMEM/F12 (1:1) supplemented with

1xB27 supplement, 1xN2 supplement, penicillin/streptomycin, and L-glutamine. Media was changed every other day.

HEK293 and hGPR55HEK cell culture

HEK293 cells over-expressing human GPR55 (HEK293 hGPR55, Kerafast, Boston, MA,

USA; Cat# EIU003, from the laboratory of Dr. Ken Mackie, Indiana University) were used as a positive control for human GPR55 expression. HEK293 cells without hGPR55

33 were used as a negative control. HEK cells were grown in DMEM containing 10% FBS and 1% penicillin/streptomycin as an adherent monolayer at 37° C in a humidified atmosphere of 95% air and 5% CO2. Cells were passaged when ~80-90% confluent.

Media was changed every 2-3 days.

Reagents

5-Methyl-4-[(1R,6R)-3-methyl-6-(1-cyclohexen-1-yl]-1,3-benzenediol (O-1602, analog of cannabidiol and potent GPR55 agonist; Tocris Bioscience), 3-[[4-(2,3- dimethylphenyl)-1-piperazinyl]carbonyl]-N,N-dimethyl-4-(1-pyrrolidinyl)- benzenesulfonamide (ML184 [CID-2440433], synthetic GPR55 agonist; Cayman

Chemical, Ann Arbor, MI, USA), N-[4-[[(3,4-dimethyl-5- isoxazolyl)amino]sulfonyl]phenyl]-6,8-dimethyl-2-(2-pyridinyl)-4-quinolinecarboxamide

(ML193 [CID-1261822], GPR55 antagonist; Cayman Chemical), 4-{4,6-dihydro-4-(3- hydroxyphenyl)-3-(4-methylphenyl)-6-oxopyrrolo[3,4-c]pyrazol-5(1H)-yl]benzoic acid

(CID-16020046, GPR55 antagonist; Tocris Bioscience). Master stock solutions of O-

1602, CID-16020046, ML184, and ML193 were prepared in 100% dimethylsulfoxide

(DMSO) according to their solubility and manufacturer’s instructions. Working concentrations were generated in culture media and further diluted to final concentrations on the day of use. For in vivo experiments, master stock solutions of O-1602 were prepared in 100% EtOH. All master stock solutions were aliquoted and stored at -20°C.

Recombinant human and murine IL-1b (PeproTech, Rocky Hill, NJ, USA) was reconstituted in 0.1% BSA in sterile saline. Cytokines were then aliquoted and stored at -

20°C.

34 Pharmacological treatments

To investigate the effects of GPR55 activation on hNSC proliferation, cells were plated on laminin-coated 6-well plates. Cells were allowed to adhere overnight and then treated with LPI (1µM), the endogenous ligand for GPR55, or synthetic agonists, O-1602 (1µM) or ML184 (1µM), in a reduced growth factor media (5% growth factor). Reduced growth factor medium was utilized to better mimic a less proliferative phenotype while still maintaining a “stemness” state. Analysis by flow cytometry showed no significant reduction of nestin+ or Sox2+ populations after 48 hours(data not shown). Cells treated with the selective GPR55 antagonist ML193 (5µM) were pre-treated for 30 minutes prior to addition of agonist. Vehicle treated cells received 0.1% DMSO in 5% growth factor media. For differentiation studies, cells were treated with either vehicle, ML184 (1µM),

ML193 (5µM), or a combination of ML184 (1µM) and ML193 (5µM) in ReNcell medium that did not contain growth factors. Differentiation studies utilizing murine NSC samples were performed in 8-well chamber slides as described above. Cells were treated with O-1602 (2µM), CID16020046 (10µM), or a combination of O-1602 (2µM) and

CID16020046 (10µM) in differentiation medium listed above.

In vitro inflammatory treatments

Recombinant human or murine IL-1b was added to cultures at a dose of either 10 ng/mL

(for chronic experiments) or 100 ng/mL (for acute experiments). Doses were chosen based on previous literature showing deficits in neurogenesis in vitro as well as acute responses by NSCs(Borsini et al. 2017; Green et al. 2012; Zonis et al. 2015).

35 Flow cytometry

Analysis of NSC markers

For flow cytometric analysis, hNSCs were detached using Accutase (Millipore), washed, and resuspended at 1x106 per 100µL in PBS containing 2% bovine serum albumin (BSA)

(Sigma-Aldrich). Cells were fixed using intracellular (IC) fixation buffer (eBiosciences,

San Diego, CA, USA). For intracellular markers, cells were permeabilized after fixation using permeabilization buffer (eBiosciences) following the manufacturer’s instructions and incubated at room temperature for 30 min in antibodies against human human Sox-2

(BD Biosciences, San Jose, CA, USA, Cat# 561610 Lot# RRID:AB_10712763), nestin

(BD Biosciences, Cat# 560393 Lot#3305985 RRID:AB_1645170), S100β (Abcam,

Cat#ab196442 Lot#GR206303-4 RRID:AB_2722596), βIII-Tubulin (BD Biosciences,

Cat# 560394 Lot# 7132512 RRID:AB_1645400), DCX (BD Biosciences, Cat# 561505

Lot# 7269884 RRID:AB_10643766), and GFAP (Biolegend, San Diego, CA, USA; Cat#

644710 Lot# B249852 RRID:AB_2566685). Cytometric acquisition was performed using a BD FACS Canto II flow cytometer and analyzed with FlowJo software (Tree Star, Inc.,

Ashland, OR, USA; RRID:SCR_008520).

Analysis of proliferation by flow cytometry

The FITC BrdU Flow Kit (BD Biosciences Cat# 559619 Lot#6097660

RRID:AB_2617060) was used to determine the proportion of S-phase hNSCs. Cells were incubated with BrdU (10 µM) for the final 1 hour of a 48 hour exposure to GPR55 selective agonist/antagonist treatment. Cells were detached using Accutase (Millipore), fixed with BD Cytofix/Cytoperm buffer and treated with DNase to expose incorporated

36 BrdU. A FITC-conjugated anti-BrdU antibody and 7-amino-actinomycin D (7-AAD) were used to determine cell cycle phases (G0/G1, S, G2+M). Fluorescent signals from

FITC and 7-AAD/DNA complexes were detected on a BD FACS Canto II and analyzed using FlowJo (Tree Star, Inc.) software. hNSCs were first gated by cell area and width, and S-phase cells were defined as being FITC-BrdU+ and 7-AAD+. Results are displayed as percent of vehicle to control for variability between separate experiments.

RT-PCR

Total RNA from human NSCs was extracted using RNeasy® Mini kit (Qiagen, Valencia,

CA, USA) as per the manufacturer’s instructions utilizing QIAshredder spin columns and

DNase to reduce the possibility of PCR amplification of genomic DNA. Total RNA (2 mg) was converted to cDNA using high-capacity cDNA Reverse Transcription kit

(Thermo Fisher Scientific, Waltham, MA, USA). Human GPR55 was amplified using primers: 5’-ATC CAT GGC TTC AGC ACC TT -3’ and 5’-ATG GTG CAG ATC CCA

AA-3’, which yielded a product of 311bp. Human GAPDH was amplified using primers:

5’-TCC ACC CAT GGC AAA TTC CA-3’and 5’-TGG TTC ACA CCC ATG ACG AA-

3’ which yielded a product of 260 bp. Mouse GPR55 was amplified using primers: 5’-

AGA CCT TTG GGA TCT GCT GC-3’ and 5’- AGC TGA TGC CCT GCT TCA TT-3’ which yielded a product of 420bp. Mouse GAPDH was amplified using primers: 5’- CTC

ACT GGC ATG GCC TTC CG-3’ and 5’- ACC ACC CTG TTG CTG TAG CC-3’ which yielded a product of 292bp. The template was first denatured at 95°C for 2 minutes followed by 35 cycles (95°C for 45 sec, 57°C for 30 sec, and 72°C for 30 sec), then at

72°C for 2 min in an Eppendorf vapo protect thermal cycler. Aliquots (20 µL) of the PCR products were run on a 2% agarose gel containing 0.5 mg/ml ethidium bromide.

37 qPCR

Total RNA from human NSCs was extracted using RNeasy® Mini kit (Qiagen) as per manufacturer’s instructions. Total RNA (2 mg) was converted to cDNA using high- capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific). Specific primers and probes (TaqMan) for human and mouse genes were purchased from Life

Technologies (Thermo Fisher Scientific) and analyses were performed using the

StepOnePlus real-time PCR system (Thermo Fisher Scientific). For a full list of primer and probe sets see Table 1 (human) and Table 2 (mouse). Data for human samples were normalized to GAPDH while data for mouse samples were normalized to Rn18s.

Characterization data utilizing qPCR are reported as DCT (threshold cycle (CT) of the gene of interest minus the CT of the reference gene) in which a DCT of -1 corresponds to a doubling of the mRNA. qPCR analysis for in vitro differentiation studies is represented

-DDCt as fold change using the 2 method. Statistics for these studies were performed on DCT values. Samples were normalized to GAPDH while data for mouse samples were

38 normalized to Rn18s. Differentiation samples treated with vehicle were used as control for mRNA levels for each gene of interest.

Table 1: Human qPCR primer/probe sets

Target Assay ID Amplicon Length

CNR1 (CB1R) Hs00275634_m1 83

CNR2 (CB2R) Hs00952004_m1 108 EGFR Hs01076090_m1 57 FGFR1 Hs00241111_m1 81 GAPDH Hs02786624_g1 157 GFAP Hs00909236_m1 59 GPR55 Hs00271662_s1 80 GRM1 (mGluR1) Hs00168250_m1 102 IL-10Ra Hs00155485_m1 72

IL-1R1 Hs00991002_m1 152 IL-1R2 Hs01030384_m1 77 IL-6st Hs00174360_m1 72 MAP2 Hs00258900_m1 98 NES (Nestin) Hs00707120_s1 81 S100b Hs00902901_m1 96

Sox2 Hs01053049_s1 91 TNFR1 Hs01042313_m1 150 TNFR2 Hs00961749_m1 82 TUBB3 (bIII Tubulin) Hs00801390_s1 134

39 Table 2: Mouse qPCR primer/probe sets

Target Assay ID Amplicon Length

CNR1 (CB1R) Mm01212171_s1 66

CNR2 (CB2R) Mm00438286_m1 91 EGFR Mm01187858_m1 101 FGFR1 Mm00438930_m1 76 GPR55 Mm02621622_s1 102 GRM5 (mGluR5) Mm00690332_m1 97 IL-1R1 Mm00434237_m1 63 IL-1R2 Mm00439629_m1 65 IL-6st Mm00439665_m1 80 NES (Nestin) Mm00450205_m1 72 Rn18s Mm04277571_s1 115 SOX2 Mm03053810_s1 86 TNFR1 Mm00441883_g1 82 TNFR2 Mm00441889_m1 64

Animals

GPR55-/- mice were obtained from the Texas A&M Institute for Genomic Medicine (Dr.

Andrei Golovko). Generation of these animals is described in detail by Wu et al. (Wu et al. 2010). These animals have been backcrossed onto a C57BL/6 backbone for at least 10 generations. In short, mice heterozygous for the GPR55 deletion were inter-crossed to produce homozygous C57BL/6 GPR55-/- pups. A colony of all homozygous GPR55-/- animals was maintained at Temple University. All wild type C57BL/6 mice used in this

40 study were purchased from the Jackson Laboratory (Bar Harbor, ME). We did not detect significant differences in neural stem cell markers (Nestin, Sox2), extracellular signaling receptors (EGFR, FGFR1, GRM5), or cannabinoid receptors (CNR1, CNR2). All mice were 12-15 weeks of age at the beginning of the study and single-housed on a 12:12 light/dark cycle (7:00am – 7:00pm) at 22.1 ± 1°C with ad libitum access to food and water. Animals were randomly assigned to treatment group. Based on power analyses from previous experiments conducted by the laboratory we determined that a minimum of 6 animals per group would provide sufficient power to detect a significant difference with 95% confidence. All in vivo experiments were approved by the Temple University

Institutional Animal Care and Use Committee in accordance with guidelines based on the

National Institutes of Health (NIH) guide for care and use of laboratory animals and

ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines

(www.nc3rs.org.uk/arrive-guidelines).

Animal genotyping

For genotyping, DNA extraction from tail clips was obtained using the REDExtract-N-

Amp tissue PCR kit (Sigma-Aldrich) as per the manufacturer’s instructions. Samples were stored at -20°C. Supernatants were used as DNA templates for PCR reactions.

Samples were tested for 2 separate sets of primers to detect either the WT GPR55 allele

(Forward 5’-GCC ATC CAG TAC CCG ATC C-3’, Reverse 5’-GTC CAA GAT AAA

GCG GTT CC-3’; 441bp product) or the GPR55 mutant allele (Forward 5’-TCA AGC

TAC GTT TTG GGT T-3’, Reverse 3’-GCA GCG CAT CGC CTT CTA TC-3’; 301bp product). PCR cycle conditions were: 5 min at 95°C, 35 cycles of 30 sec at 95°C, 45 sec at 58°C, 1 min at 68°C, then 2 min at 68°C using standard PCR reagents.

41 Intrahippocampal injections

Animals were anesthetized with isoflourane and placed in a stereotaxic frame.

Stereotactic coordinates were calculated from Bregma (in mm) -2.0 anterio-posterior,

+1.5 medial-lateral, -1.5 dorso-ventral. Vehicle (artificial cerebral spinal fluid (ACSF,

Tocris Bioscience), 0.05% EtOH) or O-1602 (O-1602 diluted in 100% EtOH. Agonist was further diluted in ACSF with a final concentration of no more than 0.05% EtOH) was injected via stainless-steel cannula (Alzet Durect, Cupertino, CA, USA; brain infusion kit

3) connected through a polyvinyl tube to an osmotic mini-pump (Alzet Durect, model

1002). Pumps were primed in sterile saline overnight at 37° C to ensure infusion would begin upon implantation. Infusion period was a continuous 14 days. Infusion doses were set to 4 µg/kg/day. O-1602 was chosen for treatment in vivo due to ML184 being a piperazine and structurally similar to another benzoylpiperazine GPR55 agonist from

GlaxoSmithKline (GSK494581A) which is active at human GPR55 but not rodent

GPR55 (Brown et al. 2011).

Systemic LPS administration

LPS (O111:B4; Sigma Aldrich, St. Louis, MO, USA) was administered systemically via osmotic mini pump (Alzet Durect, model 1002) at a dose of 0.2 mg/kg/day for a total of

14 continuous days. Animals in the no insult group were implanted with osmotic mini pumps filled with sterile saline. Pumps with LPS or saline were implanted at the same time as pumps connected to cannulae and were set for subdermal administration. Low- dose continuous dispersion was chosen to better mimic a chronic, low-level systemic infectious state rather than an acute, high-level infection.

42 BrdU injections

Animals were injected with 100mg.kg-1 5-bromo-2’-deoxyuridine (BrdU; Sigma-Aldrich) i.p. twice per day for days 1 and 2 of the experimental schedule and once per day for days

3 and 4 for a total of six injections.

Immunofluorescent staining

Hippocampal samples were sectioned coronally on a Leica CM1860 cryostat (Leica

Biosystems, Wetzlar, Germany) into 30 µM sections. Samples were serially sectioned into 6 wells. All groups of sections were washed 3x with PBS to remove residual cryoprotectant. In vitro hNSCs attached to laminin on coverslips were fixed with 4% paraformaldehyde solution for 15 minutes and washed 3 times with PBS to remove residual paraformaldehyde. All samples were permeabilized with 0.1% Triton X-100

(Sigma-Aldrich) in PBS and blocked in 1% BSA (Sigma-Aldrich), 5% normal donkey serum (NDS; Jackson ImmunoResearch, West Grove, PA, USA) in PBS/0.1% Triton X-

100 for 2 hours at room temperature before they were incubated with primary antibodies.

Primary antibodies were diluted in blocking solution and samples were incubated overnight at 4°C. Sections or cells were then washed 4 times with PBS/0.1% Triton X-

100 and incubated with Alexa Fluor chromophore conjugated secondary antibodies

(1:400, Thermo Fisher Scientific) in blocking solution for 2 hours at room temperature in the dark. Samples were then washed 3 times in PBS/0.1% Triton X-100, once in PBS and mounted onto slides using antifade reagent with DAPI (4’,6-diamidino-2-phenylinodole,

Dilactate; Vector Laboratories, Burlingame, CA, USA Cat# H-1200 Lot#ZC0814

RRID:AB_233679). For BrdU labeling, free floating brain sections were pre-treated with ice cold 1 N HCl at 4°C for 10 minutes, 2 N HCl at 37°C for 30 minutes then washed

43 twice in 0.1 M borate buffer (pH 8.5) for 5 minutes each before beginning wash steps and blocking. Antibodies used were anti-Ki67 (Abcam, Cambridge, UK; Cat# ab15580 Lot#

GR264777 RRID:AB_443209), anti-DCX (Millipore Cat# AB2253 Lot# 2828588

RRID:AB_1586992), anti-BrdU (Abcam Cat# ab6326 Lot# GR267766-1

RRID:AB_305426), anti-βIII-tubulin (Sigma Aldrich Cat# T2200 Lot#028M4759V

RRID:AB_262133), anti-GFAP (Aves Labs, Tigard, OR, USA; Cat# GFAP

Lot#87867983 RRID:AB_2313547), and anti-IBA-1 (Wako Chemicals, Richmond, VA,

USA; Cat# 019-19741 Lot#WDK2121 RRID:AB_839504). Secondary antibodies used were Alexa-488® goat anti-guinea pig (Thermo Fisher Scientific, Waltham, MA, USA

Cat# A-11073 Lot#1841755 RRID:AB_2534117), Alexa-594® donkey anti-rat(Thermo

Fisher Scientific Cat# A-21209 Lot#45081A RRID:AB_2535795), Alexa-488® donkey anti-rabbit (Thermo Fisher Scientific Cat# A-21206 Lot#1608521 RRID:AB_2535792 ),

Alexa-488® goat anti-chicken (Thermo Fisher Scientific Cat# A-11039 Lot #1937504

RRID:AB_142924), Alexa-594® goat anti-chicken (Thermo Fisher Scientific Cat# A-

11042 Lot#1899511 RRID:AB_142803). Imaging for hNSCs was performed using a

CoolSNAP EZ CCD camera (Photometrics, Tucson, AZ, USA) coupled to a Nikon i80

Eclipse (Nikon Instruments Inc., Melville, NY, USA). Confocal images were taken on a

Nikon A1R confocal microscope (Nikon).

Cell counts and quantification

Based on a modified unbiased stereology protocol (Efstathopoulos et al., 2015; Malberg et al., 2000; Rossi et al., 2006), one out of every six adjacent sections was chosen and processed for immunofluorescence staining. The number of Ki67+, BrdU+, or DCX+ cells was then counted under a fluorescent microscope (CoolSNAP EZ CCD camera

44 (Photometrics) coupled to a Nikon i80 Eclipse (Nikon) in the area of the SGZ (defined as a two-cell layer in the borders of the granular cell layer, omitting those in the outermost focal planes) for a total of 12 coronal sections through the hippocampus. The total number of positive cells in the SGZ of the hippocampal dentate gyrus was estimated by multiplying the total positive cells per section by 6 and taking the sum of all 12 sections.

All counts were performed blinded to treatment group or genotype. The percentage of

BrdU+ cells also expressing DCX was determined by dividing the total number of

DCX+/BrdU+ cells per animal by the total number of BrdU+ cells and multiplying by

100.

Microglial activation and morphology analysis

Hippocampal sections were immunostained for IBA-1 as described above. Confocal 3D z-stack images were taken on a Nikon A1R confocal microscope at 0.5µM steps over a range of 24-26 µM per section. Z-stacks were analyzed using Imaris 8.1.2 (Bitplane;

Zurich, Switzerland RRID:SCR_007370) surface generation module to determine average microglial volume and surface area. Objects were identified with surface area detail of 0.2 µM and absolute threshold intensity of 1100. Results were filtered to exclude objects with surface area below 1000 µm2 and volume below 600 µm3 or above 1.6x104

µm3. Results are presented as means of surface area (µM2) or volume (µM3).

RNAseq

C57BL/6 or GPR55-/- mice were chronically treated with a low dose LPS (0.2 mg/kg/day) or saline via osmotic mini-pump for 14 days as outlined previously in methods. These animals were not implanted with cannula to determine immune responses within the

45 hippocampus without any disruption of the CNS. Animal numbers were as follows:

C57BL/6 + saline n=5, C57BL/6 + LPS n=4, GPR55-/- + saline n=5, GPR55-/- + LPS n=4.

At the end of the treatment schedule, animals were perfused with saline and brains were harvested. RNA was prepared using the tissue from the hippocampal region and gene expression was analyzed by RNAseq using the mouse inflammation and immunity transcriptome array (RMM005Z) from Qiagen. Data from primary analysis from Qiagen was uploaded onto GeneGlobe Data Analysis Center (Qiagen). Data was normalized for

Total Unique Molecular Index Count in each sample. This method was used to normalize the Unique Molecular Index count of every gene by the Total Unique Molecular Index count in each sample. Heatmaps were generated using this data to visualize changes in the proinflammatory profile. Specific genes within the LPS proinflammatory pathway were selected and bar graphs were plotted using the normalized Total Unique Molecular

Index Count for each gene.

Statistics

For all analyses, data was processed randomly and quantification was performed by experimenters who were blinded to experimental groups. All values are expressed as mean ± SEM. Analyses were performed using GraphPad Prism 7.0 software (San Diego,

CA, USA; RRID:SCR_002798). The significance of the differences between groups was evaluated by one-way analysis of variance (ANOVA) (followed by Tukey’s post-hoc test) or two-way ANOVA (followed by Tukey’s post-hoc test). Post hoc tests were only performed when F achieved p<0.05 and there was no significant variance in the homogeneity. p<0.05 values were determined to be statistically significant. Statistics for

46 qPCR data were performed on DCT values. Confidence intervals and p values for each are denoted for each statistically significant event.

47 CHAPTER 3 – RESULTS

Expression of GPR55 in human neural stem cells

First, we examined the expression of GPR55 mRNA in hNSCs by RT-PCR (Figure 1A). hNSCs were found to express GPR55 mRNA. HEK293 cells with stable overexpression of human GPR55 were used as a positive control while HEK293 without overexpression were used as negative control. Expression of GAPDH was used as a loading control to determine that equal amounts of cDNA were generated. We then sought to determine

GPR55 mRNA expression from hNSCs under both undifferentiating and differentiation

Figure 1. Expression of GPR55 in human neural stem cells. RNA from hNSCs was extracted and cDNA and PCR amplification were performed. GPR55 was amplified and yielded a product of 311 bp. GAPDH was used as control and yielded a product of 260 bp (A). HEK293 cells overexpressing human GPR55 were used as a positive control while HEK293 cells were used as a negative control. RNA for qPCR analysis (B-E) was isolated from whole cells of cultures either in undifferentiation conditions or after 10 days of differentiation conditions (removal of growth factors from culture medium). Shown are individual DCt values (n=5, each individual point refers to RNA extracted from a separate culture) for neural stem cell markers (B), extracellular signal receptors (C), and cannabinoid receptors (D). Expression of GPR55 in undifferentiated and differentiation conditions (10 days) was compared to expression of GPR55 by HEK293 hGPR55 overexpressing cells and HEK293 cells (E).

48 conditions in comparison to neural stem cell markers nestin and Sox2 (Figure 1B), extracellular signal receptors EGFR, FGFR1, GRM1 (mGluR1) (Figure 1C), and cannabinoid receptors CNR1 and CNR2 (Figure 1D). hNSC GPR55 mRNA was also analyzed in comparison to both hGPR55 HEK293 overexpressing cells and HEK293 without hGPR55 (Figure 1E). GPR55 mRNA expression did become detectable within

HEK293 samples but it should be noted that all raw CT values were above 36 (36 cycles).

GPR55 mRNA within undifferentiated hNSCs was found to be expressed at lower levels than EGFR, FGFR1, and CNR1 and at higher levels than mGluR1 and CNR2. After 10 days of culture in differentiating conditions cells showed reduced expression of nestin,

Sox2, EGFR, CNR2, and GPR55. Expression of CNR1 and GRM1 (mGluR1) were increased after 10 days of differentiation. These data suggest that hNSCs express GPR55 and that levels of expression of GPR55 mRNA are reduced upon differentiation.

Primary murine hippocampal NSCs express GPR55

The expression of GPR55 mRNA in hippocampus-derived primary NSCs was confirmed by RT-PCR (Fig. 2A). NSCs derived from the hippocampus of GPR55-/- mice showed no expression of GPR55 and were used as a negative control. GAPDH was used as a loading control for RT-PCR reactions. We further confirmed the lack of GPR55 in GPR55-/- mice using DNA derived from tail clips of both C57BL/6 and GPR55-/- mice (representative image in Fig. 2B). Genotyping data utilized a primer set for the WT GPR55 allele and

GPR55-/- mutant allele as describe by Wu et al (Wu et al. 2010). C57BL/6 mice displayed expression of the WT GPR55 allele while GPR55-/- mice did not. The opposite was found for expression of the mutant GPR55 allele. We subsequently characterized primary

49 hippocampal-derived NSC cultures to determine any differences between cultures from

C57BL/6 and GPR55-/- mice. qPCR analysis of GPR55-/- NSCs displayed no differences in NSC markers (Nestin, Sox2; Fig. 2C), extracellular signal receptors (EGFR, FGFR1,

GRM5 [mGluR5]; Fig 2D), or cannabinoid receptors (CNR1, CNR2; Fig 2E) as compared to C57BL/6 cultures (n=5 separate NSC harvests). GPR55 was not found via qPCR in samples derived from GPR55-/- animals.

Figure 2. Expression of GPR55 in primary murine hippocampal neural stem cells. RNA from primary hippocampal NSC cultures from C57BL/6 and GPR55-/- was extracted and cDNA and PCR amplification were performed. GPR55 was amplified and yielded a product of 420 bp. GAPDH was used as control and yielded a product of 292 bp (A). DNA was also extracted from tail clips of C57BL/6 and GPR55-/- mice to determine genotype (B). Two sets of primers were used to detect amplification of the WT GPR55 allele (441bp) or GPR55-/- mutant allele (301bp). RNA for qPCR analysis was isolated from whole cells of primary hippocampal NSC cultures derived from C57BL/6 and GPR55-/- animals. Individual data points represent separate NSC harvests; n=5. Shown are individual DCt values of neural stem cell markers (C), extracellular signal receptors (D), and cannabinoid receptors (E) for samples from C57BL/6 and GPR55-/- cultures.

Activation of GPR55 increases proliferation of human neural stem cells in vitro

Since cannabinoid compounds increase NSC proliferation rates (Palazuelos et al. 2012;

Molina-Holgado et al. 2007; Xapelli et al. 2013), we sought to determine if selective activation of GPR55 by its endogenous ligand LPI or synthetic cannabinoid compounds

Figure 3. Activation of GPR55 increases proliferation of human neural stem cells in vitro. hNSCs were plated on T25 flasks coated with laminin. Cells were allowed to adhere overnight prior to vehicle, agonist, or antagonist treatment. Vehicle refers to medium with 0.01% DMSO. Cells were then treated for 48 hours with GPR55 agonists (O-1602 1 µM, LPI 1 µM, ML184 1 µM), GPR55 antagonist ML193 (5 µM), or both. ML193 was added to cells 30 minutes prior to addition of agonists. BrdU (10 µM) was added for the final 1 hour of treatment. Cells were then collected and stained for BrdU incorporation and total DNA (7-AAD) for flow cytometric analysis. (A) Representative dot plots of vehicle, agonist, and antagonist treatment. Treatment of hNSCs with GPR55 agonists ML184 (B), O-1602 (C), and LPI (D) increased proliferation rates while pre- treatment with ML193 attenuated these effects. ML193 treatment alone did not alter proliferation rates. Data are displayed as percentage of cells in S phase for the last 1 hour of treatment as compared to vehicle control. Results are expressed as mean ± SEM; n=7 experiments. One-way ANOVA with Dunnet’s post hoc test were used to detect statistical significance.

O-1602 and ML184 (CID2440433) would elicit similar responses. hNSCs were treated with 1 µM LPI, O-1602, or ML184 for 48 hours either with or without the GPR55

51 antagonist ML193 (5µM). During the last hour of treatment, cells were pulsed with BrdU

(10 µM) to ascertain the total number of cells actively in the S-phase of the cell cycle.

Cells were collected, stained for BrdU and 7-AAD, run through a BD FACS Canto II flow cytometer, and analyzed using FlowJo software. Cells were determined to be actively within the S-phase if they showed positive staining for BrdU. G0/1, S-phase, and

G2 populations were determined by 7-AAD staining (Figure 3A). Activation of GPR55 by ML184 (Figure 3B), O-1602 (Figure 3C), and LPI (Figure 3D) significantly increased proliferation rates of hNSCs as compared to vehicle control (ML184, 142.9% ±5.1; O-

1602, 117.4% ±3.2; LPI, 116.3% ±2.8 as compared to vehicle control; n=7). These effects were attenuated by concurrent treatment with the selective GPR55 antagonist

ML193. ML193 treatment alone (CID1261822, 5µM; 102.9%±1.4; n=7), or in combination with agonist treatment (ML184+ML193, 98.2%±2.8; O-1602+ML193,

96.0%±4.1; LPI+ML193, 99.6%±1.6; n=7), did not significantly alter proliferation rates as compared to vehicle control.

GPR55 increases neuronal differentiation and reduces astrogliosis of neural stem cells in vitro

Next, we sought to determine the effects of GPR55 activation on hNSC differentiation in vitro. Previous reports indicate that cannabinoid signaling can induce neuronal differentiation through the CB1R or CB2R(Jin et al. 2004; Jiang et al. 2005; Rodrigues et al. 2017). hNSCs were cultured in differentiation medium with either vehicle, ML184 (1

µM), ML193 (5 µM) or ML184+ML193 for 10 days. Representative images of βIII- tubulin positive staining are shown in Figure 4 [vehicle (A), ML184 (B), ML193 (C),

ML184+ML193 (D), undifferentiated (E)]. Cells were also collected and stained for flow

Figure 4. GPR55 increases neuronal differentiation and reduces astrogliosis of neural stem cells in vitro. hNSCs were cultured under differentiating conditions (maintenance medium without bFGF and EGF) and treated with either vehicle (0.01% DMSO), the selective GPR55 agonist ML184 (1 µM), selective antagonist ML193 (5 µM), or a combination of ML184 and ML193 for 10 days. Representative images of vehicle- treated (A), ML184 (B), ML193 (C), ML184+ML193 (D) and undifferentiated (E) cells stained for βIII-tubulin (red), and DAPI (blue). Scale bars are 100 µM. (F, H) Representative histograms of flow cytometric analysis of βIII-tubulin and S100β, respectively. For flow cytometric analysis, cells were incubated with antibodies against human βIII-tubulin (APC) and S100β (Alexa-fluor 488). Cytometric acquisition was performed using a BD FACS Canto II flow cytometer and analyzed with FlowJo software. Treatment with ML184 increased expression of βIII-tubulin after 10 days, while ML193 attenuated these effects. ML193 treatment decreased the formation of neurons as compared to differentiated vehicle control. Quantitative analysis of βIII-tubulin+ cells (G) and S100β+ cells (I) as compared to differentiated control from flow cytometric analysis of five experiments. mRNA was derived from whole cells collected from each experiment and analyzed via qPCR for neuronal markers (βIII-tubulin, J; Map2, K) and astrocytic markers (S100β, L; GFAP, M). Data are represented as fold change of vehicle- treated cells under differentiating conditions for 10 days. Individual data points represent separate, individual experiments. One-way ANOVA and Dunnet’s post hoc test. cytometric analysis. Results showed a significant increase in the number of cells expressing βIII-tubulin after treatment with ML184 (117.4% ±3.3 as compared to vehicle control, n=5). These effects were mediated through GPR55 as concurrent treatment with

53 the GPR55 antagonist ML193 attenuated the increases in neuronal differentiation by

ML184(71.2% ±3.8, n=5). Interestingly, treatment with ML193 alone significantly reduced βIII-tubulin expression as compared to vehicle control (49.2% ±1.9, n=5). To determine effects of GPR55 activation on glial differentiation cells were analyzed via flow cytometry for the astrocytic marker S100b. Treatment with ML184 showed no significant changes in S100b from vehicle (101.1%±1.4, n=5). Treatment with ML193 showed a slight increase in S100b (105.5%±2.2, n=5) as did combination treatment of

ML184 with ML193 (108.7%±4.3, n=5), yet results were not significant. Representative histograms and quantitative analysis of these experiments is shown in Figure 4 (F,G, H, and I). Treatment with ML184 during differentiation also resulted in significant increases in expression of bIII-Tubulin (CI 0.075 – 1.622; p=0.029, n=5) and MAP2 (CI 0.225 –

1.586; p=0.007, n=5) mRNA (Figure 3 J, K), while treatment with ML193 or the combination ML184 plus ML193 showed no differences compared to vehicle control.

Treatment with ML184 was also significantly different from ML193 treatment for bIII-

Tubulin (CI -1.602 – -0.055; p=0.034, n=5) and MAP2 (CI -1.738 - -0.377; p=0.002).

ML184 treated samples also had significantly increased MAP2 expression as compared to ML184 plus ML193 samples (CI -1.763 - -0.402; p=0.001, n=5). Treatments did not significantly alter astrocytic marker mRNA expression (GFAP, S100b; n=5). These results suggest that signaling through GPR55 plays a significant role in the formation of new neurons from the NSC population without affecting glial differentiation.

54 Administration of O-1602 promotes hippocampal neural stem cell proliferation in vivo

12 - 15 week-old C57BL/6 (WT) and GPR55-/- mice were treated for 14 days with either vehicle (0.05% EtOH in ACSF) or O-1602 (4µg/kg/day) directly administered into the left hippocampus via cannula attached to an osmotic pump. Experimental time points are

Figure 5. Effects of O-1602, a GPR55 agonist, on murine NSC proliferation in vivo. C57BL/6(WT) and GPR55-/- mice were implanted with osmotic pumps sub-dermally set for a dispersion time of 14 days. Pumps were connected to brain infusion kits for direct infusion into the left hippocampus via continuous cannulation. Mice were treated with artificial cerebral spinal fluid + 0.05% EtOH (vehicle) or O-1602 (4 µg/kg/day). BrdU (100 mg/kg) was injected i.p. 2x per day for the first 2 days and 1x per day for days 3 and 4. Brain tissue was harvested at day 14. Schematic representation of experimental time points is displayed in (A). (B) Quantitative analysis of total Ki67 positive cells per dentate gyrus per mouse. Statistical analysis showed significant increases in Ki67+ cells within the SGZ of the dentate gyrus in O- 1602 treated WT animals as compared to vehicle controls; n=8 per group. Vehicle-treated GPR55-/- animals (n=6) displayed significantly lower Ki67+ cells in the SGZ as compared to WT vehicle animals. O-1602 treatment had no significant effect on GPR55-/- animals; n=7; one-way ANOVA and Tukey’s multiple comparisons test. (C) Representative confocal microscopy images depicting localization of Ki67 (green) within the dentate gyrus from 30 µm brain sections of WT and GPR55-/- mice. Scale bar = 200 µm. represented in Figure 5A. NSC proliferation within the SGZ of the dentate gyrus in the hippocampus after 14 days of continuous agonist treatment was determined by positive

55 staining for Ki67, a nuclear antigen expressed during all stages of the cell cycle except

G0(Fisher et al. 2002). The total number of Ki67+ cells in WT animals averaged 2567

±119.8 (n=8). Treatment with the GPR55 agonist O-1602 significantly increased the total number of Ki67 positive cells in WT mice (3648 ±330.7; n=8). Vehicle- treated GPR55-/- mice displayed a significantly reduced rate of proliferation as compared to vehicle-treated

WT animals (844 ±134.8; n=6). Agonist treatment had no effect on GPR55-/- mice as total Ki67 positive cells were not significantly different from vehicle-treated GPR55-/- animals (818 ±126.6). Quantitative analysis is shown in Figure 5B. Representative images of in vivo Ki67 staining are shown in Figure 5C. These data suggest that chronic treatment with O-1602 increases NSC proliferation within the dentate gyrus of the hippocampus after 14 days and that this effect is mediated by GPR55.

Neural stem cell survival and neuroblast formation are increased due to O-1602 administration within the hippocampus in vivo

To investigate if GPR55 agonist treatment can increase adult hippocampal immature neuron formation in vivo, 12-15 week-old C57BL/6 and GPR55-/- mice were treated with either vehicle (0.05% EtOH in ACSF) or O-1602 (4 µg/kg/day) as outlined in the proliferation study. A series of BrdU injections (100 mg/kg) were given for the first four days of the treatment schedule. Injections were given two times per day for the first two days and one time per day for days 3 and 4. After 14 days, immature neuronal markers and proliferating NSC survival were measured in the SGZ of the dentate gyrus in the hippocampus. Representative images of DCX+ and BrdU + staining within the dentate gyrus are shown in Figure 6A. As hypothesized, the total number of DCX-positive cells

Figure 6. Effects of O-1602 on adult murine hippocampal neurogenesis in vivo. C57BL/6(WT) and GPR55-/- mice were implanted with osmotic pumps sub-dermally and set for a dispersion time of 14 days. Experimental methods were the same as described for Figure 5. (A) Representative confocal microscopy images depicting localization of BrdU (red) and DCX (green) within the dentate gyrus of the hippocampus from 30 µm brain sections of WT and GPR55-/- mice. Scale bar = 100 µm. Arrows indicate BrdU+/DCX+ double labeled cells. Quantitative analysis of DCX (B), BrdU (C), DCX/BrdU (D) total positive cells per dentate gyrus per mouse. Analysis shows significant increases in DCX+, BrdU+, and DCX+/BrdU+ cells within the SGZ of the dentate gyrus after treatment with O- 1602 as compared to vehicle control; n=8 per group. Vehicle treated GPR55-/- animals (n=6) exhibit significant reduction in the number of DCX+, BrdU+, and DCX+/BrdU+ cells in the SGZ as compared to WT vehicle control animals. Treatment with O-1602 in GPR55-/- animals had no significant effect on the total number of DCX+, BrdU+, or DCX+/BrdU+ cells in the SGZ. (E) Analysis also determined that the percentage of total BrdU+ cells that were also DCX+ was increased in WT animals treated with O-1602, yet results were not significant; p=0.07. GPR55-/- animals demonstrated a significantly reduced rate of BrdU+ cells becoming DCX+ as compared to WT vehicle control. O-1602 treatment had no effect on this percentage as compared to vehicle-treated GPR55-/- mice. Bars represent mean ±SEM; one-way ANOVA and Tukey’s multiple comparisons test. were significantly increased after continuous treatment with O-1602 (12131 ±1077, n=8) as compared to vehicle treated WT mice (8139 ±603.4, n=8) (Figure 6B) BrdU+ cells were significantly increased in WT animals treated with O-1602 (2144 ±155, n=8) as compared to vehicle control mice (1553 ±144.2, n=8) (Figure 6C). Analysis of double labelled DCX+/BrdU+ cells in WT animals demonstrated a significant increase due to O-

57 1602 treatment (1217 ±72.92, n=8) as compared to vehicle (783 ±65.48, n=8)(Figure

6D). The percentage of total BrdU+ cells also expressing DCX, though not significant, was trending to increase as well (WT 51.00% ±1.71, n=8; O-1602 57.7% ±3.00, n=8, p=0.073) (Figure 6E). Meanwhile, vehicle treated GPR55-/- mice displayed significantly reduced BrdU+ (600.7 ±82.23, n=6), DCX+ (2808 ±834.4, n=6) and DCX+/BrdU+ cells

(190.0 ±37.36, n=6) as compared to WT vehicle treated animals. The percentage of

BrdU+ cells also expressing DCX was also significantly reduced in GPR55-/- mice

(31.67% ±3.923, n=6). O-1602 treatment had no significant effect on generation of immature neurons within GPR55-/- animals (BrdU+, 483.4 ±106.5; DCX+, 1793 ±382.1;

DCX+/BrdU+, 164.6 ±62.83, n=7) nor the percentage of BrdU+ cells also expressing

DCX (28.32% ±4.831, n=7). These data suggest that activation of GPR55 in vivo increases adult NSC differentiation along a neuronal lineage within the hippocampus.

Activation of GPR55 is protective against reduced neuron formation due to IL-1b in vitro

To determine the protective effects of GPR55 activation during neuroinflammation, we treated hNSCs for 10 days with 10 ng/mL recombinant IL-1b under differentiation conditions with or without ML184 (1 µM), a selective agonist for GPR55. To ensure that effects seen with ML184 were through activation of GPR55, we also treated cells with the GPR55 antagonist ML193 (5 µM). We determined the number of cells positive for the neuronal marker bIII-tubulin or glial marker GFAP using immunofluorescent staining and calculated the percentage of positive cells as the number of positively marked cells divided by the total number of DAPI nuclei in each image. Representative images are

Figure 7: Activation of GPR55 is protective against reduced neuron formation due to IL- 1b in vitro. hNSCs (ReNcell VM) were cultured under differentiating conditions for 10 days. Cells were treated with IL-1b (10 ng/mL) and either the GPR55 agonist ML184 (1 µM), GPR55 antagonist ML193 (5 µM), or both. Cells were then stained for neuronal (bIII- Tubulin; green) and glial (GFAP; Red) differentiation. Representative imaging is presented in A. Quantification of neurons and astrocytes derived from hNSC cultures are shown in B and C, respectively. For flow cytometry studies, after 10 days, cells were collected and stained for bIII-Tubulin (APC), DCX (PE), GFAP (Pacific Blue), and S100b (Alexa-488). Quantification of positive cells and representative histograms for bIII-Tubulin (D), GFAP (E), DCX (F), and S100b (G) are displayed. Bars represent mean ±SEM. Analysis was One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001; n=5 for IFA studies, n=6 for flow cytometry studies. Scale bar = 50 µM. presented in Figure 7A. Treatment with IL-1b significantly reduced the number of cells

59 positive for bIII-tubulin (0.994% ± 0.15) as compared to vehicle controls (3.918% ± 0.41;

F(4,20)=14.85, p<0.0001, n=5). Pre-treatment with ML184 significantly protected against reduction in bIII-tubulin positive cells (2.709% ± 0.36), while treatment with IL-

1b + ML193 (1.267% ± 0.24) or the combination IL-1b + ML184 + ML193 (1.218% ±

0.183) had no effect as compared to IL-1b treatment alone (Figure 7B). Unexpectedly, treatment with IL-1b reduced the number of GFAP positive cells (5.834% ± 0.44) as compared to vehicle treated controls (7.387% ± 0.51; F(4,20)=19.61, p<0.0001, n=5) yet this reduction was not significant (Figure 7C). ML184 treatment with IL-1b significantly diminished the number of GFAP positive cells (4.525% ± 0.31) as compared to vehicle.

IL-1b + ML193 (6.342% ± 0.46) and IL-1b + ML184 + ML193 (7.23% ± 0.22) did not differ from vehicle controls. Using flow cytometry, we obtained similar results as in the immunofluorescence studies and normalized separate experiments as percent of vehicle control to account for inter-experiment variability. For bIII-tubulin (Figure 7D), IL-1b significantly reduced the percent positive cells (26.63% ± 3.37; F(4,25) = 54.65, p<0.0001, n=6) as compared to vehicle controls, while treatment with ML184 significantly protected against this reduction (49.97% ± 6.64). Treatment of IL-1b +

ML193 (20.77% ± 2.26) or the combination IL-1b + ML184 + ML193 (22.21% ± 3.49) had no effect as compared to IL-1b treatment alone. GFAP positive cells trended in a similar manner to the IFA studies, yet results were not significantly different between groups (Figure 7E). Results for DCX (Figure 7F) showed similar trends as bIII-tubulin which is not surprising as DCX is a marker for immature, rather than mature, neurons.

DCX positive cells were significantly reduced with IL-1b treatment (33.28% ± 1.49;

60 F(4,25) = 41.07, p<0.0001, n=6) when compared to vehicle control. ML184 application protected against this reduction (60.04% ± 4.70) while treatment with IL-1b + ML193

(42.74% ± 3.93) or the combination IL-1b + ML184 + ML193 (42.92% ± 6.02) had no effect as compared to IL-1b treatment alone. As with GFAP, staining for the mature astrocyte marker S100b showed reduced numbers of positive cells for all treatments as compared to vehicle-treated controls. Yet unlike GFAP, results for S100b were statistically significant (F(4,25) = 20.25, p<0.0001, n=6) as represented in Figure 7G.

Treatment with ML184 (57.51% ± 2.49) also reduced the number of S100b positive cells significantly as compared to IL-1b treatment alone (74.63% ± 4.63).

Human neural stem cell cytokine receptor expression is altered by GPR55 activation after inflammatory insult with IL-1b

We subsequently investigated what, if any, effects activation of GPR55 had on pro- or anti-inflammatory cytokine receptor mRNA expression in hNSC. We chose to target receptors known to be expressed by NSCs within the hippocampus: IL-1R1, IL-1R2,

TNFR1, TNFR2, IL-6st (gp130), and IL-10Ra. hNSC cultures were treated with the

GPR55 agonist ML184 (1 µM) or antagonist ML193 (5 µM) 30 minutes prior to insult with IL-1b (100 ng/mL). For samples treated with the combination ML184 + ML193, antagonist was added to culture medium 30 minutes prior to agonist. Cells were collected

4 hours and 24 hours after addition of IL-b to the media. Neither treatment with ML184,

ML193, nor ML184 + ML193, had any significant effects on cytokine receptor mRNA levels at 4 hours or 24 hours, suggesting that activation of GPR55 does not have any

61 effect on immune responses of hNSCs in the absence of inflammatory mediators.

Statistics for hNSC inflammatory response experiments are listed in Table 3, n = 3 independent experiments. After treatment with IL-1b for 4 hours, no treatment

Figure 8: hNSC cytokine receptor expression is altered by GPR55 activation after inflammatory insult with IL-1b. hNSCs were treated with GPR55 agonist ML184 (1 µM), GPR55 antagonist ML193 (5 µM) or both 30 minutes prior to inflammatory insult with IL-1b (100 ng/mL) under proliferative conditions. Cells were collected after 4 or 24 hour exposure to IL-1b. Expression of cytokine receptor IL-1RI (A), IL-1RII (B), TNFR1 (C), TNFR2 (D), IL-6st (E), and IL-10Ra (F) mRNA was assessed by qPCR. The data are represented as fold difference (mean ± SEM) of expression where expression of specific receptors on vehicle-treated cells was assigned a value of 1. Statistics were performed on DCT values, Two-way ANOVA followed by Tukey’s post hoc test; *p<0.05, ** p<0.01, *** p<0.001 as compared to vehicle treated control; #p<0.05 as compared to IL-1b treated samples; n=3 independent experiments. combination showed a significant change in comparison to vehicle-treated samples for any of the cytokine receptors selected, with the exception of TNFR1 after treatment with

IL-1b + ML184 treatment. Results from 24-hour samples showed significant changes in receptor expression. After 24 hours of IL-1b, pre-treatment with ML184 significantly attenuated increased expression of mRNA for IL-1R1 and IL-6st as compared to IL-1b-

62 treated samples while at the same time increased expression of TNFR2 and IL-10Ra.

These results were mediated through GPR55 as concurrent treatment with ML193 attenuated all effects seen with administration of ML184, thus suggesting that activation of GPR55 during inflammatory insult with IL-1b in vitro is neuroprotective through

Table 3: Statistics for human inflammatory cytokine receptor expression (Figure 8)

Two-way Interaction Pharmacological Inflammatory treatment ANOVA (Pharmacological treatment (vehicle vs (saline vs IL-1b) treatment vs Inflammatory agonist) treatment) F(1,16) F(3,16) F(3,16)

IL-1R1 4 hr F = 0.0495, p=0.9849 F = 0.0484, p=0.9854 F = 0.9355, p=0.3478

IL-1R1 24 hr F = 3.414, p=0.0431 F = 2.735, p=0.0779 F = 24.98, p<0.0001

IL-1R2 4 hr F = 0.5996, p=0.6245 F = 0.7969, p=0.5135 F = 7.8, p=0.0130

IL-1R2 24 hr F = 0.4889, p=0.6948 F = 0.0349, p=0.9909 F = 2.494, p=0.1339

TNFR1 4 hr F = 0.729, p=0.5495 F = 0.7627, p=0.5314 F = 12.04, p=0.0032

TNFR1 24 hr F = 0.8552, p=0.8552 F = 1.041, p=0.4014 F = 0.0045, p=0.9475

TNFR2 4 hr F = 0.1745, p=0.9121 F = 0.694, p=0.5690 F = 7.684, p=0.0136

TNFR2 24 hr F = 2.729, p=0.0783 F = 4.28, p=0.0213 F = 26.03, p<0.0001

IL-6st 4 hr F = 0.5328, p=0.6663 F = 0.6336, p=0.6041 F = 0.0026, p=0.9599

IL-6st 24 hr F = 2.113, p=0.1387 F = 3.405, p=0.0434 F = 27.52, p<0.0001

IL-10Ra 4 hr F = 0.1841, p=0.9056 F = 0.0784, p=0.9708 F = 45.07, p<0.0001

IL-10Ra 24 hr F = 3.807, p=0.0311 F = 5.21, p=0.0106 F = 80.09, p=<0.0001

immune mechanisms not directly related to neuronal differentiation.

63 Activation of GPR55 on murine neural stem cells increases neurogenesis and is protective against reduced neuron formation due to IL-1b in vitro

We next sought to determine if GPR55 activation is protective against IL-1b-induced reduction in neurogenesis in mouse primary hippocampal NSC cultures. Hippocampal

NSCs were dissected out of 4-6 week-old C57BL/6 mice and cultured as an adherent monolayer. To determine if activation of GPR55 on mNSCs had effects similar to that seen with hNSCs, murine hippocampal cultures under differentiating conditions were treated with O-1602 (2 µM) for 5 days. Different pharmacological agents were utilized in murine experiments because ML184 has a piperazine structure which precludes this compound from activating the rodent receptor (Brown et al. 2011; Lingerfelt et al. 2017).

Samples were immunostained for bIII-tubulin and GFAP (Figure 9A) and assessed for total percentage of cells positively stained for either marker as performed with hNSC samples. O-1602 treatment significantly increased the percentage of bIII-tubulin positive cells (20.46% ± 0.92) as compared to vehicle treated samples (12.83% ±1.09; F(3,12) =

24.91, p<0.0001, n=4) as seen in Figure 9B. Consistent with results with hNSCs, blocking activation by the GPR55 antagonist CID-16020046 (10µM) significantly reduced bIII-tubulin positive cells (7.69% ± 0.76) as compared to vehicle. Combination treatment with O-1602 + CID-16020046 also resulted in reduced bIII-tubulin positive cells as compared to vehicle yet these results did not reach statistical significance. GFAP positive cells were reduced with O-1602 treatment (26.81% ± 1.15) in comparison to vehicle (32.94% ± 2.25; F(3,12) = 5.139, p=0.0163, n=4) yet not to a significant degree

(Figure 9C). Cells treated with O-1602 did demonstrate significant differences in GFAP positive cells when compared to both CID-16020046 treatment alone (37.39% ± 3.24) or

Figure 9: Activation of GPR55 on murine neural stem cells increases neurogenesis and is protective against reduced neuron formation due to IL-1b in vitro. Primary neural stem cells were harvested from the hippocampus of C57BL/6 mice and cultured under proliferative conditions as an adherent monolayer. To determine rates of neurogenesis, cells were plated in 8 well chamber slides under differentiating conditions (removal of bFGF, EGF, addition of N2 supplement) and treated with vehicle (0.01% DMSO in medium), the GPR55 agonist O-1602 (2 µM), GPR55 antagonist CID16020046 (10 µM), or both. For inflammatory conditions, murine IL-1b (10 ng/mL) was added. Antagonist was added 30 minutes prior to agonist, and agonist was added 30 minutes prior to IL-1b. Half media was replaced every other day for a total of 5 days of differentiation. Cells were then stained for neuronal (bIII-Tubulin; red) and glial (GFAP; green) differentiation. Representative imaging is presented in (A). Quantification of neurons and astrocytes derived from mNSC cultures under homeostatic conditions is shown in B and C, respectively. Quantification of neurons and astrocytes derived from mNSC cultures under inflammatory conditions is shown in D and E, respectively. Bars represent mean ±SEM. One-way ANOVA followed by Tukey’s post hoc test; *p<0.05, ** p<0.01, *** p<0.001 as compared to vehicle treated control; n=4 independent experiments. Scale bar = 50 µM. combination treatment with O-1602 + CID-16020046 (40.16% ± 2.22), yet these conditions did not differ significantly from vehicle-treated samples. Inflammatory insult

65 (10 ng/mL of IL-1b) decreased bIII-tubulin expression (4.93% ± 0.64; F(4,15) = 60.84, p<0.0001, n=4) while pre-treatment with O-1602 attenuated this reduction (10.84% ±

0.66)(Figure 9D). Pharmacological blockade of GPR55 during insult displayed similar results to IL-1b treatment alone (4.52% ± 0.39) or treatment with IL-1b plus agonist

(4.31% ± 0.41), suggesting that the neuroprotective effects of O-1602 are GPR55- dependent. As expected, treatment with IL-1b significantly increased the number of

GFAP positive cells (50.59% ± 1.75; F(4,15) = 20.56, p<0.0001, n=4) in comparison to vehicle, while GPR55 activation attenuated these effects (27.86% ± 1.76) as seen in

Figure 9E. Administration of CID-16020046 alone (47.84% ± 2.79) or in combination with O-1602 (43.84% ± 1.69) did not alter GFAP positive cell numbers in comparison to

IL-1b, yet was significant in comparison to IL-1b + O-1602 treated samples. In summary, we demonstrate for the first time that GPR55 activation in cultured murine hippocampal

NSCs increases neuronal differentiation under homeostatic conditions and is protective against reduced neurogenesis caused by insult with IL-1b.

mNSC cytokine receptor expression is altered by GPR55 activation after inflammatory insult with IL-1b

We next determined whether murine NSC cytokine receptor mRNA expression was altered by GPR55 activation as seen in the hNSC experiments. Again, we treated mNSC cultures with O-1602 (2 µM) or CID-16020046 (10 µM) 30 minutes prior to inflammatory insult with IL-1b (100 ng/mL). In the case of treatment with both O-1602 and CID-16020046, CID-16020046 treatment was applied to cells 30 minutes prior to O-

Figure 10: mNSC cytokine receptor expression is altered by GPR55 activation after inflammatory insult with IL-1b. mNSCs were treated with GPR55 agonist O-1602 (2 µM), GPR55 antagonist CID16020046 (10 µM) or both 30 minutes prior to inflammatory insult with IL-1b (100 ng/mL) under proliferative conditions as an adherent monolayer. Cells were collected after 4 or 24 hours of exposure to IL-1b. Expression of cytokine receptor IL-1RI (A), IL-1RII (B), TNFR1 (C), TNFR2 (D), and IL-6st (E) mRNA was assessed by qPCR. The data are represented as fold difference (mean ± SEM) of expression where expression of specific receptors on vehicle-treated cells was assigned a value of 1. Statistics were performed on DCT values; Two-way ANOVA followed by Tukey’s post hoc test; *p<0.05, *** p<0.001, **** p<0.0001 as compared to vehicle treated control; #p<0.05, ##p<0.01 as compared to IL-1b treated samples; n=3 independent experiments.

1602. Cells were collected at 4 or 24 hours post IL-1b treatment. In mNSC samples, I found a more rapid response in inflammatory cytokine mRNA upregulation due to IL-1b treatment as we did in the hNSC experiments. At the 4-hour time point, there was significant upregulation of all inflammatory cytokine receptor mRNA tested (IL-1R1, IL-

1R2, TNFR1, TNFR2, IL-6st) (Figure 10 A-E) with IL-1b insult as compared to non- insulted vehicle control samples. Without inflammatory insult, neither O-1602 nor CID-

67 Table 4: Statistics for murine inflammatory cytokine receptor expression (Figure 10)

Two-way Interaction Pharmacological Inflammatory treatment ANOVA (Pharmacological treatment (vehicle, (saline vs IL-1b) treatment vs Inflammatory agonist, antagonist) treatment) F(1,16) F(3,16) F(3,16)

IL-1R1 4 hr F = 2.769, p=0.0755 F = 3.304, p=0.0473 F = 136.6, p<0.0001

IL-1R1 24 hr F = 1.027, p=0.3957 F = 1.757, p=0.1782 F = 105.9, p<0.0001

IL-1R2 4 hr F = 0.1844, p=0.9054 F = 0.1499, p=0.9282 F = 666.1, p<0.0001

IL-1R2 24 hr F = 0.4742, p=0.7027 F = 0.1797, p=0.9092 F = 12.55, p=0.0014

TNFR1 4 hr F = 1.033, p=0.4043 F = 2.748, p=0.0770 F = 395.5, p<0.0001

TNFR1 24 hr F = 0.1306, p=0.9411 F = 0.0552, p=0.9826 F = 10.93, p=0.0026

TNFR2 4 hr F = 4.107, p=0.0297 F = 6.422, p=0.0067 F = 68.76, p<0.0001

TNFR2 24 hr F = 1.11, p=0.3904 F = 1.607, p=0.2490 F = 4.315, p=0.0645

IL-6st 4 hr F = 4.673, p=0.0158 F = 5.296, p=0.0100 F = 333.6, p<0.0001

IL-6st 24 hr F = 0.346, p=0.7923 F = 0.5819, p=0.6318 F = 40.25, p<0.0001

16020046 administration had any significant effect on inflammatory receptor mRNA expression suggesting, like with human cells, activation of GPR55 during non- inflammatory conditions either at 4- or 24-hour time points does not affect NSC immune responses. Interestingly, activation of GPR55 with O-1602 significantly attenuated the increase in IL-1R1 at 4 hours and 24 hours as compared to IL-1b treatment with or without antagonist. O-1602 treatment also attenuated IL-1b-induced increases in mRNA for IL-6st at 4 hours, but did not have significant effects at 24 hours even though there was a trend toward attenuated responses. Moreover, O-1602 significantly increased

TNFR2 mRNA as compared to IL-1b treated samples. CID-16020046 abrogated the effects seen with O-1602, again suggesting that these responses are due to GPR55

68 activation. All statistics for mNSC mRNA studies can be found in Table 4. Together, these data suggest that during insult with IL-1b, activation of GPR55 suppresses pro- inflammatory responses within NSCs.

Direct intrahippocampal infusion of GPR55 agonist O-1602 is neuroprotective during chronic inflammation in vivo

Next, we wanted to test not only if administration of O-1602 would be neuroprotective during a state of inflammation, but also if a chronic low dose of LPS administered systemically would be sufficient to alter hippocampal neurogenesis. We treated male and female 12-15 week old C57BL/6 and GPR55-/- mice with LPS via osmotic mini-pump at a dose of 0.2 mg/kg/day for 14 days. Animals were also implanted with a second mini- pump connected to a stainless-steel cannula which administered O-1602 directly into the left hippocampus at a dose of 4 µg/kg/day. BrdU was administered twice per day on treatment days 1 and 2 and once per day on days 3 and 4. Animals were harvested after

14 days of continuous treatment and the hippocampus was sectioned for immunohistological analysis. Hippocampal NSC proliferation was assessed by positive staining for Ki67, a nuclear antigen expressed during all stages of the cell cycle except

G0(Fisher et al. 2002). Survival of NSCs was assessed by positive staining for BrdU and neuroblast formation was determined by staining for doublecortin (DCX) within the SGZ of the dentate gyrus within the hippocampus. Representative images can be seen in

Figure 11A. Our results replicated data from our previous findings concerning administration of O-1602 into the hippocampus, i.e., O-1602 increased NSC proliferation

(Figure 11B; Interaction F(1,28) = 0.028, p=0.869; Inflammatory treatment F(1,28) =

Figure 11: Direct intrahippocampal infusion of GPR55 agonist O-1602 is neuroprotective during chronic inflammation in vivo. C57BL/6(WT) and GPR55-/- mice had osmotic pumps (Alzet) implanted sub-dermally for a continuous dispersion time of 14 days. Two pumps were implanted. The first pump contained vehicle or O-1602 and was connected to a stainless-steel cannula which was implanted into the left hippocampus. The second pump contained saline or LPS (0.2 mg/kg/day) and was implanted for systemic administration. (A) Representative images are shown depicting the dentate gyrus from 30 µm brain sections of 12-15-week-old WT and GPR55-/- mice immunostained with anti-BrdU(green) and anti-DCX antibodies(red). Scale bar = 50 µm. Quantitative analysis of Ki67 (B), BrdU (C), DCX (D), and DCX/BrdU (E) total positive cells per dentate gyrus per mouse. Bars represent mean ±SEM, n=6-8 mice per group. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 as compared to WT vehicle saline group; ##, p<0.01; ### p<0.001 as compared to WT vehicle LPS group; †, p<0,05; ††, p<0.01as compared to GPR55-/- vehicle saline group by Two-way ANOVA followed by Tukey’s post hoc test.

4.506, p=0.043; Agonist treatment F(1,28) = 21.9, p<0.0001), survival (Figure 11C

Interaction F(1,28) = 1.518, p=0.228; Inflammatory treatment F(1,28) = 7.341, p=0.011;

Agonist treatment F(1,28) = 30.57, p<0.0001), and immature neurons (Figure 11D

Interaction F(1,28) = 0.1577, p=0.694; Inflammatory treatment F(1,28) = 19.93, p=0.0001; Agonist treatment F(1,28) = 28.06, p<0.0001). There were also significant increases in the number of formed neuroblasts (Interaction F(1,28) = 1.023, p=0.321;

Inflammatory treatment F(1,28) = 12.02, p=0.0017; Agonist treatment F(1,28) = 28.91, p<0.0001). Interestingly, a low dose of chronic, systemic LPS negatively regulated neuronal differentiation and survival of NSCs. Animals treated with LPS had

70 significantly reduced cell numbers that were DCX+ (7218 ± 399.1), BrdU+ (1243 ±

87.79), and DCX+/BrdU+ (643.5 ± 57.76)(n=8) as compared to saline treated animals

(DCX+ (9133 ± 445.5), BrdU+ (1680 ± 89.56), and DCX+/BrdU+ (1176 ± 90.84) (n=8).

To our surprise, the number of Ki67+ cells within the SGZ were not significantly reduced due to LPS treatment (Saline 1734 ± 138.4; LPS 1410 ± 96.26). This suggests that animals treated with LPS become tolerant to the dose of LPS; data presented later corroborate this idea. Administration of O-1602 had significant neuroprotective effects when given to animals also receiving LPS. There were significant increases in the number of DCX+ (9525 ± 292.3), BrdU+ (2032 ± 182.1), and DCX+/BrdU+ (1403 ±

167) (n=8) cells as compared to animals given LPS with vehicle administration into the hippocampus. Similar to our previous results, GPR55-/- animals displayed significantly reduced rates of NSC proliferation (Ki67+, 890 ± 110.5), BrdU incorporation (BrdU+,

390.8 ± 65.5), immature neurons (DCX+, 1859 ± 220.7), and neuroblast formation

(DCX+/BrdU+, 209.6 ± 35.9)(n=6) as compared to C57BL/6 animals. This current experiment also shows that O-1602 treatment did not alter neurogenesis markers as compared to saline-treated GPR55-/- animals (Ki67+, 837 ± 55.1; BrdU+, 417.1 ± 60.2;

DCX+, 1758 ± 94.7; DCX+/BrdU+, 231.1 ± 28.7)(n=7). LPS treatment of GPR55-/- mice showed significant reductions in immature neurons (DCX+, 1282 ± 52.3; Interaction

F(1,22) = 0.0012, p=0.972; Inflammatory treatment F(1,22) = 19.03, p=0.0002; Agonist treatment F(1,22) = 0.631, p=0.435) and neuroblast formation (DCX+/BrdU+, 81.2 ± 9.1;

Interaction F(1,22) = 0.242, p=0.627; Inflammatory treatment F(1,22) = 42.02, p<0.0001;

Agonist treatment F(1,22) = 0.156, p=0.6968), yet results for proliferation (Ki67, 560.3 ±

102; Interaction F(1,22) = 0.104, p=0.749; Inflammatory treatment F(1,22) = 11.66,

71 p=0.0025; Agonist treatment F(1,22) = 0.1197, p=0.733) and survival (BrdU+, 285.6 ±

40.3; Interaction F(1,22) = 0.030, p=0.864; Inflammatory treatment F(1,22) = 5.837, p=0.0244; Agonist treatment F(1,22) = 0.157, p=0.696)(n=6), although reduced, were not significant as compared to saline-treated GPR55-/- animals. O-1602 also did not have any effect in any conditions during LPS treatment (Ki67+, 558.3 ± 78.9; BrdU+, 296.7 ±

29.8; DCX+, 1182 ± 85.2; DCX+/BrdU+, 80.6 ± 9.6)(n=7). Thus, administration of the

GPR55 agonist O-1602 displayed significant neuroprotective effects on hippocampal

NSC survival and neuroblast formation. This effect was GPR55-dependent due to lack of effect seen in GPR55-/- animals.

Microglial activation is not attenuated by intrahippocampal administration of GPR55 agonist O-1602 during chronic inflammation in vivo

Previous reports indicated that administration of LPI to hippocampal slice cultures from rats protected against neuronal damage due to excitotoxic lesion in a microglial- dependent manner(Kallendrusch et al. 2013). Based on this report, as well as data showing GPR55 expression on microglia, we sought to determine if treatment with O-

1602 in vivo had any effect on microglial activation. In order to visualize morphological changes of resident microglia, hippocampal sections were stained for IBA-1 and visualized by confocal microscopy. Z-stack images were acquired encompassing the entire DG and 3D volumetric surfaces were generated using Imaris 8.1.2 software.

Representative images are shown in Figure 12A. Saline- treated C57BL/6 and GPR55-/- animals display microglia with small cell bodies and long processes throughout the hippocampal area. Interestingly, there were no significant differences in microglial

Figure 12: Microglial activation is not attenuated by intrahippocampal administration of GPR55 agonist O-1602 during chronic inflammation in vivo. C57BL/6(WT) and GPR55-/- mice had osmotic pumps (Alzet) implanted sub-dermally for a continuous dispersion time of 14 days. Two pumps were implanted. The first pump contained vehicle or O-1602 and was connected to a stainless-steel cannula which was implanted into the left hippocampus. The second pump contained saline or LPS (0.2 mg/kg/day) and was implanted for systemic administration. (A) Representative images depicting the hippocampal area from 30 µm brain sections of 12-15-week-old WT and GPR55-/- mice immunostained with anti-IBA-1(green). Scale bar = 100 µm. Quantitative morphometric analysis of microglial volume (B) and surface area (C) depicting activated state of resident microglia due to systemic inflammation. Morphology was assessed using Imaris software. 3D surfaces were created on positive immunostained microglia to create representative whole cells. Insets in upper right quadrant of close up images are examples of single cell 3D surface renderings from Imaris software and are located in the white box of image. Volume of cells and surface area were determined from 3D representations. Bars represent mean ±SEM. *, p<0.05; **, p<0.01 as compared to vehicle saline animals; #, p<0.05; ##, p<0.01 as compared to agonist saline animals within genotype by Two-way ANOVA followed by Tukey’s post hoc test. n= 4 animals per group and analyzing a minimum of 300 positive cells per animal. volume (Figure 12B) or surface area (Figure 12C) between C57BL/6 and GPR55-/- animals. LPS-treated animals showed significantly increased microglial volume as compared to saline-treated animals; this effect was not altered by administration of O-

1602(C57BL/6 Interaction F(1,12) = 0.026, p=0.875; Inflammatory treatment F(1,12) =

21.48, p=0.0006; Agonist treatment F(1,12) = 0.274, p=0.610; GPR55-/- Interaction

73 F(1,12) = 0.281, p=0.606; Inflammatory treatment F(1,12) = 23.03, p=0.0004; Agonist treatment F(1,12) = 0.276, p=0.609). Microglial surface area was not significantly altered in C57BL/6 animals yet GPR55-/- mice showed significant differences between vehicle + saline-treated animals and vehicle + LPS or O-1602 + LPS-treated animals(C57BL/6

Interaction F(1,12) = 1.251, p=0.285; Inflammatory treatment F(1,12) = 3.166, p=0.101;

Agonist treatment F(1,12) = 5.717e-011, p>0.9999; GPR55-/- Interaction F(1,12) =

0.00309, p=0.9566; Inflammatory treatment F(1,12) = 16.45, p=0.0016; Agonist treatment F(1,12) = 0.5531, p=0.471). Together, these results suggest that the neuroprotective effects of O-1602 treatment on NSCs were not due to reduced microglial activation within the hippocampus because O-1602 treatment did not alter microglial activation after 14 days of low-level, systemic inflammation.

Lack of GPR55 induces altered inflammatory responses after 14 days of chronic, low- level systemic inflammation in vivo

We next determined if lack of GPR55 has an effect on the immune response to chronic, low-level LPS administration. For this experiment, osmotic mini-pumps containing either

LPS (0.2 mg/kg/day) or saline were implanted sub-dermally in C57BL/6 and GPR55-/- mice. These animals did not receive a second pump connected to cannula so as to not have any breach or trauma to the CNS. Brains were harvested after 14 days of treatment and the hippocampal region was dissected out for RNA isolation. Samples were then analyzed for gene expression via the QIAseq mouse inflammation and immunity transcriptome array (RMM005Z, Qiagen). Heatmaps were generated (Figure 13A) from the resulting analysis and graphical analysis of molecular targets were grouped based on

74 A BC* ** C57BL/6- saline 1.50 10-4 2 10-3 **

Saline LPS -3 C57BL/6--LPS

-/- -/- 1 10 1.25 10-4 * -4 -4 1 10 5.0 10 GPR55-/- -saline C57BL/6 - Saline C57BL/6 - LPS GPR55 GPR55 * * ** GPR55-/--LPS * ** * * 5 10-5 2.5 10-4 * index count index count * Normalized total unique molecular Normalized total unique molecular

0 0.0

IL6 C3 IL1b TNF Bst2 IL12a IL!2b CD86 Icam1 Ticam2 *** H2-DMa

DE1 10-3 2.5 10-3

5.0 10-4

1.5 10-3 1 10-3 ** * *

2.5 10-4 index count index count

5 10-4 Normalized total unique molecular Normalized total unique molecular

0.0 0

Min Max K-1 Ccl3 Ccl5 Ccl25 Ccr6 TLR4 yD88 Cxcr6 M IRA TRAF6 Figure 13: Lack of GPR55 induces altered inflammatory responses after 14 days of chronic, low-level systemic inflammation in vivo. C57BL/6 and GPR55-/- mice were chronically treated with LPS (0.2 mg/kg/day) or saline (C57BL/6 + saline, n=5; C57BL/6 + LPS, n=4; GPR55-/- + saline, n=5; GPR55-/- + LPS, n=4) for 14 days. After harvest, the hippocampi were removed and processed for RNA. The expression of inflammation-related genes in the hippocampus was analyzed using the Qiagen mouse Inflammation and Immunity Transcriptome array. (A) Heat map analysis of inflammatory array data showing differentially expressed genes between groups. Normalized Total Unique Molecular Index Count for each gene was compared between groups. Results are displayed as pro-inflammatory cytokines (B), co-stimulators (C), chemokines (D), and LPS related signaling molecules (E). Bars represent mean ±SEM. p<0.05 was considered significant, represented as *p<0.05; ** p <0.01; ***p<0.001. Analysis was by Two-way ANOVA followed by Tukey’s post hoc test. type of target; cytokines (Figure 13B), co-stimulators (Figure 13C), chemokines (Figure

13D), and LPS-related signaling (Figure 13E). C57BL/6 and GPR55-/- animals treated with saline showed no differences in any of the targets analyzed, except for significant reductions in GPR55-/- samples of the chemokine CCL3 and co-stimulator Bst2 and significant upregulation of CCL25. TLR4 in GPR55-/- animals was also upregulated, but results were not significant. LPS treatment in C57BL/6 mice showed a significant

75 Table 5: Statistics for hippocampal expression of inflammatory mediators (Figure 13)

Two-way Interaction Genotype Inflammatory treatment ANOVA (Genotype vs (C57BL/6 vs GPR55-/-) (saline vs LPS) F(1,14) Inflammatory treatment)

IL1B F = 4.435, p=0.0537 F = 10.11, p=0.0067 F = 2.226, p=0.1579

TNF F = 3.307, p=0.0904 F = 7.342, p=0.0169 F = 0.063, p=0.8060

IL6 F = 2.367, p=0.1462 F = 21.8, p=0.0004 F = 3.582, p=0.0793

IL12A F = 4.301, p=0.0570 F = 6.363, p=0.0244 F = 0.4474, p=0.5144

IL12B F = 4.287, p=0.0574 F = 14.13, p=0.0021 F = 1.327, p=0.2686

CD86 F = 14.56, p=0.0019 F = 0.5926, p=0.4542 F = 0.0928, p=0.7651

H2-DMa F = 4.42, p=0.0541 F = 0.8652, p=0.3680 F = 13.93, p=0.0022

C3 F = 0.0284, p=0.8686 F = 0.467, p=0.5055 F = 5.616, p=0.0327

BST2 F = 0.7985, p=0.3867 F = 41.06, p<0.0001 F = 0.4318, p=0.5217

ICAM1 F = 8.191, p=0.0125 F = 5.344, p=0.0365 F = 20.43, p=0.0005

TICAM2 F = 4.997, p=0.0422 F = 0.0014, p=0.9705 F = 3.966, p=0.0663

CCL3 F = 0.0014, p=0.9711 F = 17.48, p=0.0009 F = 0.192, p=0.6680

CCL5 F = 8.035, p=0.0132 F = 0.0722, p=0.7921 F = 1.543, p=0.2346

CCL25 F = 0.8894, p=0.3616 F = 61.16, p<0.0001 F = 0.2272, p=0.6410

CCR6 F = 1.698, p=0.2135 F = 15.31, p=0.0016 F = 0.9721, p=0.3409

CXCR6 F = 1.54, p=0.2351 F = 0.0212, p=0.8864 F = 4.253, p=0.0582

TLR4 F = 1.176, p=0.2965 F = 1.992, p=0.1800 F = 0.8237, p=0.3795

MYD88 F = 0.1872, p=0.6719 F = 0.7739, p=0.3939 F = 3.634, p=0.0773

IRAK1 F = 0.1718, p=0.6848 F = 2.633, p=0.1270 F = 0.5287, p=0.4792

TRAF6 F = 7.701, p=0.0149 F = 11.11, p=0.0049 F = 0.067, p=0.7993

increase in CD86 while also displaying a trend in reduction of inflammatory cytokines

(IL-1b, TNFa, IL-6, IL-12A, IL-12B), Myd88, and TRAF6. GPR55-/- animals treated with LPS exhibited significantly higher levels of inflammatory cytokines and the co-

76 stimulators ICAM-1 and TICAM2 with a trend in upregulation of TRAF6. These animals also displayed significant reduction of co-stimulators Bst2 and H2-Dma. Statistics for hippocampal RNA expression data are listed in Table 5. Taken together, these data suggest that C57BL/6 mice were becoming tolerant to the low-level inflammation being administered, while GPR55-/- animals had an altered immune response resulting in higher levels of inflammatory mediators within the hippocampus.

77 CHAPTER 4 – GENERAL DISCUSSION

Recent studies have shown novel roles for cannabinoid receptors and cannabinoid-like ligands on NSC physiology and pathophysiology (Palazuelos et al.

2006; Aguado et al. 2005; Karanian et al. 2005; Avraham et al. 2014; Rodrigues et al.

2017; Xapelli et al. 2013). Expression of GPR55, an emerging potential member of the cannabinoid family (Yang, Zhou, and Lehmann 2016), has been demonstrated within numerous regions of the adult brain including the hippocampus, a known NSC niche

(Wu, Chen, et al. 2013). However, comprehensive studies concerning GPR55 on NSC biology and function are lacking.

This study shows for the first time that human and murine NSCs do in fact express GPR55. Our results indicate that GPR55 mRNA levels are comparable to other known extracellular signal receptors [EGFR, FGFR1, GRM1(human), GRM5(mouse)] as well as cannabinoid receptors (CNR1, CNR2) within the NSC population. Interestingly, levels of GPR55 mRNA decreased after culture of hNSC samples for 10 days in differentiating conditions suggesting that as NSCs differentiate the levels of GPR55 expression are reduced. Further studies are needed to ascertain exact expression characteristics of GPR55 as NSCs differentiate along neuronal or glial lineages. To further assess GPR55 in the murine hippocampal NSC population, we harvested primary

NSCs from the hippocampus of GPR55-/- mice and compared these samples to those of

C57BL/6 animals. GPR55-/- NSCs showed no difference in neural stem cell markers, extracellular signal receptors, or cannabinoid receptors as compared to C57BL/6 NSCs.

These results suggest that GPR55 may be a potent target for pharmacological activation on NSCs with possible therapeutic potential.

78 Regarding hNSC proliferation, our results indicate that activation of GPR55 by endogenous (LPI) and synthetic agonists (ML184, O-1602) increase NSC proliferation in a low-proliferating culture of human NSCs. These actions were attenuated by concurrent treatment with the selective GPR55 antagonist ML193 suggesting that the increase in proliferation seen with GPR55 agonists is dependent on signaling through GPR55.

Several reports show that activation of the cannabinoid system through both CB1 and CB2 receptors increases NSC proliferation in primary murine NSC cultures. The synthetic cannabinoids HU210 and WIN-55,212-2 promote NSC proliferation through the CB1 receptor yet, interestingly, did not increase differentiation of these cells (Jiang et al. 2005;

Aguado et al. 2005). CB1 activation has also recently been found to induce self-renewal of murine-derived SVZ NSC cultures and increase proliferation via ERK and PI3K dependent mechanisms indicating a necessary role of CB1 in the maintenance of NSC self-renewal and proliferation (Xapelli et al. 2013). Further study of cannabinoid receptor signaling on NSC proliferation showed differences between cultures of murine SVZ and

DG NSCs; SVZ cells had a significant increase in proliferation with CB1 receptor activation while DG cells only had increases with a combination of CB1 and CB2 receptor activation (Rodrigues et al. 2017). Interestingly, all proliferative effects seen by

Rodrigues et al. were blocked by either CB1 or CB2 antagonism suggesting that both CB1 and CB2 receptors are necessary for NSC proliferation through possible CB1 / CB2 heteromer formation. It may be that GPR55 activation and signaling also has similar actions as GPR55 has also been shown to form heteromers with both CB1 and CB2 receptors (Balenga et al. 2014; Martinez-Pinilla et al. 2014). Of note, treatment of hNSCs with ML184 showed the greatest increase in actively proliferating hNSCs during BrdU

79 administration. It is noteworthy that all three GPR55 agonists used in the proliferation experiments have different pharmacological structures: ML184 is a piperazine; O-1602 is an atypical cannabinoid (derivative of abnormal cannabidiol); LPI is a phospholipid.

Recent studies have begun to elucidate specific amino acids critical for activation of

GPR55 by ML184 and LPI (Lingerfelt et al. 2017). The study by Lingerfelt et al. found conserved amino acid residues necessary for both ML184 and LPI activation of GPR55, yet they also found another residue (F6.55) that was only necessary for ML184. It may be that activation of GPR55 within hNSCs by these compounds and subsequent different ligand interactions within the receptor binding pocket is facilitating altered responses which may explain differences in proliferation data.

In fact, it has also been reported that there are species differences concerning some compounds that activate GPR55. It was found that two benzoylpiperazines

(GSK598945A, GSK522372A) failed to activate mouse GPR55 while GSK494581A failed to activate rat GPR55 (Brown et al. 2011). It was determined that there are two residues that vary between human and rodent GPR55 and that this alteration precludes benzoylpiperazines from interacting with a residue (K2.60) necessary for GPR55 activation and is therefore a devastating mutation (Lingerfelt et al. 2017). Therefore, our in vivo experiments utilized O-1602 as a synthetic GPR55 ligand as O-1602 has been shown to activate mouse GPR55. Our results indicate that chronic O-1602 treatment directly into the hippocampus elevates the number of Ki67+ cells, a marker for cell proliferation, within the SGZ in comparison to vehicle treatment after 14 days. These results suggest that chronic administration of O-1602 increases proliferation of NSCs within the hippocampus. Furthermore, GPR55-/- animals displayed reduced numbers of

80 Ki67+ cells within the SGZ as compared to WT controls. Treatment with O-1602 in these animals had no effect on Ki67+ NSCs suggesting a necessary role of GPR55 activation in

NSC proliferation similar to findings with CB1 and CB2 receptor studies. Of note, studies

-/- -/- utilizing CB1 or CB2 mice also have displayed reduced rates of hippocampal NSC proliferation as compared to WT controls (Palazuelos et al. 2006; Jin et al. 2004).

Interestingly, our data may help explain some of the enigmatic results seen in recent studies with the CB1 receptor and TRPV1 antagonist, SR141716A. This antagonist has been shown to increase NSC proliferation both in the SGZ and SVZ of the mouse brain,

-/- yet CB1 mice also showed similar increases in proliferation rates suggesting that this compound has another target (Jin et al. 2004). Intensive studies into the pharmacology of

GPR55 have recently revealed that SR141716A acts as an antagonist at GPR55 at low concentrations (2 µM) yet produces robust agonist activity at higher concentrations (10-

30 µM) implying that GPR55 may play a yet unknown role in the effects seen in studies utilizing SR141716A (Kapur et al. 2009; Lauckner et al. 2008).

Upregulation of neuronal differentiation has also been reported as a key aspect of cannabinoid action on the NSC population. Recent reports have suggested that activation of either CB1 or CB2 upregulate neuronal differentiation. CB1 receptor activation by the eCB AEA significantly promoted NSC differentiation into neurons while decreasing the percentage of astrocytes and having no effect on the oligodendrocyte population in culture (Compagnucci et al. 2013). Therefore, we sought to determine if activation of

GPR55 had an impact on differentiation of hNSCs. We chose to utilize only ML184 for differentiation studies in vitro due to this compound having the most robust results in the proliferation studies discussed earlier. Our results indicate that activation of GPR55

81 promotes neuronal differentiation in hNSC culture as evidenced by increases in bIII- tubulin positive cells treated with ML184 for 10 days under differentiating conditions as compared to vehicle treated samples. Interestingly, we also observed significant reduction of bIII-tubulin positive cells after treatment with ML193 as compared to vehicle treated samples. Concurrent treatment of both ML184 and ML193 still showed reduction in bIII- tubulin positive cells yet not to the same degree as ML193 alone. To further assess alterations in differentiation upon GPR55 activation, we determined population differences of S100b, a marker for astrocytic differentiation. Flow cytometric analysis showed no significant differences between samples treated with ML184, ML193, or concurrent treatment of ML184 with ML193 as compared to vehicle treated samples. To confirm these results, we determined mRNA expression of neuronal markers (bIII- tubulin, MAP2) and astrocytic markers (S100b, GFAP). Similar to flow cytometric analysis, I observed significant increases in bIII-tubulin and MAP2 mRNA from cells treated for 10 days under differentiating conditions with ML184 as compared to vehicle treated samples. ML193 treatment decreased mRNA expression as compared to vehicle but results did not reach significance. S100b and GFAP mRNA was not significantly changed yet there was a trend of increased expression by samples treated with ML193.

Based on these data, we assert that activation of GPR55 plays a key role in neuron formation in vitro. To our knowledge, this is the first report of GPR55 activation having direct involvement in the differentiation state of NSCs.

Moreover, we observed an increase in the number of immature neurons (DCX+) and BrdU+ cells within the hippocampus of C57BL/6 animals chronically treated with O-

1602 for 14 days. In addition, we also determined that the number of surviving,

82 proliferating cells at the start of treatment (BrdU+) that subsequently differentiated along a neuronal lineage was also increased with O-1602 treatment as compared to vehicle treated animals (BrdU+/DCX+). These data are the first to demonstrate the role of

GPR55 activation on hippocampal NSC differentiation and survival of proliferating cells.

Furthermore, I found that GPR55-/- animals have diminished basal rates of immature neuron generation within the hippocampus, implying that signaling through GPR55 enhances early neurogenesis in vivo. We also observed that GPR55-/- animals have a significantly reduced percentage of BrdU+ cells differentiating along a neuronal lineage as compared to WT controls, suggesting that lack of GPR55 impairs neuronal differentiation in vivo in terms of the total rate of neurogenesis and percentage of proliferating cells actually differentiating along a neuronal lineage. Importantly, recent evidence suggests that GPR55 activation by LPI or O-1602 transiently increases the frequency of CA1 excitatory postsynaptic currents and enhances hippocampal CA1 LTP and synaptic integrity (Hurst et al. 2017; Sylantyev et al. 2013). It may be possible that

GPR55 ligands are strengthening the synaptic connections of newly generated progenitors into the hippocampal circuitry and allowing these cells to survive and integrate into the existing circuitry at a higher rate.

It should be noted that we have not yet performed studies regarding the mechanistic aspects as to how GPR55 signaling influences neurogenesis. Interestingly, effects of GPR55 activation on NSCs are similar to those seen by targeting CB1 and CB2, yet the known downstream signaling cascades between GPR55 and CB1/CB2 receptors differ vastly. GPR55 is known to utilize Gq or G12/13, as opposed to Gi/O in CB1R/CB2R, for signal transduction ultimately leading to increased intracellular Ca2+, ERK1/2

83 phosporylation, NFAT activation, nuclear translocation of NFκB, and activation of

CREB (Henstridge et al. 2009; Lauckner et al. 2008; Kapur et al. 2009; Henstridge et al.

2010). Each of these downstream signaling targets has been implicated in adult neurogenesis, necessitating further studies to fully elucidate the signaling mechanisms mediated by GPR55 on NSCs.

Dysregulation of adult hippocampal neurogenesis is implicated in numerous pathological conditions including AD, HIV-1 associated neurocognitive disorder, virus- induced memory dysfunction, and depression (Mu and Gage 2011; Okamoto et al. 2007;

Garber et al. 2018; Mahar et al. 2014). A common trait of each of these conditions is a chronic upregulation of inflammatory mediators leading to a neuroinflammatory state.

To continue the present study, we showed that reduced neuron formation in vitro induced by IL-1b was ameliorated by activation of GPR55 in both human and mouse cultures of NSCs. Pre-treatment with GPR55 agonists also blocked upregulation of inflammatory cytokine receptor mRNA (IL-1R1, IL-6st) while increasing mRNA for anti-inflammatory or neuroprotective cytokine receptors (IL-10Ra, TNFR2) within

NSCs. This was apparent in both human and mouse cells. We also show for the first time that GPR55 activation induces neuronal differentiation of murine primary hippocampal

NSCs similar to results seen with human NSCs. In vivo, we sought to determine if a chronic, low-level inflammation (systemic administration of LPS) would elicit negative effects on hippocampal neurogenesis similar to those seen after high level, acute infection. Interestingly, a dose of 0.2 mg/kg/day of LPS was sufficient to significantly reduce NSC survival (as measured by BrdU) and neuroblast formation within the SGZ of the hippocampus. Direct intrahippocampal administration of O-1602, a potent GPR55

84 agonist, protected against reduced NSC survival and neuroblast formation during insult with chronic, low-level infusion of LPS. We determined that neuroprotective effects were elicited by direct involvement on NSCs as microglial activation (detected by comprehensive image analysis) did not differ between LPS animals that received vehicle as compared to O-1602 treatment. To further assess GPR55 function during the immune response, we treated animals with a low-level, chronic LPS paradigm and assessed inflammatory mRNA levels within the hippocampus showing an altered immune response in GPR55-/- animals. The presented data suggest that GPR55 can protect NSCs against inflammatory insult and poses a potent target with therapeutic potential.

Inflammatory insult with IL-1b on NSCs has been extensively studied showing reduced proliferation and neuronal differentiation rates both in vitro and in vivo (Wu,

Montgomery, et al. 2013; Zhang et al. 2013; Crampton et al. 2012). We chose to focus on

IL-1b for in vitro studies since insult with IL-1b has also been studied in the context of cannabinoid signaling and neuroprotection(Garcia-Ovejero et al. 2013). Our results indicate that pre-treatment with the GPR55 agonists ML184 or O-1602 on human or mouse NSCs, respectively, is neuroprotective against reduced neuronal formation caused by insult with IL-1b. This effect was attenuated by concurrent treatment with GPR55 antagonist, suggesting that these effects are GPR55-dependent. These results are consistent with other studies of the effects of CB1 and CB2 receptors on protection against insult by IL-1b implicating GPR55 as another target for therapeutic intervention against

NSC injury. O-1602 has also been implicated in protection of neuronal cell viability, yet the exact receptor necessary for this effect was not elucidated (Janefjord et al. 2014).

85 The number of GFAP positive cells was also decreased in samples treated with

GPR55 agonist in combination with IL-1b indicating that this was not simply a blunting of gliogenesis, but rather a protective effect of GPR55 activation. Interestingly, we found that treatment of human NSCs with IL-1b reduced the number of GFAP positive cells in comparison to vehicle- treated samples. This observation goes against the notion that gliogenesis is increased during insult with inflammatory mediators. We speculate that this may be due to slowing of proliferation rates, thus resulting in fewer positive cells. Our studies in primary murine NSCs did show increases in GFAP-positive cells with IL-1b treatment similar to findings from other studies suggesting to us that our methods for quantification of neurogenesis were not faulty (Green and Nolan 2012; Crampton et al.

2012). Numerous mechanisms have also been linked to IL-1b-induced reductions in neurogenesis directly within NSCs, including regulation of the transcription factor TLX and p53-dependent apoptosis (Ryan et al. 2013; Guadagno et al. 2015). We did not examine directly intracellular mechanisms in which GPR55 activation protected against reduced neurogenesis facilitating a need for more in-depth study of the exact mechanisms necessary for the neuroprotective effects of GPR55.

In order for cytokines to exert any effect on the NSC population, they must first bind to specific receptors on the cell surface. Numerous pro- and anti-inflammatory cytokines are able to exert effects on the NSC population, including IL-1b through IL-

1R1 and IL-1R2, TNFa through TNFR1 and TNFR2, IL-6 through the surface co- receptor IL-6st (gp130), and the anti-inflammatory cytokine IL-10 through IL-10Ra

(Green and Nolan 2012; Ben-Hur et al. 2003; Chen and Palmer 2013; Kotasova,

Prochazkova, and Pachernik 2014; Perez-Asensio et al. 2013). To investigate if activation

86 of GPR55 had any effect on the acute immune response by NSCs to IL-1b, we measured mRNA expression of known cytokine receptors expressed by NSCs (IL-1R1, IL-1R2,

TNFR1, TNFR2, IL-6st, IL-10Ra) at 4 and 24 hours post insult. We found that activation of GPR55 reduced increases in pro-inflammatory receptor mRNA (IL-1R1, IL-6st) while further promoting anti-inflammatory receptor mRNA (IL-10Ra, TNFR2) during insult with IL-1b. It is interesting that activation of GPR55 induced effects on some specific cytokine receptors while not affecting others. IL-1R1 is the primary receptor through which IL-1b signals and GPR55 significantly attenuated increases due to IL-1b treatment while having no effect on IL-1R2, a decoy receptor with no signaling capacity (Wang et al. 2007; Koo and Duman 2008; Colotta et al. 1993). Activation of TNFR1 is known to induce detrimental effects on neurogenesis. GPR55 agonist treatment of NSCs during insult did not alter TNFR1 mRNA levels; however, TNFR2, which has been shown to be neuroprotective, was significantly upregulated as compared to IL-1b treatment alone

(Chen and Palmer 2013). IL-6st increases were also attenuated by GPR55 activation yet this signal transducer functions not only for IL-6 mediated signaling, but also for IL-11, leukemia inhibitory factor (LIF), and oncostatin M (OSM), which are all part of the IL-6 family of cytokines (Kwak et al. 2010). Insult with IL-1b upregulated IL-10Ra mRNA in human samples and pre-treatment with ML184 significantly increased this upregulation.

It should be noted that we could not determine expression of IL-10Ra on murine hippocampal NSC samples so analysis of effects of GPR55 activation could not be performed. Results from inflammatory response experiments are indicative of an anti- inflammatory, or neuroprotective, mechanism through which GPR55 is actively attenuating the ability of pro-inflammatory cytokines (IL-1b, IL-6) to signal while

87 increasing the effect of anti-inflammatory and neuroprotective signals (TNFa via

TNFR2, IL-10). It is unclear how activation of GPR55 affected changes to mRNA levels.

The genes for these receptors, either human or mouse, are on different chromosomes

(except IL-1R1 and IL-1R2; human-chromosome 2, mouse-chromosome 1) so it is unlikely that there is only one specific loci or promoter where these effects are taking place, yet treatment with GPR55 may be inducing upregulation of transcription factors necessary for cytokine receptor transcription. Another possibility is that GPR55 signaling increases post-transcriptional degradation of IL-1R1 and IL-6st mRNA through a yet unknown mechanism. IL-1R1 mRNA degradation has been shown during inflammatory states by microRNAs through targeting of the 3’UTR suggesting that the process by which GPR55 activation regulates inflammatory cytokine receptor mRNA expression could be microRNA-dependent (Skinner et al. 2017; Halappanavar et al. 2013).

Regulation of microRNA by the cannabinoid system, specifically the CB2 receptor, results in protective effects against acute liver failure caused by d-galactosamine/LPS, further supporting the possibility that GPR55 may act in a similar fashion (Tomar et al.

2015). Further study is needed to fully elucidate these mechanisms.

A major facet of neurodegenerative disorders and viral infection is a chronic upregulation of neuroinflammatory mediators and subsequent reductions in hippocampal neurogenesis leading to cognitive impairment (Ferguson et al. 2016; Zonis et al. 2015;

Ferrell and Giunta 2014; Belarbi et al. 2012). We wanted to understand if low levels of chronic, systemic inflammation could exert negative effects on neurogenesis similar to studies utilizing acute, high dose, or direct administration of inflammatory mediators to the CNS. We therefore utilized osmotic mini-pumps to deliver LPS (0.2 mg/kg/day) sub-

88 dermally for 14 days and found significant reductions in NSC survival and immature neuron formation within the hippocampus. These results indicate that even very low levels of systemic inflammation can, over time, induce negative regulation of hippocampal neurogenesis. Interestingly, NSC proliferation within the SGZ (Ki67+) was not significantly reduced 14 days after pump implantation. Taken together with data showing significant reduction of BrdU+ cells (BrdU was administered on days 1-4 of

LPS treatment), these data suggest that the animals may be acclimating to the levels of

LPS being administered and attenuating their immune responses. Tolerance of LPS- induced signals impairs the production of pro-inflammatory cytokines without inhibiting the expression of anti-inflammatory mediators and occurs through numerous mechanisms including reduction in TLR4, MyD88, IRAK-1, and TRAF6 (Nomura et al. 2000; Oak et al. 2006; Li, Wang, and Redmond 2006; Xiong et al. 2011). Indeed, we did see trends in downregulation of mRNA for MyD88 and TRAF6 in the hippocampus of C57BL/6 animals chronically treated with LPS suggesting an increase in tolerance. We did not see changes in mRNA expression of TLR4 or IRAK-1 in C57BL/6 animals, but that does not rule out changes in protein levels because LPS tolerance has been shown to decrease protein levels while maintaining mRNA expression (Li, Wang, and Redmond 2006).

Moreover, we observed a trend in reduction of mRNA for major cytokines including

IL1b, TNFa, IL6, IL12A, and IL12B, further suggesting LPS tolerance in C57BL/6 mice. GPR55-/- mice showed some reduction of TLR4 and MyD88, yet these animals also displayed higher levels of TLR4 in the hippocampus compared to C57BL/6 animals. All other tolerance-like effects due to LPS treatment in C57BL/6 mice were altered in

GPR55-/- animals. GPR55-/- mice also had increased mRNA expression of pro-

89 inflammatory cytokines (IL1b, TNFa, IL12A) and increased IRAK-1 and TRAF6, suggesting that lack of GPR55 in these animals is altering the chronic immune response due to LPS administration. It is important to note that the animals tested for hippocampal mRNA expression did not receive cannula implantation nor any agonist treatment. The exact contribution GPR55 has on this immune response is still not fully understood and requires further study.

To combat the detrimental effects of neuropathological conditions, additional therapeutic interventions that either dampen chronic neuroinflammation or attenuate reductions in hippocampal neurogenesis are critically needed. The cannabinoid system has already been shown to rescue impaired neurogenesis and reduce inflammation due to the HIV-1 protein gp120 through activation of CB2 receptors (Avraham et al. 2014).

Using this chronic, systemic inflammatory paradigm, we investigated if treatment with the GPR55 agonist O-1602 could protect against LPS-induced dysregulation of hippocampal neurogenesis. O-1602 significantly protected against reduction in hippocampal NSC survival (BrdU+), the number of immature neurons (DCX+), and proliferating NSC that ultimately became neuroblasts (DCX+/BrdU+) within the SGZ of the hippocampus. GPR55-/- animals showed reduced rates of NSC proliferation, survival, and neuroblast formation under non-inflammatory conditions. LPS treatment in GPR55-/- animals further reduced the number of immature neurons (DCX+ and DCX+/BrdU+) as compared to saline treated animals. The number of Ki67+ and BrdU+ cells were also diminished yet these results were not significant. It may be that GPR55-/- animals had such low rates of NSC proliferation and immature neuron formation that, although LPS did have a trend in reduction, significance could not be achieved. Administration of O-

90 1602 had no effect in GPR55-/- animals, either under control or inflammatory conditions, further demonstrating that the neuroprotective results seen in C57BL/6 mice were due to

GPR55. These results demonstrate for the first time that activation of GPR55 elicits neuroprotective effects within the SGZ of the hippocampus during chronic neuroinflammatory conditions. It should be noted that O-1602 was administered at the same time as LPS suggesting that pre-treatment is therapeutic under inflammatory conditions which may be beneficial as a therapy for people predisposed to inflammatory diseases or at high risk for systemic inflammation. It is therefore necessary to determine if GPR55 is an effective therapeutic target after inflammatory conditions are already present.

Since GPR55 is expressed by primary microglia, it is possible that the neuroprotective properties of O-1602 in vivo during inflammatory insult may not have a direct NSC effect (Pietr et al. 2009). Indeed, activation of GPR55 by its endogenous ligand LPI shows neuroprotective effects in hippocampal slice cultures after glutamate induced excitotoxic lesion and on CA1 and CA3 hippocampal neurons after ischemic stroke (Kallendrusch et al. 2013; Blondeau et al. 2002). Protective effects in hippocampal slice cultures were found to be microglia-dependent, suggesting that targeting GPR55 reduces microglial activation under inflammatory conditions. Here we found that chronic

LPS administration at a low dose induces microglial activation in both C57BL/6 and

GPR55-/- mice as determined by microglial volume. O-1602 had no effect on either microglial volume or surface area suggesting that the neuroprotective outcomes seen on

NSCs were not mediated by reductions in microglial activation. Moreover, we did not detect any differences in microglia between C57BL/6 and GPR55-/- animals. The lack of

91 effect on microglia may not necessarily be due to lack of GPR55 signaling but may be due to the agonist used. LPI was seen to reduce microglial activation in the study by

Kallendrusch et al., but LPI and O-1602 have different chemical structures and therefore may not act similarly on microglia. Another recent study by McHugh et al. describes an increase of microglial migration due to O-1602 on BV-2 microglia in vitro, but this action was mediated through another candidate cannabinoid receptor, GPR18 (McHugh et al. 2012). This other receptor may be present on microglia in the C57BL/6 and GPR55-

/- CNS, but we do not believe that O-1602 is having any significant effect through activation of GPR18 because, again, we did not see any differences in microglial activation between LPS-treated animals that received either vehicle or O-1602.

In summary, these findings support the hypothesis that NSCs express functional

GPR55, targeting GPR55 with selective agonists increases NSC proliferation and early neurogenesis both in vitro and in vivo, and offers protection against deficits in neurogenesis induced by inflammatory insult through direct targeting of NSCs. Better understanding of the mechanisms by which GPR55 provides neuroprotection is critically necessary and may provide a more directed target for future therapeutics.

92 SUMMARY OF CONCLUSIONS

Adult hippocampal neurogenesis is a necessary facet of proper CNS function in terms of memory formation and mood regulation (Deng, Aimone, and Gage 2010; Sahay and Hen 2007). The generation of therapeutics that alleviate negative modulation of hippocampal neurogenesis and NSC dysfunction by inflammatory conditions is in great need. Recent evidence suggests that the cannabinoid system, specifically the cannabinoid receptors CB1 and CB2, plays not only a functional role in regulating NSC proliferation and neuronal differentiation but can be targeted with therapeutic potential within this cellular population (Avraham et al. 2014; Rodrigues et al. 2017; Xapelli et al. 2013). Yet not all effects of cannabinoid-like compounds can be attributed to either of the classically characterized receptors (CB1 or CB2). It is therefore probable that these compounds are acting through other receptors that have yet to be fully characterized. The recently de- orphaned GPCR GPR55 is thought to be a candidate cannabinoid receptor due to the ability of numerous cannabinoid and cannabinoid-like compounds to activate this receptor and elicit functional molecular, cellular, and behavioral outcomes. In this dissertation, we performed comprehensive evaluation of the expression of GPR55 by

NSCs, and the possible functional regulation by GPR55 of neurogenesis either under homeostatic conditions or during inflammatory insult.

To start, we confirmed the expression of GPR55 in human and murine samples of

NSCs to establish not only the existence of GPR55 within this population but also that the abundance of receptor, in contrast to known functional receptors, was sufficient to elicit responses. We determined that GPR55 is expressed at lower levels than that of the gene for the CB1 receptor (CNR1) but higher than that of the CB2 receptor (CNR2)

93 leading to the thought that this receptor may indeed have functional importance on NSCs.

We demonstrate that treatment of human NSC samples with GPR55 selective agonists significantly increases proliferation of these cells and that this effect is GPR55 dependent as concurrent treatment with a GPR55 antagonist attenuates these responses. We then investigated the effects of activation of GPR55 on neuronal differentiation both in vitro and in vivo and determined that signaling through GPR55 actively pushes NSCs along a

Table 6: Effects of GPR55 activation in human NSCs Analysis Model Compound Effect seen

Proliferation In vitro LPI, O-1602, ML184 Increases proliferation rate as assessed by BrdU

Increases the number of cells differentiating along a neuronal Differentiation In vitro ML184 lineage, decreases the number of glia generated

Activation of GPR55 attenuated reductions in neurogenesis due to Neuroprotection In vitro ML184, IL-1b inflammatory insult

Concurrent treatment with GPR55 agonist reduces the expression of pro-inflammatory cytokine receptors (IL-1R1, IL-6st), Cytokine receptor In vitro ML184, IL-1b increases the expression of pro- expression neurogenic receptors (TNFR2, IL- 10Ra) after insult with IL-1b

neuronal differentiation lineage while inhibition of GPR55 reduces neuron formation. To date, these are the first reports suggesting that GPR55 plays a functional role on neurogenesis under homeostatic conditions. We then investigated the therapeutic potential of GPR55 under inflammatory conditions. Selectively targeting GPR55 in murine and human NSC cultures that were insulted with the inflammatory cytokine IL-1b

94 showed attenuated reductions in the number of new neurons formed during insult. The number of cells actively differentiating along a glial lineage was also reduced suggesting that activation of GPR55 has neuroprotective effects. We further investigated the effects of GPR55 activation during insult with IL-1b on cytokine receptor expression within the cells at both 4 hours and 24 hours post insult. Interestingly, we found that GPR55 agonist treatment significantly reduced that expression of mRNA for the inflammatory cytokine receptors IL-1R1 (IL-1b) and IL-6st (IL-6) while also significantly increasing the mRNA expression of known pro-neurogenic cytokine receptors TNFR2 (TNFa) and IL-10Ra

(IL-10) which confirms a neuroprotective role of GPR55 activation on NSCs. We then determined that systemic treatment with a chronic, low dose of LPS in mice is sufficient to reduce hippocampal neurogenesis rates similar to that seen by larger, acute doses of

LPS. This paradigm can now be used to better mimic a chronic, mild infectious state.

Concurrent administration of the GPR55 selective agonist O-1602 directly into the hippocampus alleviated reductions due to LPS in terms of proliferation, survival, and neuroblast formation. We concluded that these effects were not due to attenuating microglial responses within the hippocampal region by O-1602 since microglial activation was not altered between animals treated with vehicle versus those treated with

O-1602 in either WT or GPR55-/- animals. Lastly, we performed a thorough evaluation of immune response mediators and inflammatory components within the hippocampus after chronic, low-level systemic administration of LPS to determine differences between WT and GPR55-/- mice. An analysis of this kind has been nonexistent until our study. Results suggest that WT mice were becoming tolerant to the amount of LPS being administered in that there were decreased levels of inflammatory cytokines, chemokines, and TLR4

95 Table 7: Effects of GPR55 activation in mouse NSCs

Analysis Model Compound Effect seen

Proliferation In vivo O-1602 Increases proliferation rates of NSCs within the SGZ as evidenced by Ki67

Increases the number of cells differentiating along a neuronal lineage, decreases the Differentiation In vitro O-1602 number of glia generated

Increases the survival of proliferating NSCs and neuroblast formation in the SGZ of the Differentiation In vivo O-1602 hippocampus

Activation of GPR55 attenuates reductions in neurogenesis due to inflammatory insult Neuroprotection In vitro O-1602, IL-1b

Administration of GPR55 agonist into the hippocampus attenuates reductions in Neuroprotection In vivo O-1602, LPS hippocampal NSC proliferation, survival, and neuroblast formation

Concurrent treatment with GPR55 agonist reduces the expression of pro-inflammatory Cytokine receptor In vitro O-1602, IL-1b cytokine receptors (IL-1R1, IL-6st), increases expression the expression of pro-neurogenic receptors

(TNFR2,)

O-1602 treatment did not alter microglial activation as compared to vehicle after 14 Microglial In vivo O-1602, LPS days of administration activation

Immune responses GPR55-/- animals display prolonged to chronic, inflammatory responses to LPS and an systemic In vivo LPS inability to properly regulate this response inflammation signaling modulators. GPR55-/- animals displayed increased levels of inflammatory cytokines and TLR4 signaling components further suggesting that these animals have an altered immune response and that GPR55 is responsible for these effects. All results for effects of GPR55 activation on human and murine NSC samples are summarized in

Tables 6 and 7 respectively.

96 Taken together, these studies highlight the importance of GPR55 activation in

NSC proliferation, differentiation, and immune responses. Our findings indicate that

GPR55 has a functional role in maintaining neurogenesis under homeostatic conditions, and that targeting of GPR55 during inflammatory insult may have therapeutic efficacy.

These studies herein underscore the potential therapeutic value of targeting GPR55, and modulating its function for neuroprotection and positive therapeutic outcomes.

97 FUTURE DIRECTIONS

To determine the full therapeutic potential of GPR55 activation on the NSC population, further investigation into the mechanistic aspects of how GPR55 exerts its effects is much needed. To date, ligand binding to GPR55 is known to activate a number of signaling pathways including phosphorylation of ERK1/2, CREB, activation of the

NFκB pathway, and translocation of NFAT to the nucleus (Henstridge et al. 2010;

Henstridge et al. 2009). These effects are highly dependent upon the specific ligand being used as well as the type of cell being activated. Phosphorylation of CREB, MAPKs including ERK1/2, and NFκB activation have all proven to control the proliferation and terminal differentiation of NSCs leading to a multitude of possibilities of how GPR55 regulates NSC function (Jagasia et al. 2009; Tocharus et al. 2014; Zhang and Hu 2012).

In addition, it may be that different downstream signaling cascades are utilized under different conditions (differentiation conditions, inflammatory insult, etc.) to produce the full range of effects seen from this study. Future in vitro analysis of human and murine

NSC samples using different agonists should shed light on the signaling mechanisms necessary for the increases in proliferation and neuronal differentiation, and neuroprotection from inflammatory insult seen by this study.

Proliferation studies suggest that major selective agonists of GPR55 (LPI, O-

1602, ML184) all increase hNSC proliferation rates. Unfortunately, we did not perform necessary experiments to determine which stage, or all stages, of precursor cells that are being targeted by activation of GPR55. Radial type 1 cells are considered “true” stem cells according to the most stringent criteria, yet a second class of type 1 precursor stem cell with similar self-renewing and multipotent characteristics has been identified

98 (Bonaguidi et al. 2011; Lugert et al. 2010). These cells are morphologically characterized by short, horizontal processes and divide more quickly than the radial type 1 cells which are mostly quiescent and divide very slowly (Lugert et al. 2010). These type 1 cells give rise to consecutive stages of dividing progenitor cells, namely type 2a, type 2b, and type

3 neuroblasts (Kempermann et al. 2004). Future studies are still needed to elucidate if activation of GPR55 is simply increasing proliferation of all NSCs or if it is selective to a single stage (i.e. type 2b or type 3 neuroblasts).

We also determined that activation of GPR55 is neuroprotective of human and murine NSC samples during insult with IL-1b. These studies were imperative since IL-1b has deleterious consequences on neurogenesis both in vitro and in vivo. Yet, IL-1b is not the only inflammatory cytokine, or inflammatory mediator, that has negative consequences on neuronal differentiation rates. Others include TNFa, IL-6, Type 1 IFNs

(IFNa, IFNb), Type 2 IFNs (IFNg), and HIV-1 neurotoxic proteins (gp120, tat).

Extensive studies using these inflammatory mediators to elicit negative effects on the

NSC population are needed to determine if activation of GPR55 is neuroprotective regardless of which cytokine/mediator is used or if results are cytokine or context dependent.

It is well known that systemic inflammation from LPS administration can induce anxiety-like behaviors in rodents (Ming, Sawicki, and Bekar 2015; Hritcu and Gorgan

2014; Bassi et al. 2012). Due to studies showing anxiolytic effects of GPR55 agonists it will be necessary to elucidate if administration of GPR55 agonists can alleviate increased anxiety due to LPS treatment (Shi et al. 2017; Romero-Zerbo et al. 2017; Rahimi,

Hajizadeh Moghaddam, and Roohbakhsh 2015). These studies determined that different

99 compounds could elicit similar anxiolytic responses in mice yet the only region of the rodent CNS that was specifically targeted was the medial orbital cortex (Shi et al. 2017).

To date, there is no data connecting GPR55 activation within the hippocampus to anxiolytic-like effects. There is, however, data suggesting that increasing adult hippocampal neurogenesis is sufficient to reduce anxiety-like behaviors which may be a possible mechanism as to which GPR55 elicits its anxiolytic effects (Hill, Sahay, and

Hen 2015). Future studies are needed to determine if altering hippocampal neurogenesis by activating GPR55 can also elicit anxiolytic-like effects after LPS administration.

Our in vivo studies also determined that GPR55-/- animals showed significantly reduced rates of hippocampal NSC proliferation and neuroblast formation as compared to

WT controls. Reduced rates of neurogenesis are also seen in aged populations resulting in cognitive decline and memory impairments (Seib and Martin-Villalba 2015). It is possible that deletion of GPR55 lead to either a committed fate and subsequent depletion of the stem cell pool or a complete halt of proper neurogenesis resulting in the reduced rates seen in our study. In either case it will be imperative to determine what constitutive deletion of GPR55 has on neurogenesis as these animals age.

Our data has provided valuable insight into proper homeostatic regulation of adult neurogenesis, and has identified a potential candidate with therapeutic potency to alleviate negative effects of inflammation on neuronal differentiation. Targeting GPR55 during inflammatory insult may help mediate neuroinflammatory responses, and prevent cognitive decline associated with reductions in hippocampus neurogenesis. Further characterization of GPR55 signaling and neuroprotection during insult with inflammatory

100 mediators will be applicable to conditions seen in other neuroinflammatory conditions such as Alzheimer’s disease of HAND.

101 REFERENCES

Ables, J. L., J. J. Breunig, A. J. Eisch, and P. Rakic. 2011. 'Not(ch) just development: Notch signalling in the adult brain', Nat Rev Neurosci, 12: 269-83.

Aguado, T., K. Monory, J. Palazuelos, N. Stella, B. Cravatt, B. Lutz, G. Marsicano, Z. Kokaia, M. Guzman, and I. Galve-Roperh. 2005. 'The endocannabinoid system drives neural progenitor proliferation', Faseb j, 19: 1704-6.

Ahmed, S., H. T. Gan, C. S. Lam, A. Poonepalli, S. Ramasamy, Y. Tay, M. Tham, and Y. H. Yu. 2009. 'Transcription factors and neural stem cell self-renewal, growth and differentiation', Cell Adh Migr, 3: 412-24.

Ahn, J., J. Lee, and S. Kim. 2015. 'Interferon-gamma inhibits the neuronal differentiation of neural progenitor cells by inhibiting the expression of Neurogenin2 via the JAK/STAT1 pathway', Biochem Biophys Res Commun, 466: 52-9.

Aid, S., and F. Bosetti. 2011. 'Targeting cyclooxygenases-1 and -2 in neuroinflammation: Therapeutic implications', Biochimie, 93: 46-51.

Alboni, S., L. Gibellini, C. Montanari, C. Benatti, S. Benatti, F. Tascedda, N. Brunello, A. Cossarizza, and C. M. Pariante. 2013. 'N-acetyl-cysteine prevents toxic oxidative effects induced by IFN-alpha in human neurons', Int J Neuropsychopharmacol, 16: 1849-65.

Altman, J., and G. D. Das. 1965. 'Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats', J Comp Neurol, 124: 319-35.

Andersen, J., N. Urban, A. Achimastou, A. Ito, M. Simic, K. Ullom, B. Martynoga, M. Lebel, C. Goritz, J. Frisen, M. Nakafuku, and F. Guillemot. 2014. 'A transcriptional mechanism integrating inputs from extracellular signals to activate hippocampal stem cells', Neuron, 83: 1085-97.

Arguello, A. A., S. J. Fischer, J. R. Schonborn, R. W. Markus, R. A. Brekken, and A. J. Eisch. 2009. 'Effect of chronic morphine on the dentate gyrus neurogenic microenvironment', Neuroscience, 159: 1003-10.

102 Arvidsson, A., T. Collin, D. Kirik, Z. Kokaia, and O. Lindvall. 2002. 'Neuronal replacement from endogenous precursors in the adult brain after stroke', Nat Med, 8: 963-70.

Austin, C. P., B. Ky, L. Ma, J. A. Morris, and P. J. Shughrue. 2004. 'Expression of Disrupted-In- Schizophrenia-1, a schizophrenia-associated gene, is prominent in the mouse hippocampus throughout brain development', Neuroscience, 124: 3-10.

Avraham, H. K., S. Jiang, Y. Fu, E. Rockenstein, A. Makriyannis, A. Zvonok, E. Masliah, and S. Avraham. 2014. 'The cannabinoid CB(2) receptor agonist AM1241 enhances neurogenesis in GFAP/Gp120 transgenic mice displaying deficits in neurogenesis', Br J Pharmacol, 171: 468-79.

Baker, D., G. Pryce, W. L. Davies, and C. R. Hiley. 2006. 'In silico patent searching reveals a new cannabinoid receptor', Trends Pharmacol Sci, 27: 1-4.

Balenga, N. A., E. Aflaki, J. Kargl, W. Platzer, R. Schroder, S. Blattermann, E. Kostenis, A. J. Brown, A. Heinemann, and M. Waldhoer. 2011. 'GPR55 regulates cannabinoid 2 receptor- mediated responses in human neutrophils', Cell Res, 21: 1452-69.

Balenga, N. A., E. Martinez-Pinilla, J. Kargl, R. Schroder, M. Peinhaupt, W. Platzer, Z. Balint, M. Zamarbide, I. G. Dopeso-Reyes, A. Ricobaraza, J. M. Perez-Ortiz, E. Kostenis, M. Waldhoer, A. Heinemann, and R. Franco. 2014. 'Heteromerization of GPR55 and cannabinoid CB2 receptors modulates signalling', Br J Pharmacol, 171: 5387-406.

Barbeau, D., J. J. Liang, Y. Robitalille, R. Quirion, and L. K. Srivastava. 1995. 'Decreased expression of the embryonic form of the neural cell adhesion molecule in schizophrenic brains', Proc Natl Acad Sci U S A, 92: 2785-9.

Bassi, G. S., A. Kanashiro, F. M. Santin, G. E. de Souza, M. J. Nobre, and N. C. Coimbra. 2012. 'Lipopolysaccharide-induced sickness behaviour evaluated in different models of anxiety and innate fear in rats', Basic Clin Pharmacol Toxicol, 110: 359-69.

Bedse, G., A. Romano, S. Cianci, A. M. Lavecchia, P. Lorenzo, M. R. Elphick, F. M. Laferla, G. Vendemiale, C. Grillo, F. Altieri, T. Cassano, and S. Gaetani. 2014. 'Altered expression of the CB1 cannabinoid receptor in the triple transgenic mouse model of Alzheimer's disease', J Alzheimers Dis, 40: 701-12.

Begbie, J., P. Doherty, and A. Graham. 2004. 'Cannabinoid receptor, CB1, expression follows neuronal differentiation in the early chick embryo', J Anat, 205: 213-8.

103 Belarbi, K., C. Arellano, R. Ferguson, T. Jopson, and S. Rosi. 2012. 'Chronic neuroinflammation impacts the recruitment of adult-born neurons into behaviorally relevant hippocampal networks', Brain Behav Immun, 26: 18-23.

Ben-Hur, T., O. Ben-Menachem, V. Furer, O. Einstein, R. Mizrachi-Kol, and N. Grigoriadis. 2003. 'Effects of proinflammatory cytokines on the growth, fate, and motility of multipotential neural precursor cells', Mol Cell Neurosci, 24: 623-31.

Benito, C., E. Nunez, R. M. Tolon, E. J. Carrier, A. Rabano, C. J. Hillard, and J. Romero. 2003. 'Cannabinoid CB2 receptors and fatty acid amide hydrolase are selectively overexpressed in neuritic plaque-associated glia in Alzheimer's disease brains', J Neurosci, 23: 11136-41.

Blanco-Calvo, E., P. Rivera, S. Arrabal, A. Vargas, F. J. Pavon, A. Serrano, E. Castilla-Ortega, P. Galeano, L. Rubio, J. Suarez, and F. Rodriguez de Fonseca. 2014. 'Pharmacological blockade of either cannabinoid CB1 or CB2 receptors prevents both cocaine-induced conditioned locomotion and cocaine-induced reduction of cell proliferation in the hippocampus of adult male rat', Front Integr Neurosci, 7: 106.

Blankman, J. L., G. M. Simon, and B. F. Cravatt. 2007. 'A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol', Chem Biol, 14: 1347-56.

Blazquez, C., C. Sanchez, A. Daza, I. Galve-Roperh, and M. Guzman. 1999. 'The stimulation of ketogenesis by cannabinoids in cultured astrocytes defines carnitine palmitoyltransferase I as a new ceramide-activated enzyme', J Neurochem, 72: 1759-68.

Blondeau, N., I. Lauritzen, C. Widmann, M. Lazdunski, and C. Heurteaux. 2002. 'A potent protective role of lysophospholipids against global cerebral ischemia and glutamate excitotoxicity in neuronal cultures', J Cereb Blood Flow Metab, 22: 821-34.

Boekhoorn, K., M. Joels, and P. J. Lucassen. 2006. 'Increased proliferation reflects glial and vascular-associated changes, but not neurogenesis in the presenile Alzheimer hippocampus', Neurobiol Dis, 24: 1-14.

Boldrini, M., C. A. Fulmore, A. N. Tartt, L. R. Simeon, I. Pavlova, V. Poposka, G. B. Rosoklija, A. Stankov, V. Arango, A. J. Dwork, R. Hen, and J. J. Mann. 2018. 'Human Hippocampal Neurogenesis Persists throughout Aging', Cell Stem Cell, 22: 589-99.e5.

104 Boldrini, M., R. Hen, M. D. Underwood, G. B. Rosoklija, A. J. Dwork, J. J. Mann, and V. Arango. 2012. 'Hippocampal angiogenesis and progenitor cell proliferation are increased with antidepressant use in major depression', Biol Psychiatry, 72: 562-71.

Boldrini, M., A. N. Santiago, R. Hen, A. J. Dwork, G. B. Rosoklija, H. Tamir, V. Arango, and J. John Mann. 2013. 'Hippocampal granule neuron number and dentate gyrus volume in antidepressant-treated and untreated major depression', Neuropsychopharmacology, 38: 1068-77.

Bonaguidi, M. A., M. A. Wheeler, J. S. Shapiro, R. P. Stadel, G. J. Sun, G. L. Ming, and H. Song. 2011. 'In vivo clonal analysis reveals self-renewing and multipotent adult neural stem cell characteristics', Cell, 145: 1142-55.

Borrelli, F., B. Romano, S. Petrosino, E. Pagano, R. Capasso, D. Coppola, G. Battista, P. Orlando, V. Di Marzo, and A. A. Izzo. 2015. 'Palmitoylethanolamide, a naturally occurring lipid, is an orally effective intestinal anti-inflammatory agent', Br J Pharmacol, 172: 142-58.

Borsini, A., S. Alboni, M. A. Horowitz, L. M. Tojo, G. Cannazza, K. P. Su, C. M. Pariante, and P. A. Zunszain. 2017. 'Rescue of IL-1beta-induced reduction of human neurogenesis by omega-3 fatty acids and antidepressants', Brain Behav Immun, 65: 230-38.

Bortolato, M., V. Bini, R. Frau, P. Devoto, A. Pardu, Y. Fan, and M. V. Solbrig. 2014. 'Juvenile cannabinoid treatment induces frontostriatal gliogenesis in Lewis rats', Eur Neuropsychopharmacol, 24: 974-85.

Brasier, A. R. 2010. 'The nuclear factor-kappaB-interleukin-6 signalling pathway mediating vascular inflammation', Cardiovasc Res, 86: 211-8.

Brown, A. J., D. A. Daniels, M. Kassim, S. Brown, C. P. Haslam, V. R. Terrell, J. Brown, P. L. Nichols, P. C. Staton, A. Wise, and S. J. Dowell. 2011. 'Pharmacology of GPR55 in yeast and identification of GSK494581A as a mixed-activity glycine transporter subtype 1 inhibitor and GPR55 agonist', J Pharmacol Exp Ther, 337: 236-46.

Butovsky, O., Y. Ziv, A. Schwartz, G. Landa, A. E. Talpalar, S. Pluchino, G. Martino, and M. Schwartz. 2006. 'Microglia activated by IL-4 or IFN-gamma differentially induce neurogenesis and oligodendrogenesis from adult stem/progenitor cells', Mol Cell Neurosci, 31: 149-60.

105 Cahill, S. P., R. Q. Yu, D. Green, E. V. Todorova, and J. S. Snyder. 2017. 'Early survival and delayed death of developmentally-born dentate gyrus neurons', Hippocampus, 27: 1155-67.

Campbell, S., M. Marriott, C. Nahmias, and G. M. MacQueen. 2004. 'Lower hippocampal volume in patients suffering from depression: a meta-analysis', Am J Psychiatry, 161: 598-607.

Campos, A. C., M. V. Fogaca, F. F. Scarante, S. R. L. Joca, A. J. Sales, F. V. Gomes, A. B. Sonego, N. S. Rodrigues, I. Galve-Roperh, and F. S. Guimaraes. 2017. 'Plastic and Neuroprotective Mechanisms Involved in the Therapeutic Effects of Cannabidiol in Psychiatric Disorders', Front Pharmacol, 8: 269.

Campos, A. C., Z. Ortega, J. Palazuelos, M. V. Fogaca, D. C. Aguiar, J. Diaz-Alonso, S. Ortega- Gutierrez, H. Vazquez-Villa, F. A. Moreira, M. Guzman, I. Galve-Roperh, and F. S. Guimaraes. 2013. 'The anxiolytic effect of cannabidiol on chronically stressed mice depends on hippocampal neurogenesis: involvement of the endocannabinoid system', Int J Neuropsychopharmacol, 16: 1407-19.

Cantarella, G., M. Scollo, L. Lempereur, G. Saccani-Jotti, F. Basile, and R. Bernardini. 2011. 'Endocannabinoids inhibit release of nerve growth factor by inflammation-activated mast cells', Biochem Pharmacol, 82: 380-8.

Chen, B., K. Bromley-Brits, G. He, F. Cai, X. Zhang, and W. Song. 2010. 'Effect of synthetic cannabinoid HU210 on memory deficits and neuropathology in Alzheimer's disease mouse model', Curr Alzheimer Res, 7: 255-61.

Chen, Z., and T. D. Palmer. 2013. 'Differential roles of TNFR1 and TNFR2 signaling in adult hippocampal neurogenesis', Brain Behav Immun, 30: 45-53.

Cherif, H., A. Argaw, B. Cecyre, A. Bouchard, J. Gagnon, P. Javadi, S. Desgent, K. Mackie, and J. F. Bouchard. 2015. 'Role of GPR55 during Axon Growth and Target Innervation', eNeuro, 2.

Chew, L. J., A. Takanohashi, and M. Bell. 2006. 'Microglia and inflammation: impact on developmental brain injuries', Ment Retard Dev Disabil Res Rev, 12: 105-12.

Chiu, C. T., Z. Wang, J. G. Hunsberger, and D. M. Chuang. 2013. 'Therapeutic potential of mood stabilizers lithium and valproic acid: beyond bipolar disorder', Pharmacol Rev, 65: 105- 42.

106 Chiurchiu, V., M. Lanuti, M. De Bardi, L. Battistini, and M. Maccarrone. 2015. 'The differential characterization of GPR55 receptor in human peripheral blood reveals a distinctive expression in monocytes and NK cells and a proinflammatory role in these innate cells', Int Immunol, 27: 153-60.

Christian, K. M., H. Song, and G. L. Ming. 2014. 'Functions and dysfunctions of adult hippocampal neurogenesis', Annu Rev Neurosci, 37: 243-62.

Coe, C. L., M. Kramer, B. Czeh, E. Gould, A. J. Reeves, C. Kirschbaum, and E. Fuchs. 2003. 'Prenatal stress diminishes neurogenesis in the dentate gyrus of juvenile rhesus monkeys', Biol Psychiatry, 54: 1025-34.

Colotta, F., F. Re, M. Muzio, R. Bertini, N. Polentarutti, M. Sironi, J. G. Giri, S. K. Dower, J. E. Sims, and A. Mantovani. 1993. 'Interleukin-1 type II receptor: a decoy target for IL-1 that is regulated by IL-4', Science, 261: 472-5.

Compagnucci, C., S. Di Siena, M. B. Bustamante, D. Di Giacomo, M. Di Tommaso, M. Maccarrone, P. Grimaldi, and C. Sette. 2013. 'Type-1 (CB1) cannabinoid receptor promotes neuronal differentiation and maturation of neural stem cells', PLoS One, 8: e54271.

Costola-de-Souza, C., A. Ribeiro, V. Ferraz-de-Paula, A. S. Calefi, T. P. Aloia, J. A. Gimenes-Junior, V. I. de Almeida, M. L. Pinheiro, and J. Palermo-Neto. 2013. 'Monoacylglycerol lipase (MAGL) inhibition attenuates acute lung injury in mice', PLoS One, 8: e77706.

Crampton, S. J., L. M. Collins, A. Toulouse, Y. M. Nolan, and G. W. O'Keeffe. 2012. 'Exposure of foetal neural progenitor cells to IL-1beta impairs their proliferation and alters their differentiation - a role for maternal inflammation?', J Neurochem, 120: 964-73.

Cravatt, B. F., D. K. Giang, S. P. Mayfield, D. L. Boger, R. A. Lerner, and N. B. Gilula. 1996. 'Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides', Nature, 384: 83-7.

Cunningham, C., D. C. Wilcockson, S. Campion, K. Lunnon, and V. H. Perry. 2005. 'Central and systemic endotoxin challenges exacerbate the local inflammatory response and increase neuronal death during chronic neurodegeneration', J Neurosci, 25: 9275-84.

Das, S., and A. Basu. 2008. 'Inflammation: a new candidate in modulating adult neurogenesis', J Neurosci Res, 86: 1199-208.

107

Dayer, A. G., A. A. Ford, K. M. Cleaver, M. Yassaee, and H. A. Cameron. 2003. 'Short-term and long-term survival of new neurons in the rat dentate gyrus', J Comp Neurol, 460: 563-72.

De Marchi, N., L. De Petrocellis, P. Orlando, F. Daniele, F. Fezza, and V. Di Marzo. 2003. 'Endocannabinoid signalling in the blood of patients with schizophrenia', Lipids Health Dis, 2: 5.

de Oliveira, R. W., C. L. Oliveira, F. S. Guimaraes, and A. C. Campos. 2018. 'Cannabinoid signalling in embryonic and adult neurogenesis: possible implications for psychiatric and neurological disorders', Acta Neuropsychiatr: 1-16.

Deng, W., J. B. Aimone, and F. H. Gage. 2010. 'New neurons and new memories: how does adult hippocampal neurogenesis affect learning and memory?', Nat Rev Neurosci, 11: 339-50.

Dev, A., S. Iyer, B. Razani, and G. Cheng. 2011. 'NF-kappaB and innate immunity', Curr Top Microbiol Immunol, 349: 115-43.

Diaz-Alonso, J., T. Aguado, A. de Salas-Quiroga, Z. Ortega, M. Guzman, and I. Galve-Roperh. 2015. 'CB1 Cannabinoid Receptor-Dependent Activation of mTORC1/Pax6 Signaling Drives Tbr2 Expression and Basal Progenitor Expansion in the Developing Mouse Cortex', Cereb Cortex, 25: 2395-408.

Dillon, S. E., D. Tsivos, M. Knight, B. McCann, C. Pennington, A. I. Shiel, M. E. Conway, M. A. Newson, R. A. Kauppinen, and E. J. Coulthard. 2017. 'The impact of ageing reveals distinct roles for human dentate gyrus and CA3 in pattern separation and object recognition memory', Sci Rep, 7: 14069.

Dirikoc, S., S. A. Priola, M. Marella, N. Zsurger, and J. Chabry. 2007. 'Nonpsychoactive cannabidiol prevents prion accumulation and protects neurons against prion toxicity', J Neurosci, 27: 9537-44.

Donato, R., E. A. Miljan, S. J. Hines, S. Aouabdi, K. Pollock, S. Patel, F. A. Edwards, and J. D. Sinden. 2007. 'Differential development of neuronal physiological responsiveness in two human neural stem cell lines', BMC Neurosci, 8: 36.

108 Duan, X., J. H. Chang, S. Ge, R. L. Faulkner, J. Y. Kim, Y. Kitabatake, X. B. Liu, C. H. Yang, J. D. Jordan, D. K. Ma, C. Y. Liu, S. Ganesan, H. J. Cheng, G. L. Ming, B. Lu, and H. Song. 2007. 'Disrupted-In-Schizophrenia 1 regulates integration of newly generated neurons in the adult brain', Cell, 130: 1146-58.

Ekdahl, C. T., J. H. Claasen, S. Bonde, Z. Kokaia, and O. Lindvall. 2003. 'Inflammation is detrimental for neurogenesis in adult brain', Proc Natl Acad Sci U S A, 100: 13632-7.

El Marroun, H., J. J. Hudziak, H. Tiemeier, H. Creemers, E. A. Steegers, V. W. Jaddoe, A. Hofman, F. C. Verhulst, W. van den Brink, and A. C. Huizink. 2011. 'Intrauterine cannabis exposure leads to more aggressive behavior and attention problems in 18-month-old girls', Drug Alcohol Depend, 118: 470-4.

Elbegdorj, O., R. B. Westkaemper, and Y. Zhang. 2013. 'A homology modeling study toward the understanding of three-dimensional structure and putative pharmacological profile of the G-protein coupled receptor GPR55', J Mol Graph Model, 39: 50-60.

Elsohly, M. A., and D. Slade. 2005. 'Chemical constituents of marijuana: the complex mixture of natural cannabinoids', Life Sci, 78: 539-48.

Eriksson, P. S., E. Perfilieva, T. Bjork-Eriksson, A. M. Alborn, C. Nordborg, D. A. Peterson, and F. H. Gage. 1998. 'Neurogenesis in the adult human hippocampus', Nat Med, 4: 1313-7.

Esposito, G., C. Scuderi, M. Valenza, G. I. Togna, V. Latina, D. De Filippis, M. Cipriano, M. R. Carratu, T. Iuvone, and L. Steardo. 2011. 'Cannabidiol reduces Abeta-induced neuroinflammation and promotes hippocampal neurogenesis through PPARgamma involvement', PLoS One, 6: e28668.

Fakhoury, M. 2017. 'Role of the Endocannabinoid System in the Pathophysiology of Schizophrenia', Mol Neurobiol, 54: 768-78.

Farina, C., F. Aloisi, and E. Meinl. 2007. 'Astrocytes are active players in cerebral innate immunity', Trends Immunol, 28: 138-45.

Felger, J. C., O. Alagbe, F. Hu, D. Mook, A. A. Freeman, M. M. Sanchez, N. H. Kalin, E. Ratti, C. B. Nemeroff, and A. H. Miller. 2007. 'Effects of interferon-alpha on rhesus monkeys: a nonhuman primate model of cytokine-induced depression', Biol Psychiatry, 62: 1324-33.

109 Ferguson, D., S. Clarke, N. Berry, and N. Almond. 2016. 'Attenuated SIV causes persisting neuroinflammation in the absence of a chronic viral load and neurotoxic antiretroviral therapy', Aids, 30: 2439-48.

Ferrell, D., and B. Giunta. 2014. 'The impact of HIV-1 on neurogenesis: implications for HAND', Cell Mol Life Sci, 71: 4387-92.

Fisher, B. J., E. Naumova, C. C. Leighton, G. N. Naumov, N. Kerklviet, D. Fortin, D. R. Macdonald, J. G. Cairncross, G. S. Bauman, and L. Stitt. 2002. 'Ki-67: a prognostic factor for low-grade glioma?', Int J Radiat Oncol Biol Phys, 52: 996-1001.

Fredriksson, R., P. J. Hoglund, D. E. Gloriam, M. C. Lagerstrom, and H. B. Schioth. 2003. 'Seven evolutionarily conserved human rhodopsin G protein-coupled receptors lacking close relatives', FEBS Lett, 554: 381-8.

Fried, P. A. 2002. 'Conceptual issues in behavioral teratology and their application in determining long-term sequelae of prenatal marihuana exposure', J Child Psychol Psychiatry, 43: 81-102.

Gao, Y., D. V. Vasilyev, M. B. Goncalves, F. V. Howell, C. Hobbs, M. Reisenberg, R. Shen, M. Y. Zhang, B. W. Strassle, P. Lu, L. Mark, M. J. Piesla, K. Deng, E. V. Kouranova, R. H. Ring, G. T. Whiteside, B. Bates, F. S. Walsh, G. Williams, M. N. Pangalos, T. A. Samad, and P. Doherty. 2010. 'Loss of retrograde endocannabinoid signaling and reduced adult neurogenesis in diacylglycerol lipase knock-out mice', J Neurosci, 30: 2017-24.

Garber, C., M. J. Vasek, L. L. Vollmer, T. Sun, X. Jiang, and R. S. Klein. 2018. 'Astrocytes decrease adult neurogenesis during virus-induced memory dysfunction via IL-1', Nat Immunol, 19: 151-61.

Garcia-Gutierrez, M. S., J. M. Perez-Ortiz, A. Gutierrez-Adan, and J. Manzanares. 2010. 'Depression-resistant endophenotype in mice overexpressing cannabinoid CB(2) receptors', Br J Pharmacol, 160: 1773-84.

Garcia-Ovejero, D., A. Arevalo-Martin, B. Navarro-Galve, E. Pinteaux, E. Molina-Holgado, and F. Molina-Holgado. 2013. 'Neuroimmmune interactions of cannabinoids in neurogenesis: focus on interleukin-1beta (IL-1beta) signalling', Biochem Soc Trans, 41: 1577-82.

110 Gil, J. M., P. Mohapel, I. M. Araujo, N. Popovic, J. Y. Li, P. Brundin, and A. Petersen. 2005. 'Reduced hippocampal neurogenesis in R6/2 transgenic Huntington's disease mice', Neurobiol Dis, 20: 744-51.

Gil-Mohapel, J., J. M. Simpson, M. Ghilan, and B. R. Christie. 2011. 'Neurogenesis in Huntington's disease: can studying adult neurogenesis lead to the development of new therapeutic strategies?', Brain Res, 1406: 84-105.

Godbout, J. P., J. Chen, J. Abraham, A. F. Richwine, B. M. Berg, K. W. Kelley, and R. W. Johnson. 2005. 'Exaggerated neuroinflammation and sickness behavior in aged mice following activation of the peripheral innate immune system', Faseb j, 19: 1329-31.

Goncalves, J. T., S. T. Schafer, and F. H. Gage. 2016. 'Adult Neurogenesis in the Hippocampus: From Stem Cells to Behavior', Cell, 167: 897-914.

Gould, E., P. Tanapat, B. S. McEwen, G. Flugge, and E. Fuchs. 1998. 'Proliferation of granule cell precursors in the dentate gyrus of adult monkeys is diminished by stress', Proc Natl Acad Sci U S A, 95: 3168-71.

Green, H. F., and Y. M. Nolan. 2012. 'Unlocking mechanisms in interleukin-1beta-induced changes in hippocampal neurogenesis--a role for GSK-3beta and TLX', Transl Psychiatry, 2: e194.

Green, H. F., E. Treacy, A. K. Keohane, A. M. Sullivan, G. W. O'Keeffe, and Y. M. Nolan. 2012. 'A role for interleukin-1beta in determining the lineage fate of embryonic rat hippocampal neural precursor cells', Mol Cell Neurosci, 49: 311-21.

Guadagno, J., P. Swan, R. Shaikh, and S. P. Cregan. 2015. 'Microglia-derived IL-1beta triggers p53-mediated cell cycle arrest and apoptosis in neural precursor cells', Cell Death Dis, 6: e1779.

Halappanavar, S., J. Nikota, D. Wu, A. Williams, C. L. Yauk, and M. Stampfli. 2013. 'IL-1 receptor regulates microRNA-135b expression in a negative feedback mechanism during cigarette smoke-induced inflammation', J Immunol, 190: 3679-86.

Hall, W., and L. Degenhardt. 2009. 'Adverse health effects of non-medical cannabis use', Lancet, 374: 1383-91.

111 Harkany, T., E. Keimpema, K. Barabas, and J. Mulder. 2008. 'Endocannabinoid functions controlling neuronal specification during brain development', Mol Cell Endocrinol, 286: S84-90.

Heckers, S. 2001. 'Neuroimaging studies of the hippocampus in schizophrenia', Hippocampus, 11: 520-8.

Henstridge, C. M., N. A. Balenga, L. A. Ford, R. A. Ross, M. Waldhoer, and A. J. Irving. 2009. 'The GPR55 ligand L-alpha-lysophosphatidylinositol promotes RhoA-dependent Ca2+ signaling and NFAT activation', Faseb j, 23: 183-93.

Henstridge, C. M., N. A. Balenga, R. Schroder, J. K. Kargl, W. Platzer, L. Martini, S. Arthur, J. Penman, J. L. Whistler, E. Kostenis, M. Waldhoer, and A. J. Irving. 2010. 'GPR55 ligands promote receptor coupling to multiple signalling pathways', Br J Pharmacol, 160: 604- 14.

Hepgul, N., A. Cattaneo, K. Agarwal, S. Baraldi, A. Borsini, C. Bufalino, D. M. Forton, V. Mondelli, N. Nikkheslat, N. Lopizzo, M. A. Riva, A. Russell, M. Hotopf, and C. M. Pariante. 2016. 'Transcriptomics in Interferon-alpha-Treated Patients Identifies Inflammation-, Neuroplasticity- and Oxidative Stress-Related Signatures as Predictors and Correlates of Depression', Neuropsychopharmacology, 41: 2502-11.

Hermanson, D. J., J. C. Gamble-George, L. J. Marnett, and S. Patel. 2014. 'Substrate-selective COX-2 inhibition as a novel strategy for therapeutic endocannabinoid augmentation', Trends Pharmacol Sci, 35: 358-67.

Heynen-Genel, S., R. Dahl, S. Shi, L. Milan, S. Hariharan, E. Sergienko, M. Hedrick, S. Dad, D. Stonich, Y. Su, M. Vicchiarelli, A. Mangravita-Novo, L. H. Smith, T. D. Y. Chung, H. Sharir, M. G. Caron, L. S. Barak, and M. E. Abood. 2010. 'Screening for Selective Ligands for GPR55 - Antagonists.' in, Probe Reports from the NIH Molecular Libraries Program (National Center for Biotechnology Information (US): Bethesda (MD)).

Hill, A. S., A. Sahay, and R. Hen. 2015. 'Increasing Adult Hippocampal Neurogenesis is Sufficient to Reduce Anxiety and Depression-Like Behaviors', Neuropsychopharmacology, 40: 2368-78.

Hill, J. D., V. Zuluaga-Ramirez, S. Gajghate, M. Winfield, and Y. Persidsky. 2018. 'Activation of GPR55 increases neural stem cell proliferation and promotes early adult hippocampal neurogenesis', Br J Pharmacol, 175: 3407-21.

112

Hill, M. N., and B. B. Gorzalka. 2005. 'Is there a role for the endocannabinoid system in the etiology and treatment of melancholic depression?', Behav Pharmacol, 16: 333-52.

Hill, M. N., G. E. Miller, E. J. Carrier, B. B. Gorzalka, and C. J. Hillard. 2009. 'Circulating endocannabinoids and N-acyl ethanolamines are differentially regulated in major depression and following exposure to social stress', Psychoneuroendocrinology, 34: 1257-62.

Hill, M. N., G. E. Miller, W. S. Ho, B. B. Gorzalka, and C. J. Hillard. 2008. 'Serum endocannabinoid content is altered in females with depressive disorders: a preliminary report', Pharmacopsychiatry, 41: 48-53.

Hill, M. N., A. K. Titterness, A. C. Morrish, E. J. Carrier, T. T. Lee, J. Gil-Mohapel, B. B. Gorzalka, C. J. Hillard, and B. R. Christie. 2010. 'Endogenous cannabinoid signaling is required for voluntary exercise-induced enhancement of progenitor cell proliferation in the hippocampus', Hippocampus, 20: 513-23.

Hillard, C. J., and Q. S. Liu. 2014. 'Endocannabinoid signaling in the etiology and treatment of major depressive illness', Curr Pharm Des, 20: 3795-811.

Hoglinger, G. U., P. Rizk, M. P. Muriel, C. Duyckaerts, W. H. Oertel, I. Caille, and E. C. Hirsch. 2004. 'Dopamine depletion impairs precursor cell proliferation in Parkinson disease', Nat Neurosci, 7: 726-35.

Hollands, C., N. Bartolotti, and O. Lazarov. 2016. 'Alzheimer's Disease and Hippocampal Adult Neurogenesis; Exploring Shared Mechanisms', Front Neurosci, 10: 178.

Hornung, V., S. Rothenfusser, S. Britsch, A. Krug, B. Jahrsdorfer, T. Giese, S. Endres, and G. Hartmann. 2002. 'Quantitative expression of toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides', J Immunol, 168: 4531-7.

Howlett, A. C., F. Barth, T. I. Bonner, G. Cabral, P. Casellas, W. A. Devane, C. C. Felder, M. Herkenham, K. Mackie, B. R. Martin, R. Mechoulam, and R. G. Pertwee. 2002. 'International Union of Pharmacology. XXVII. Classification of cannabinoid receptors', Pharmacol Rev, 54: 161-202.

113 Hritcu, L., and L. D. Gorgan. 2014. 'Intranigral lipopolysaccharide induced anxiety and depression by altered BDNF mRNA expression in rat hippocampus', Prog Neuropsychopharmacol Biol Psychiatry, 51: 126-32.

Hurst, K., C. Badgley, T. Ellsworth, S. Bell, L. Friend, B. Prince, J. Welch, Z. Cowan, R. Williamson, C. Lyon, B. Anderson, B. Poole, M. Christensen, M. McNeil, J. Call, and J. G. Edwards. 2017. 'A putative lysophosphatidylinositol receptor GPR55 modulates hippocampal synaptic plasticity', Hippocampus.

Iadecola, C., and J. Anrather. 2011. 'The immunology of stroke: from mechanisms to translation', Nat Med, 17: 796-808.

Iosif, R. E., C. T. Ekdahl, H. Ahlenius, C. J. Pronk, S. Bonde, Z. Kokaia, S. E. Jacobsen, and O. Lindvall. 2006. 'Tumor necrosis factor receptor 1 is a negative regulator of progenitor proliferation in adult hippocampal neurogenesis', J Neurosci, 26: 9703-12.

Jagasia, R., K. Steib, E. Englberger, S. Herold, T. Faus-Kessler, M. Saxe, F. H. Gage, H. Song, and D. C. Lie. 2009. 'GABA-cAMP response element-binding protein signaling regulates maturation and survival of newly generated neurons in the adult hippocampus', J Neurosci, 29: 7966-77.

Janefjord, E., J. L. Maag, B. S. Harvey, and S. D. Smid. 2014. 'Cannabinoid effects on beta amyloid fibril and aggregate formation, neuronal and microglial-activated neurotoxicity in vitro', Cell Mol Neurobiol, 34: 31-42.

Jenniches, I., S. Ternes, O. Albayram, D. M. Otte, K. Bach, L. Bindila, K. Michel, B. Lutz, A. Bilkei- Gorzo, and A. Zimmer. 2016. 'Anxiety, Stress, and Fear Response in Mice With Reduced Endocannabinoid Levels', Biol Psychiatry, 79: 858-68.

Jessberger, S., N. Toni, G. D. Clemenson, Jr., J. Ray, and F. H. Gage. 2008. 'Directed differentiation of hippocampal stem/progenitor cells in the adult brain', Nat Neurosci, 11: 888-93.

Jiang, W., Y. Zhang, L. Xiao, J. Van Cleemput, S. P. Ji, G. Bai, and X. Zhang. 2005. 'Cannabinoids promote embryonic and adult hippocampus neurogenesis and produce anxiolytic- and antidepressant-like effects', J Clin Invest, 115: 3104-16.

114 Jin, K., L. Xie, S. H. Kim, S. Parmentier-Batteur, Y. Sun, X. O. Mao, J. Childs, and D. A. Greenberg. 2004. 'Defective adult neurogenesis in CB1 cannabinoid receptor knockout mice', Mol Pharmacol, 66: 204-8.

Johns, D. G., D. J. Behm, D. J. Walker, Z. Ao, E. M. Shapland, D. A. Daniels, M. Riddick, S. Dowell, P. C. Staton, P. Green, U. Shabon, W. Bao, N. Aiyar, T. L. Yue, A. J. Brown, A. D. Morrison, and S. A. Douglas. 2007. 'The novel endocannabinoid receptor GPR55 is activated by atypical cannabinoids but does not mediate their vasodilator effects', Br J Pharmacol, 152: 825-31.

Johnstone, M., A. J. Gearing, and K. M. Miller. 1999. 'A central role for astrocytes in the inflammatory response to beta-amyloid; chemokines, cytokines and reactive oxygen species are produced', J Neuroimmunol, 93: 182-93.

Jung, K. M., G. Astarita, C. Zhu, M. Wallace, K. Mackie, and D. Piomelli. 2007. 'A key role for diacylglycerol lipase-alpha in metabotropic glutamate receptor-dependent endocannabinoid mobilization', Mol Pharmacol, 72: 612-21.

Kallendrusch, S., S. Kremzow, M. Nowicki, U. Grabiec, R. Winkelmann, A. Benz, R. Kraft, I. Bechmann, F. Dehghani, and M. Koch. 2013. 'The G protein-coupled receptor 55 ligand l- alpha-lysophosphatidylinositol exerts microglia-dependent neuroprotection after excitotoxic lesion', Glia, 61: 1822-31.

Kandasamy, M., S. Couillard-Despres, K. A. Raber, M. Stephan, B. Lehner, B. Winner, Z. Kohl, F. J. Rivera, H. P. Nguyen, O. Riess, U. Bogdahn, J. Winkler, S. von Horsten, and L. Aigner. 2010. 'Stem cell quiescence in the hippocampal neurogenic niche is associated with elevated transforming growth factor-beta signaling in an animal model of Huntington disease', J Neuropathol Exp Neurol, 69: 717-28.

Kano, M., T. Ohno-Shosaku, Y. Hashimotodani, M. Uchigashima, and M. Watanabe. 2009. 'Endocannabinoid-mediated control of synaptic transmission', Physiol Rev, 89: 309-80.

Kapur, A., P. Zhao, H. Sharir, Y. Bai, M. G. Caron, L. S. Barak, and M. E. Abood. 2009. 'Atypical responsiveness of the orphan receptor GPR55 to cannabinoid ligands', J Biol Chem, 284: 29817-27.

Karanian, D. A., Q. B. Brown, A. Makriyannis, and B. A. Bahr. 2005. 'Blocking cannabinoid activation of FAK and ERK1/2 compromises synaptic integrity in hippocampus', Eur J Pharmacol, 508: 47-56.

115

Kempermann, G., S. Jessberger, B. Steiner, and G. Kronenberg. 2004. 'Milestones of neuronal development in the adult hippocampus', Trends Neurosci, 27: 447-52.

Kerr, D. M., B. Harhen, B. N. Okine, L. J. Egan, D. P. Finn, and M. Roche. 2013. 'The monoacylglycerol lipase inhibitor JZL184 attenuates LPS-induced increases in cytokine expression in the rat frontal cortex and plasma: differential mechanisms of action', Br J Pharmacol, 169: 808-19.

Kim, K., D. H. Moore, A. Makriyannis, and M. E. Abood. 2006. 'AM1241, a cannabinoid CB2 receptor selective compound, delays disease progression in a mouse model of amyotrophic lateral sclerosis', Eur J Pharmacol, 542: 100-5.

Kim, S. J., T. G. Son, K. Kim, H. R. Park, M. P. Mattson, and J. Lee. 2007. 'Interferon-gamma promotes differentiation of neural progenitor cells via the JNK pathway', Neurochem Res, 32: 1399-406.

Kizil, C., N. Kyritsis, and M. Brand. 2015. 'Effects of inflammation on stem cells: together they strive?', EMBO Rep, 16: 416-26.

Knoth, R., I. Singec, M. Ditter, G. Pantazis, P. Capetian, R. P. Meyer, V. Horvat, B. Volk, and G. Kempermann. 2010. 'Murine features of neurogenesis in the human hippocampus across the lifespan from 0 to 100 years', PLoS One, 5: e8809.

Kohler, S. J., N. I. Williams, G. B. Stanton, J. L. Cameron, and W. T. Greenough. 2011. 'Maturation time of new granule cells in the dentate gyrus of adult macaque monkeys exceeds six months', Proc Natl Acad Sci U S A, 108: 10326-31.

Konat, G. W., T. Kielian, and I. Marriott. 2006. 'The role of Toll-like receptors in CNS response to microbial challenge', J Neurochem, 99: 1-12.

Koo, J. W., and R. S. Duman. 2008. 'IL-1beta is an essential mediator of the antineurogenic and anhedonic effects of stress', Proc Natl Acad Sci U S A, 105: 751-6.

Kotasova, H., J. Prochazkova, and J. Pachernik. 2014. 'Interaction of Notch and gp130 signaling in the maintenance of neural stem and progenitor cells', Cell Mol Neurobiol, 34: 1-15.

116 Krakauer, T. 2004. 'Molecular therapeutic targets in inflammation: cyclooxygenase and NF- kappaB', Curr Drug Targets Inflamm Allergy, 3: 317-24.

Kuhn, H. G., T. Toda, and F. H. Gage. 2018. 'Adult hippocampal neurogenesis: a coming-of-age story', J Neurosci.

Kwak, Y. D., E. Dantuma, S. Merchant, S. Bushnev, and K. Sugaya. 2010. 'Amyloid-beta precursor protein induces glial differentiation of neural progenitor cells by activation of the IL- 6/gp130 signaling pathway', Neurotox Res, 18: 328-38.

Kyritsis, N., C. Kizil, and M. Brand. 2014. 'Neuroinflammation and central nervous system regeneration in vertebrates', Trends Cell Biol, 24: 128-35.

Lauckner, J. E., J. B. Jensen, H. Y. Chen, H. C. Lu, B. Hille, and K. Mackie. 2008. 'GPR55 is a cannabinoid receptor that increases intracellular calcium and inhibits M current', Proc Natl Acad Sci U S A, 105: 2699-704.

Lee, S., D. H. Kim, S. H. Yoon, and J. H. Ryu. 2009. 'Sub-chronic administration of rimonabant causes loss of antidepressive activity and decreases doublecortin immunoreactivity in the mouse hippocampus', Neurosci Lett, 467: 111-6.

Lehnardt, S., L. Massillon, P. Follett, F. E. Jensen, R. Ratan, P. A. Rosenberg, J. J. Volpe, and T. Vartanian. 2003. 'Activation of innate immunity in the CNS triggers neurodegeneration through a Toll-like receptor 4-dependent pathway', Proc Natl Acad Sci U S A, 100: 8514- 9.

Li, B., H. Yamamori, Y. Tatebayashi, B. Shafit-Zagardo, H. Tanimukai, S. Chen, K. Iqbal, and I. Grundke-Iqbal. 2008. 'Failure of neuronal maturation in Alzheimer disease dentate gyrus', J Neuropathol Exp Neurol, 67: 78-84.

Li, C. H., J. H. Wang, and H. P. Redmond. 2006. 'Bacterial lipoprotein-induced self-tolerance and cross-tolerance to LPS are associated with reduced IRAK-1 expression and MyD88-IRAK complex formation', J Leukoc Biol, 79: 867-75.

Li, K., J. Fichna, R. Schicho, D. Saur, M. Bashashati, K. Mackie, Y. Li, A. Zimmer, B. Goke, K. A. Sharkey, and M. Storr. 2013. 'A role for O-1602 and G protein-coupled receptor GPR55 in the control of colonic motility in mice', Neuropharmacology, 71: 255-63.

117 Li, W., B. Viengkhou, G. Denyer, P. K. West, I. L. Campbell, and M. J. Hofer. 2018. 'Microglia have a more extensive and divergent response to interferon-alpha compared with astrocytes', Glia.

Li, X., J. M. Newbern, Y. Wu, M. Morgan-Smith, J. Zhong, J. Charron, and W. D. Snider. 2012. 'MEK Is a Key Regulator of Gliogenesis in the Developing Brain', Neuron, 75: 1035-50.

Li, Y. F., H. X. Chen, Y. Liu, Y. Z. Zhang, Y. Q. Liu, and J. Li. 2006. 'Agmatine increases proliferation of cultured hippocampal progenitor cells and hippocampal neurogenesis in chronically stressed mice', Acta Pharmacol Sin, 27: 1395-400.

Lieberman, A. P., P. M. Pitha, H. S. Shin, and M. L. Shin. 1989. 'Production of tumor necrosis factor and other cytokines by astrocytes stimulated with lipopolysaccharide or a neurotropic virus', Proc Natl Acad Sci U S A, 86: 6348-52.

Lin, X. H., B. Yuece, Y. Y. Li, Y. J. Feng, J. Y. Feng, L. Y. Yu, K. Li, Y. N. Li, and M. Storr. 2011. 'A novel CB receptor GPR55 and its ligands are involved in regulation of gut movement in rodents', Neurogastroenterol Motil, 23: 862-e342.

Lingerfelt, M. A., P. Zhao, H. P. Sharir, D. P. Hurst, P. H. Reggio, and M. E. Abood. 2017. 'Identification of Crucial Amino Acid Residues Involved in Agonist Signaling at the GPR55 Receptor', Biochemistry, 56: 473-86.

Liu, L., T. Foroud, X. Xuei, W. Berrettini, W. Byerley, W. Coryell, R. El-Mallakh, E. S. Gershon, J. R. Kelsoe, W. B. Lawson, D. F. MacKinnon, M. McInnis, F. J. McMahon, D. L. Murphy, J. Rice, W. Scheftner, P. P. Zandi, F. W. Lohoff, A. B. Niculescu, E. T. Meyer, H. J. Edenberg, and J. I. Nurnberger, Jr. 2008. 'Evidence of association between brain-derived neurotrophic factor gene and bipolar disorder', Psychiatr Genet, 18: 267-74.

Liu, Y. P., H. I. Lin, and S. F. Tzeng. 2005. 'Tumor necrosis factor-alpha and interleukin-18 modulate neuronal cell fate in embryonic neural progenitor culture', Brain Res, 1054: 152-8.

Louvi, A., and S. Artavanis-Tsakonas. 2006. 'Notch signalling in vertebrate neural development', Nat Rev Neurosci, 7: 93-102.

Lu, H. C., and K. Mackie. 2016. 'An Introduction to the Endogenous Cannabinoid System', Biol Psychiatry, 79: 516-25.

118

Lucassen, P. J., M. W. Stumpel, Q. Wang, and E. Aronica. 2010. 'Decreased numbers of progenitor cells but no response to antidepressant drugs in the hippocampus of elderly depressed patients', Neuropharmacology, 58: 940-9.

Luchicchi, A., and M. Pistis. 2012. 'Anandamide and 2-arachidonoylglycerol: pharmacological properties, functional features, and emerging specificities of the two major endocannabinoids', Mol Neurobiol, 46: 374-92.

Lugert, S., O. Basak, P. Knuckles, U. Haussler, K. Fabel, M. Gotz, C. A. Haas, G. Kempermann, V. Taylor, and C. Giachino. 2010. 'Quiescent and active hippocampal neural stem cells with distinct morphologies respond selectively to physiological and pathological stimuli and aging', Cell Stem Cell, 6: 445-56.

Ma, D. K., M. C. Marchetto, J. U. Guo, G. L. Ming, F. H. Gage, and H. Song. 2010. 'Epigenetic choreographers of neurogenesis in the adult mammalian brain', Nat Neurosci, 13: 1338- 44.

Macas, J., C. Nern, K. H. Plate, and S. Momma. 2006. 'Increased generation of neuronal progenitors after ischemic injury in the aged adult human forebrain', J Neurosci, 26: 13114-9.

Mackie, K. 2005. 'Distribution of cannabinoid receptors in the central and peripheral nervous system', Handb Exp Pharmacol: 299-325.

Mahar, I., F. R. Bambico, N. Mechawar, and J. N. Nobrega. 2014. 'Stress, serotonin, and hippocampal neurogenesis in relation to depression and antidepressant effects', Neurosci Biobehav Rev, 38: 173-92.

Marichal-Cancino, B. A., A. Sanchez-Fuentes, M. Mendez-Diaz, A. E. Ruiz-Contreras, and O. Prospero-Garcia. 2016. 'Blockade of GPR55 in the dorsolateral striatum impairs performance of rats in a T-maze paradigm', Behav Pharmacol, 27: 393-6.

Martinez-Gras, I., J. Hoenicka, G. Ponce, R. Rodriguez-Jimenez, M. A. Jimenez-Arriero, E. Perez- Hernandez, I. Ampuero, J. A. Ramos-Atance, T. Palomo, and G. Rubio. 2006. '(AAT)n repeat in the cannabinoid receptor gene, CNR1: association with schizophrenia in a Spanish population', Eur Arch Psychiatry Clin Neurosci, 256: 437-41.

119 Martinez-Pinilla, E., I. Reyes-Resina, A. Onatibia-Astibia, M. Zamarbide, A. Ricobaraza, G. Navarro, E. Moreno, I. G. Dopeso-Reyes, S. Sierra, A. J. Rico, E. Roda, J. L. Lanciego, and R. Franco. 2014. 'CB1 and GPR55 receptors are co-expressed and form heteromers in rat and monkey striatum', Exp Neurol, 261: 44-52.

Mathews, K. J., K. M. Allen, D. Boerrigter, H. Ball, C. Shannon Weickert, and K. L. Double. 2017. 'Evidence for reduced neurogenesis in the aging human hippocampus despite stable stem cell markers', Aging Cell, 16: 1195-99.

McHugh, D., J. Wager-Miller, J. Page, and H. B. Bradshaw. 2012. 'siRNA knockdown of GPR18 receptors in BV-2 microglia attenuates N-arachidonoyl glycine-induced cell migration', J Mol Signal, 7: 10.

Millar, J. K., J. C. Wilson-Annan, S. Anderson, S. Christie, M. S. Taylor, C. A. Semple, R. S. Devon, D. M. St Clair, W. J. Muir, D. H. Blackwood, and D. J. Porteous. 2000. 'Disruption of two novel genes by a translocation co-segregating with schizophrenia', Hum Mol Genet, 9: 1415-23.

Ming, G. L., and H. Song. 2011. 'Adult neurogenesis in the mammalian brain: significant answers and significant questions', Neuron, 70: 687-702.

Ming, Z., G. Sawicki, and L. K. Bekar. 2015. 'Acute systemic LPS-mediated inflammation induces lasting changes in mouse cortical neuromodulation and behavior', Neurosci Lett, 590: 96-100.

Mirescu, C., J. D. Peters, and E. Gould. 2004. 'Early life experience alters response of adult neurogenesis to stress', Nat Neurosci, 7: 841-6.

Molina-Holgado, F., E. Molina-Holgado, C. Guaza, and N. J. Rothwell. 2002. 'Role of CB1 and CB2 receptors in the inhibitory effects of cannabinoids on lipopolysaccharide-induced nitric oxide release in astrocyte cultures', J Neurosci Res, 67: 829-36.

Molina-Holgado, F., A. Rubio-Araiz, D. Garcia-Ovejero, R. J. Williams, J. D. Moore, A. Arevalo- Martin, O. Gomez-Torres, and E. Molina-Holgado. 2007. 'CB2 cannabinoid receptors promote mouse neural stem cell proliferation', Eur J Neurosci, 25: 629-34.

Monje, M. L., S. Mizumatsu, J. R. Fike, and T. D. Palmer. 2002. 'Irradiation induces neural precursor-cell dysfunction', Nat Med, 8: 955-62.

120

Monje, M. L., H. Toda, and T. D. Palmer. 2003. 'Inflammatory blockade restores adult hippocampal neurogenesis', Science, 302: 1760-5.

Moreno-Navarrete, J. M., V. Catalan, L. Whyte, A. Diaz-Arteaga, R. Vazquez-Martinez, F. Rotellar, R. Guzman, J. Gomez-Ambrosi, M. R. Pulido, W. R. Russell, M. Imbernon, R. A. Ross, M. M. Malagon, C. Dieguez, J. M. Fernandez-Real, G. Fruhbeck, and R. Nogueiras. 2012. 'The L-alpha-lysophosphatidylinositol/GPR55 system and its potential role in human obesity', Diabetes, 61: 281-91.

Mossner, R., O. Mikova, E. Koutsilieri, M. Saoud, A. C. Ehlis, N. Muller, A. J. Fallgatter, and P. Riederer. 2007. 'Consensus paper of the WFSBP Task Force on Biological Markers: biological markers in depression', World J Biol Psychiatry, 8: 141-74.

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

Murataeva, N., A. Straiker, and K. Mackie. 2014. 'Parsing the players: 2-arachidonoylglycerol synthesis and degradation in the CNS', Br J Pharmacol, 171: 1379-91.

Namihira, M., and K. Nakashima. 2013. 'Mechanisms of astrocytogenesis in the mammalian brain', Curr Opin Neurobiol, 23: 921-7.

Nomura, D. K., B. E. Morrison, J. L. Blankman, J. Z. Long, S. G. Kinsey, M. C. Marcondes, A. M. Ward, Y. K. Hahn, A. H. Lichtman, B. Conti, and B. F. Cravatt. 2011. 'Endocannabinoid hydrolysis generates brain prostaglandins that promote neuroinflammation', Science, 334: 809-13.

Nomura, F., S. Akashi, Y. Sakao, S. Sato, T. Kawai, M. Matsumoto, K. Nakanishi, M. Kimoto, K. Miyake, K. Takeda, and S. Akira. 2000. 'Cutting edge: endotoxin tolerance in mouse peritoneal macrophages correlates with down-regulation of surface toll-like receptor 4 expression', J Immunol, 164: 3476-9.

Nyiri, G., C. Cserep, E. Szabadits, K. Mackie, and T. F. Freund. 2005. 'CB1 cannabinoid receptors are enriched in the perisynaptic annulus and on preterminal segments of hippocampal GABAergic axons', Neuroscience, 136: 811-22.

121 Oak, S., P. Mandrekar, D. Catalano, K. Kodys, and G. Szabo. 2006. 'TLR2- and TLR4-mediated signals determine attenuation or augmentation of inflammation by acute alcohol in monocytes', J Immunol, 176: 7628-35.

Oka, S., K. Nakajima, A. Yamashita, S. Kishimoto, and T. Sugiura. 2007. 'Identification of GPR55 as a lysophosphatidylinositol receptor', Biochem Biophys Res Commun, 362: 928-34.

Okamoto, S., Y. J. Kang, C. W. Brechtel, E. Siviglia, R. Russo, A. Clemente, A. Harrop, S. McKercher, M. Kaul, and S. A. Lipton. 2007. 'HIV/gp120 decreases adult neural progenitor cell proliferation via checkpoint kinase-mediated cell-cycle withdrawal and G1 arrest', Cell Stem Cell, 1: 230-6.

Ortega-Gutierrez, S., E. Molina-Holgado, and C. Guaza. 2005. 'Effect of anandamide uptake inhibition in the production of nitric oxide and in the release of cytokines in astrocyte cultures', Glia, 52: 163-8.

Pacher, P., S. Batkai, and G. Kunos. 2006. 'The endocannabinoid system as an emerging target of pharmacotherapy', Pharmacol Rev, 58: 389-462.

Palazuelos, J., T. Aguado, A. Egia, R. Mechoulam, M. Guzman, and I. Galve-Roperh. 2006. 'Non- psychoactive CB2 cannabinoid agonists stimulate neural progenitor proliferation', Faseb j, 20: 2405-7.

Palazuelos, J., Z. Ortega, J. Diaz-Alonso, M. Guzman, and I. Galve-Roperh. 2012. 'CB2 cannabinoid receptors promote neural progenitor cell proliferation via mTORC1 signaling', J Biol Chem, 287: 1198-209.

Palmer, T. D., P. H. Schwartz, P. Taupin, B. Kaspar, S. A. Stein, and F. H. Gage. 2001. 'Cell culture. Progenitor cells from human brain after death', Nature, 411: 42-3.

Patzke, N., M. A. Spocter, K. A. E. Karlsson, M. F. Bertelsen, M. Haagensen, R. Chawana, S. Streicher, C. Kaswera, E. Gilissen, A. N. Alagaili, O. B. Mohammed, R. L. Reep, N. C. Bennett, J. M. Siegel, A. O. Ihunwo, and P. R. Manger. 2015. 'In contrast to many other mammals, cetaceans have relatively small hippocampi that appear to lack adult neurogenesis', Brain Struct Funct, 220: 361-83.

Pekny, M., and M. Pekna. 2014. 'Astrocyte reactivity and reactive astrogliosis: costs and benefits', Physiol Rev, 94: 1077-98.

122

Perera, T. D., J. D. Coplan, S. H. Lisanby, C. M. Lipira, M. Arif, C. Carpio, G. Spitzer, L. Santarelli, B. Scharf, R. Hen, G. Rosoklija, H. A. Sackeim, and A. J. Dwork. 2007. 'Antidepressant- induced neurogenesis in the hippocampus of adult nonhuman primates', J Neurosci, 27: 4894-901.

Perez-Asensio, F. J., U. Perpina, A. M. Planas, and E. Pozas. 2013. 'Interleukin-10 regulates progenitor differentiation and modulates neurogenesis in adult brain', J Cell Sci, 126: 4208-19.

Persidsky, Y., S. H. Ramirez, J. Haorah, and G. D. Kanmogne. 2006. 'Blood-brain barrier: structural components and function under physiologic and pathologic conditions', J Neuroimmune Pharmacol, 1: 223-36.

Pertwee, R. G. 2012. 'Targeting the endocannabinoid system with cannabinoid receptor agonists: pharmacological strategies and therapeutic possibilities', Philos Trans R Soc Lond B Biol Sci, 367: 3353-63.

Petitet, F., M. Donlan, and A. Michel. 2006. 'GPR55 as a new cannabinoid receptor: still a long way to prove it', Chem Biol Drug Des, 67: 252-3.

Pietr, M., E. Kozela, R. Levy, N. Rimmerman, Y. H. Lin, N. Stella, Z. Vogel, and A. Juknat. 2009. 'Differential changes in GPR55 during microglial cell activation', FEBS Lett, 583: 2071-6.

Prenderville, J. A., A. M. Kelly, and E. J. Downer. 2015. 'The role of cannabinoids in adult neurogenesis', Br J Pharmacol, 172: 3950-63.

Qin, L. H., W. Huang, X. A. Mo, Y. L. Chen, and X. H. Wu. 2015. 'LPS Induces Occludin Dysregulation in Cerebral Microvascular Endothelial Cells via MAPK Signaling and Augmenting MMP-2 Levels', Oxid Med Cell Longev, 2015: 120641.

Qin, L., X. Wu, M. L. Block, Y. Liu, G. R. Breese, J. S. Hong, D. J. Knapp, and F. T. Crews. 2007. 'Systemic LPS causes chronic neuroinflammation and progressive neurodegeneration', Glia, 55: 453-62.

Quiroz, J. A., R. Machado-Vieira, C. A. Zarate, Jr., and H. K. Manji. 2010. 'Novel insights into lithium's mechanism of action: neurotrophic and neuroprotective effects', Neuropsychobiology, 62: 50-60.

123

Rahimi, A., A. Hajizadeh Moghaddam, and A. Roohbakhsh. 2015. 'Central administration of GPR55 receptor agonist and antagonist modulates anxiety-related behaviors in rats', Fundam Clin Pharmacol, 29: 185-90.

Raison, C. L., L. Capuron, and A. H. Miller. 2006. 'Cytokines sing the blues: inflammation and the pathogenesis of depression', Trends Immunol, 27: 24-31.

Ramirez, B. G., C. Blazquez, T. Gomez del Pulgar, M. Guzman, and M. L. de Ceballos. 2005. 'Prevention of Alzheimer's disease pathology by cannabinoids: neuroprotection mediated by blockade of microglial activation', J Neurosci, 25: 1904-13.

Ramirez, S. H., J. Hasko, A. Skuba, S. Fan, H. Dykstra, R. McCormick, N. Reichenbach, I. Krizbai, A. Mahadevan, M. Zhang, R. Tuma, Y. J. Son, and Y. Persidsky. 2012. 'Activation of cannabinoid receptor 2 attenuates leukocyte-endothelial cell interactions and blood- brain barrier dysfunction under inflammatory conditions', J Neurosci, 32: 4004-16.

Revest, J. M., D. Dupret, M. Koehl, C. Funk-Reiter, N. Grosjean, P. V. Piazza, and D. N. Abrous. 2009. 'Adult hippocampal neurogenesis is involved in anxiety-related behaviors', Mol Psychiatry, 14: 959-67.

Richardson, K. A., A. K. Hester, and G. L. McLemore. 2016. 'Prenatal cannabis exposure - The "first hit" to the endocannabinoid system', Neurotoxicol Teratol, 58: 5-14.

Rockwell, C. E., P. Raman, B. L. Kaplan, and N. E. Kaminski. 2008. 'A COX-2 metabolite of the endogenous cannabinoid, 2-arachidonyl glycerol, mediates suppression of IL-2 secretion in activated Jurkat T cells', Biochem Pharmacol, 76: 353-61.

Rodrigues, R. S., F. F. Ribeiro, F. Ferreira, S. H. Vaz, A. M. Sebastiao, and S. Xapelli. 2017. 'Interaction between Cannabinoid Type 1 and Type 2 Receptors in the Modulation of Subventricular Zone and Dentate Gyrus Neurogenesis', Front Pharmacol, 8: 516.

Rodriguez, J. J., V. C. Jones, M. Tabuchi, S. M. Allan, E. M. Knight, F. M. LaFerla, S. Oddo, and A. Verkhratsky. 2008. 'Impaired adult neurogenesis in the dentate gyrus of a triple transgenic mouse model of Alzheimer's disease', PLoS One, 3: e2935.

124 Romero-Zerbo, S. Y., I. Ruz-Maldonado, V. Espinosa-Jimenez, A. Rafacho, A. I. Gomez-Conde, L. Sanchez-Salido, N. Cobo-Vuilleumier, B. R. Gauthier, F. J. Tinahones, S. J. Persaud, and F. J. Bermudez-Silva. 2017. 'The cannabinoid ligand LH-21 reduces anxiety and improves glucose handling in diet-induced obese pre-diabetic mice', Sci Rep, 7: 3946.

Ross, C. A., R. L. Margolis, S. A. Reading, M. Pletnikov, and J. T. Coyle. 2006. 'Neurobiology of schizophrenia', Neuron, 52: 139-53.

Roy, N. S., S. Wang, L. Jiang, J. Kang, A. Benraiss, C. Harrison-Restelli, R. A. Fraser, W. T. Couldwell, A. Kawaguchi, H. Okano, M. Nedergaard, and S. A. Goldman. 2000. 'In vitro neurogenesis by progenitor cells isolated from the adult human hippocampus', Nat Med, 6: 271-7.

Rubio-Araiz, A., A. Arevalo-Martin, O. Gomez-Torres, B. Navarro-Galve, D. Garcia-Ovejero, P. Suetterlin, E. Sanchez-Heras, E. Molina-Holgado, and F. Molina-Holgado. 2008. 'The endocannabinoid system modulates a transient TNF pathway that induces neural stem cell proliferation', Mol Cell Neurosci, 38: 374-80.

Ryan, S. M., G. W. O'Keeffe, C. O'Connor, K. Keeshan, and Y. M. Nolan. 2013. 'Negative regulation of TLX by IL-1beta correlates with an inhibition of adult hippocampal neural precursor cell proliferation', Brain Behav Immun, 33: 7-13.

Ryberg, E., N. Larsson, S. Sjogren, S. Hjorth, N. O. Hermansson, J. Leonova, T. Elebring, K. Nilsson, T. Drmota, and P. J. Greasley. 2007. 'The orphan receptor GPR55 is a novel cannabinoid receptor', Br J Pharmacol, 152: 1092-101.

Safo, P. K., B. F. Cravatt, and W. G. Regehr. 2006. 'Retrograde endocannabinoid signaling in the cerebellar cortex', Cerebellum, 5: 134-45.

Sahay, A., and R. Hen. 2007. 'Adult hippocampal neurogenesis in depression', Nat Neurosci, 10: 1110-5.

Sahay, A., K. N. Scobie, A. S. Hill, C. M. O'Carroll, M. A. Kheirbek, N. S. Burghardt, A. A. Fenton, A. Dranovsky, and R. Hen. 2011. 'Increasing adult hippocampal neurogenesis is sufficient to improve pattern separation', Nature, 472: 466-70.

Sahay, A., D. A. Wilson, and R. Hen. 2011. 'Pattern separation: a common function for new neurons in hippocampus and olfactory bulb', Neuron, 70: 582-8.

125

Sanchez, C., I. Galve-Roperh, D. Rueda, and M. Guzman. 1998. 'Involvement of sphingomyelin hydrolysis and the mitogen-activated protein kinase cascade in the Delta9- tetrahydrocannabinol-induced stimulation of glucose metabolism in primary astrocytes', Mol Pharmacol, 54: 834-43.

Santarelli, L., M. Saxe, C. Gross, A. Surget, F. Battaglia, S. Dulawa, N. Weisstaub, J. Lee, R. Duman, O. Arancio, C. Belzung, and R. Hen. 2003. 'Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants', Science, 301: 805-9.

Sawzdargo, M., T. Nguyen, D. K. Lee, K. R. Lynch, R. Cheng, H. H. Heng, S. R. George, and B. F. O'Dowd. 1999. 'Identification and cloning of three novel human G protein-coupled receptor genes GPR52, PsiGPR53 and GPR55: GPR55 is extensively expressed in human brain', Brain Res Mol Brain Res, 64: 193-8.

Schicho, R., M. Bashashati, M. Bawa, D. McHugh, D. Saur, H. M. Hu, A. Zimmer, B. Lutz, K. Mackie, H. B. Bradshaw, D. M. McCafferty, K. A. Sharkey, and M. Storr. 2011. 'The atypical cannabinoid O-1602 protects against experimental colitis and inhibits neutrophil recruitment', Inflamm Bowel Dis, 17: 1651-64.

Schumacher, J., G. Laje, R. Abou Jamra, T. Becker, T. W. Muhleisen, C. Vasilescu, M. Mattheisen, S. Herms, P. Hoffmann, A. M. Hillmer, A. Georgi, C. Herold, T. G. Schulze, P. Propping, M. Rietschel, F. J. McMahon, M. M. Nothen, and S. Cichon. 2009. 'The DISC locus and schizophrenia: evidence from an association study in a central European sample and from a meta-analysis across different European populations', Hum Mol Genet, 18: 2719- 27.

Schurov, I. L., E. J. Handford, N. J. Brandon, and P. J. Whiting. 2004. 'Expression of disrupted in schizophrenia 1 (DISC1) protein in the adult and developing mouse brain indicates its role in neurodevelopment', Mol Psychiatry, 9: 1100-10.

Seib, D. R., and A. Martin-Villalba. 2015. 'Neurogenesis in the Normal Ageing Hippocampus: A Mini-Review', Gerontology, 61: 327-35.

Sharir, H., and M. E. Abood. 2010. 'Pharmacological characterization of GPR55, a putative cannabinoid receptor', Pharmacol Ther, 126: 301-13.

Sheng, W. S., S. Hu, X. Min, G. A. Cabral, J. R. Lokensgard, and P. K. Peterson. 2005. 'Synthetic cannabinoid WIN55,212-2 inhibits generation of inflammatory mediators by IL-1beta- stimulated human astrocytes', Glia, 49: 211-9.

126

Shi, Q. X., L. K. Yang, W. L. Shi, L. Wang, S. M. Zhou, S. Y. Guan, M. G. Zhao, and Q. Yang. 2017. 'The novel cannabinoid receptor GPR55 mediates anxiolytic-like effects in the medial orbital cortex of mice with acute stress', Mol Brain, 10: 38.

Shonesy, B. C., D. G. Winder, S. Patel, and R. J. Colbran. 2015. 'The initiation of synaptic 2-AG mobilization requires both an increased supply of diacylglycerol precursor and increased postsynaptic calcium', Neuropharmacology, 91: 57-62.

Sigurdsson, T., K. L. Stark, M. Karayiorgou, J. A. Gogos, and J. A. Gordon. 2010. 'Impaired hippocampal-prefrontal synchrony in a genetic mouse model of schizophrenia', Nature, 464: 763-7.

Simpson, J. M., J. Gil-Mohapel, M. A. Pouladi, M. Ghilan, Y. Xie, M. R. Hayden, and B. R. Christie. 2011. 'Altered adult hippocampal neurogenesis in the YAC128 transgenic mouse model of Huntington disease', Neurobiol Dis, 41: 249-60.

Simuni, T., and K. Sethi. 2008. 'Nonmotor manifestations of Parkinson's disease', Ann Neurol, 64 Suppl 2: S65-80.

Skinner, C. M., N. S. Ivanov, S. A. Barr, Y. Chen, and R. L. Skalsky. 2017. 'An Epstein-Barr Virus MicroRNA Blocks Interleukin-1 (IL-1) Signaling by Targeting IL-1 Receptor 1', J Virol, 91.

Snyder, J. S., A. Soumier, M. Brewer, J. Pickel, and H. A. Cameron. 2011. 'Adult hippocampal neurogenesis buffers stress responses and depressive behaviour', Nature, 476: 458-61.

Sofroniew, M. V. 2009. 'Molecular dissection of reactive astrogliosis and glial scar formation', Trends Neurosci, 32: 638-47.

Sofroniew, M. V., and H. V. Vinters. 2010. 'Astrocytes: biology and pathology', Acta Neuropathol, 119: 7-35.

Solas, M., P. T. Francis, R. Franco, and M. J. Ramirez. 2013. 'CB2 receptor and amyloid pathology in frontal cortex of Alzheimer's disease patients', Neurobiol Aging, 34: 805-8.

127 Sorrells, S. F., M. F. Paredes, A. Cebrian-Silla, K. Sandoval, D. Qi, K. W. Kelley, D. James, S. Mayer, J. Chang, K. I. Auguste, E. F. Chang, A. J. Gutierrez, A. R. Kriegstein, G. W. Mathern, M. C. Oldham, E. J. Huang, J. M. Garcia-Verdugo, Z. Yang, and A. Alvarez-Buylla. 2018. 'Human hippocampal neurogenesis drops sharply in children to undetectable levels in adults', Nature, 555: 377-81.

Spalding, K. L., O. Bergmann, K. Alkass, S. Bernard, M. Salehpour, H. B. Huttner, E. Bostrom, I. Westerlund, C. Vial, B. A. Buchholz, G. Possnert, D. C. Mash, H. Druid, and J. Frisen. 2013. 'Dynamics of hippocampal neurogenesis in adult humans', Cell, 153: 1219-27.

Staton, P. C., J. P. Hatcher, D. J. Walker, A. D. Morrison, E. M. Shapland, J. P. Hughes, E. Chong, P. K. Mander, P. J. Green, A. Billinton, M. Fulleylove, H. C. Lancaster, J. C. Smith, L. T. Bailey, A. Wise, A. J. Brown, J. C. Richardson, and I. P. Chessell. 2008. 'The putative cannabinoid receptor GPR55 plays a role in mechanical hyperalgesia associated with inflammatory and neuropathic pain', Pain, 139: 225-36.

Stefanis, N. C., P. Delespaul, C. Henquet, C. Bakoula, C. N. Stefanis, and J. Van Os. 2004. 'Early adolescent cannabis exposure and positive and negative dimensions of psychosis', Addiction, 99: 1333-41.

Surget, A., M. Saxe, S. Leman, Y. Ibarguen-Vargas, S. Chalon, G. Griebel, R. Hen, and C. Belzung. 2008. 'Drug-dependent requirement of hippocampal neurogenesis in a model of depression and of antidepressant reversal', Biol Psychiatry, 64: 293-301.

Sylantyev, S., T. P. Jensen, R. A. Ross, and D. A. Rusakov. 2013. 'Cannabinoid- and lysophosphatidylinositol-sensitive receptor GPR55 boosts neurotransmitter release at central synapses', Proc Natl Acad Sci U S A, 110: 5193-8.

Tanaka, R., K. Yamashiro, H. Mochizuki, N. Cho, M. Onodera, Y. Mizuno, and T. Urabe. 2004. 'Neurogenesis after transient global ischemia in the adult hippocampus visualized by improved retroviral vector', Stroke, 35: 1454-9.

Tang, M. M., W. J. Lin, Y. Q. Pan, X. T. Guan, and Y. C. Li. 2016. 'Hippocampal neurogenesis dysfunction linked to depressive-like behaviors in a neuroinflammation induced model of depression', Physiol Behav, 161: 166-73.

Thored, P., A. Arvidsson, E. Cacci, H. Ahlenius, T. Kallur, V. Darsalia, C. T. Ekdahl, Z. Kokaia, and O. Lindvall. 2006. 'Persistent production of neurons from adult brain stem cells during recovery after stroke', Stem Cells, 24: 739-47.

128

Tocharus, C., Y. Puriboriboon, T. Junmanee, J. Tocharus, K. Ekthuwapranee, and P. Govitrapong. 2014. 'Melatonin enhances adult rat hippocampal progenitor cell proliferation via ERK signaling pathway through melatonin receptor', Neuroscience, 275: 314-21.

Toda, T., S. L. Parylak, S. B. Linker, and F. H. Gage. 2018. 'The role of adult hippocampal neurogenesis in brain health and disease', Mol Psychiatry.

Tomar, S., E. E. Zumbrun, M. Nagarkatti, and P. S. Nagarkatti. 2015. 'Protective role of cannabinoid receptor 2 activation in galactosamine/lipopolysaccharide-induced acute liver failure through regulation of macrophage polarization and microRNAs', J Pharmacol Exp Ther, 353: 369-79.

Tortoriello, G., C. V. Morris, A. Alpar, J. Fuzik, S. L. Shirran, D. Calvigioni, E. Keimpema, C. H. Botting, K. Reinecke, T. Herdegen, M. Courtney, Y. L. Hurd, and T. Harkany. 2014. 'Miswiring the brain: Delta9-tetrahydrocannabinol disrupts cortical development by inducing an SCG10/stathmin-2 degradation pathway', Embo j, 33: 668-85.

Trapp, B. D., L. Bo, S. Mork, and A. Chang. 1999. 'Pathogenesis of tissue injury in MS lesions', J Neuroimmunol, 98: 49-56.

Ujike, H., M. Takaki, K. Nakata, Y. Tanaka, T. Takeda, M. Kodama, Y. Fujiwara, A. Sakai, and S. Kuroda. 2002. 'CNR1, central cannabinoid receptor gene, associated with susceptibility to hebephrenic schizophrenia', Mol Psychiatry, 7: 515-8.

Valero, J., G. Mastrella, I. Neiva, S. Sanchez, and J. O. Malva. 2014. 'Long-term effects of an acute and systemic administration of LPS on adult neurogenesis and spatial memory', Front Neurosci, 8: 83.

Vallieres, L., I. L. Campbell, F. H. Gage, and P. E. Sawchenko. 2002. 'Reduced hippocampal neurogenesis in adult transgenic mice with chronic astrocytic production of interleukin- 6', J Neurosci, 22: 486-92.

Van Kampen, J. M., T. Hagg, and H. A. Robertson. 2004. 'Induction of neurogenesis in the adult rat subventricular zone and neostriatum following dopamine D3 receptor stimulation', Eur J Neurosci, 19: 2377-87.

129 van Neerven, S., T. Regen, D. Wolf, A. Nemes, S. Johann, C. Beyer, U. K. Hanisch, and J. Mey. 2010. 'Inflammatory chemokine release of astrocytes in vitro is reduced by all-trans retinoic acid', J Neurochem, 114: 1511-26.

van Praag, H., A. F. Schinder, B. R. Christie, N. Toni, T. D. Palmer, and F. H. Gage. 2002. 'Functional neurogenesis in the adult hippocampus', Nature, 415: 1030-4.

Van Sickle, M. D., M. Duncan, P. J. Kingsley, A. Mouihate, P. Urbani, K. Mackie, N. Stella, A. Makriyannis, D. Piomelli, J. S. Davison, L. J. Marnett, V. Di Marzo, Q. J. Pittman, K. D. Patel, and K. A. Sharkey. 2005. 'Identification and functional characterization of brainstem cannabinoid CB2 receptors', Science, 310: 329-32.

Viscomi, M. T., S. Oddi, L. Latini, N. Pasquariello, F. Florenzano, G. Bernardi, M. Molinari, and M. Maccarrone. 2009. 'Selective CB2 receptor agonism protects central neurons from remote axotomy-induced apoptosis through the PI3K/Akt pathway', J Neurosci, 29: 4564-70.

Visintin, A., A. Mazzoni, J. H. Spitzer, D. H. Wyllie, S. K. Dower, and D. M. Segal. 2001. 'Regulation of Toll-like receptors in human monocytes and dendritic cells', J Immunol, 166: 249-55.

Vita, A., L. De Peri, C. Silenzi, and M. Dieci. 2006. 'Brain morphology in first-episode schizophrenia: a meta-analysis of quantitative magnetic resonance imaging studies', Schizophr Res, 82: 75-88.

Vivekanantham, S., S. Shah, R. Dewji, A. Dewji, C. Khatri, and R. Ologunde. 2015. 'Neuroinflammation in Parkinson's disease: role in neurodegeneration and tissue repair', Int J Neurosci, 125: 717-25.

Walter, J., S. D. Honsek, S. Illes, J. M. Wellen, H. P. Hartung, C. R. Rose, and M. Dihne. 2011. 'A new role for interferon gamma in neural stem/precursor cell dysregulation', Mol Neurodegener, 6: 18.

Walter, L., A. Franklin, A. Witting, C. Wade, Y. Xie, G. Kunos, K. Mackie, and N. Stella. 2003. 'Nonpsychotropic cannabinoid receptors regulate microglial cell migration', J Neurosci, 23: 1398-405.

130 Walton, N. M., Y. Zhou, J. H. Kogan, R. Shin, M. Webster, A. K. Gross, C. L. Heusner, Q. Chen, S. Miyake, K. Tajinda, K. Tamura, T. Miyakawa, and M. Matsumoto. 2012. 'Detection of an immature dentate gyrus feature in human schizophrenia/bipolar patients', Transl Psychiatry, 2: e135.

Wang, B., and K. Jin. 2015. 'Current perspectives on the link between neuroinflammation and neurogenesis', Metab Brain Dis, 30: 355-65.

Wang, X., S. Fu, Y. Wang, P. Yu, J. Hu, W. Gu, X. M. Xu, and P. Lu. 2007. 'Interleukin-1beta mediates proliferation and differentiation of multipotent neural precursor cells through the activation of SAPK/JNK pathway', Mol Cell Neurosci, 36: 343-54.

Weinberger, D. R. 1999. 'Cell biology of the hippocampal formation in schizophrenia', Biol Psychiatry, 45: 395-402.

Whyte, L. S., E. Ryberg, N. A. Sims, S. A. Ridge, K. Mackie, P. J. Greasley, R. A. Ross, and M. J. Rogers. 2009. 'The putative cannabinoid receptor GPR55 affects osteoclast function in vitro and bone mass in vivo', Proc Natl Acad Sci U S A, 106: 16511-6.

Willner, P. 2017. 'The chronic mild stress (CMS) model of depression: History, evaluation and usage', Neurobiol Stress, 6: 78-93.

Winner, B., Z. Kohl, and F. H. Gage. 2011. 'Neurodegenerative disease and adult neurogenesis', Eur J Neurosci, 33: 1139-51.

Wolf, Susanne A., Anika Bick-Sander, Klaus Fabel, Perla Leal-Galicia, Svantje Tauber, Gerardo Ramirez-Rodriguez, Anke Müller, Andre Melnik, Tim P. Waltinger, Oliver Ullrich, and Gerd Kempermann. 2010. 'Cannabinoid receptor CB1 mediates baseline and activity- induced survival of new neurons in adult hippocampal neurogenesis', Cell Communication and Signaling, 8: 12.

Woodward, D. F., Y. Liang, and A. H. Krauss. 2008. 'Prostamides (prostaglandin-ethanolamides) and their pharmacology', Br J Pharmacol, 153: 410-9.

Wu, C. S., H. Chen, H. Sun, J. Zhu, C. P. Jew, J. Wager-Miller, A. Straiker, C. Spencer, H. Bradshaw, K. Mackie, and H. C. Lu. 2013. 'GPR55, a G-protein coupled receptor for lysophosphatidylinositol, plays a role in motor coordination', PLoS One, 8: e60314.

131 Wu, C. S., C. P. Jew, and H. C. Lu. 2011. 'Lasting impacts of prenatal cannabis exposure and the role of endogenous cannabinoids in the developing brain', Future Neurol, 6: 459-80.

Wu, C. S., J. Zhu, J. Wager-Miller, S. Wang, D. O'Leary, K. Monory, B. Lutz, K. Mackie, and H. C. Lu. 2010. 'Requirement of cannabinoid CB(1) receptors in cortical pyramidal neurons for appropriate development of corticothalamic and thalamocortical projections', Eur J Neurosci, 32: 693-706.

Wu, M. D., S. L. Montgomery, F. Rivera-Escalera, J. A. Olschowka, and M. K. O'Banion. 2013. 'Sustained IL-1beta expression impairs adult hippocampal neurogenesis independent of IL-1 signaling in nestin+ neural precursor cells', Brain Behav Immun, 32: 9-18.

Xapelli, S., F. Agasse, L. Sarda-Arroyo, L. Bernardino, T. Santos, F. F. Ribeiro, J. Valero, J. Braganca, C. Schitine, R. A. de Melo Reis, A. M. Sebastiao, and J. O. Malva. 2013. 'Activation of type 1 cannabinoid receptor (CB1R) promotes neurogenesis in murine subventricular zone cell cultures', PLoS One, 8: e63529.

Xiong, Y., F. Qiu, W. Piao, C. Song, L. M. Wahl, and A. E. Medvedev. 2011. 'Endotoxin tolerance impairs IL-1 receptor-associated kinase (IRAK) 4 and TGF-beta-activated kinase 1 activation, K63-linked polyubiquitination and assembly of IRAK1, TNF receptor- associated factor 6, and IkappaB kinase gamma and increases A20 expression', J Biol Chem, 286: 7905-16.

Yang, H., J. Zhou, and C. Lehmann. 2016. 'GPR55 - a putative "type 3" cannabinoid receptor in inflammation', J Basic Clin Physiol Pharmacol, 27: 297-302.

Yirmiya, R., and I. Goshen. 2011. 'Immune modulation of learning, memory, neural plasticity and neurogenesis', Brain Behav Immun, 25: 181-213.

Zhang, K., H. Xu, L. Cao, K. Li, and Q. Huang. 2013. 'Interleukin-1beta inhibits the differentiation of hippocampal neural precursor cells into serotonergic neurons', Brain Res, 1490: 193- 201.

Zhang, X., Y. Maor, J. F. Wang, G. Kunos, and J. E. Groopman. 2010. 'Endocannabinoid-like N- arachidonoyl serine is a novel pro-angiogenic mediator', Br J Pharmacol, 160: 1583-94.

Zhang, Y., and W. Hu. 2012. 'NFkappaB signaling regulates embryonic and adult neurogenesis', Front Biol (Beijing), 7.

132

Zhang, Z., W. Wang, P. Zhong, S. J. Liu, J. Z. Long, L. Zhao, H. Q. Gao, B. F. Cravatt, and Q. S. Liu. 2015. 'Blockade of 2-arachidonoylglycerol hydrolysis produces antidepressant-like effects and enhances adult hippocampal neurogenesis and synaptic plasticity', Hippocampus, 25: 16-26.

Zheng, L. S., S. Hitoshi, N. Kaneko, K. Takao, T. Miyakawa, Y. Tanaka, H. Xia, U. Kalinke, K. Kudo, S. Kanba, K. Ikenaka, and K. Sawamoto. 2014. 'Mechanisms for interferon-alpha-induced depression and neural stem cell dysfunction', Stem Cell Reports, 3: 73-84.

Zonis, S., R. N. Pechnick, V. A. Ljubimov, M. Mahgerefteh, K. Wawrowsky, K. S. Michelsen, and V. Chesnokova. 2015. 'Chronic intestinal inflammation alters hippocampal neurogenesis', J Neuroinflammation, 12: 65.

Zoppi, S., J. L. Madrigal, J. R. Caso, M. S. Garcia-Gutierrez, J. Manzanares, J. C. Leza, and B. Garcia-Bueno. 2014. 'Regulatory role of the cannabinoid CB2 receptor in stress-induced neuroinflammation in mice', Br J Pharmacol, 171: 2814-26.

Zunszain, P. A., N. Hepgul, and C. M. Pariante. 2013. 'Inflammation and depression', Curr Top Behav Neurosci, 14: 135-51.

133