Histamine Is the Macrophage-Derived Factor That Plays a Crucial Role In

Histamine is the Macrophage-derived factor that plays a crucial role in

promoting Axon Regeneration of Retinal Explants

Khyati Walia

A thesis submitted in conformity with the requirements for the degree of MSc.

Department of Physiology

University of Toronto

2020 Histamine is the Macrophage-derived factor that plays a crucial role in promoting Axon

Regeneration of Retinal Explants

Khyati Walia Master of Science Department of Physiology University of Toronto 2020

Abstract

The role of macrophages in promoting axon regeneration in the injured mammalian

CNS is still strongly debated. While there have been past studies that reported regenerative effects induced by inflammation following injuries to the lens or the optic nerve, there are still several gaps in our understanding of the underlying mechanisms behind achieving that successful axon regeneration. With the use of three different primary cell lines, we show that histamine is the active macrophage-derived factor that is responsible for neurite growth- promoting effects observed in the retinal explant cultures. We also report that histamine is mediating these effects by binding to the H1 receptors found on the astroglial cells.

Furthermore, our findings indicate that histamine is influencing the exosome secretion from astroglial cells in eliciting these effects. Therefore, our results address the role of macrophages and show the mechanism whereby they are promoting the axon regrowth at the

CNS injury site.

Acknowledgements

First and foremost, I would like to express my deepest gratitude to my supervisor Dr.

Monnier for making me a part of his team and for giving me the opportunity to carry out this project. I would also like to thank my committee members, Dr. Zhong Ping Feng and Dr.

Koeberle for providing their feedback on my progress during the committee meetings.

I would also like to specifically thank Nahal Farhani and Amy from Dr. Wrana’s lab for training me on specific lab techniques. My sincere thanks go to the entire team of Dr. Wrana for allowing me to use their Zetaview Particle Meter and for helping with the mRNA

Sequencing analysis. I would also like to extend my gratitude to Hidekiyo Harada for helping with the mRNA Sequencing. Besides, I am grateful to all my teammates for providing me with such a positive atmosphere in these past couple years.

Lastly, I would like to thank my parents, my brother and my partner for being that major support system for me throughout this time. Without you, I never would have even imagined coming this far. It was your love and encouragement that allowed me to stay committed. I would also like to thank my mentor, Dr. Krepinsky. If it was not for your advice, I may not even have pursued graduate studies.

I would also like to sincerely thank Vision Science Research Program for supporting this project.

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Table of Contents

Abstract………………………………………………………………………………………ii Acknowledgements………………………………………………………………………….iii List of Figures …...………………………………………………………………………....vii List of Tables ……..…………………………………………………………………………ix List of Abbreviations ……………………………………………………………………...... x

Chapter 1: Introduction……………………………………………………………………..1 1.1 Injury to the Central Nervous System …………………………………………………1 1.2 Astrocytes in CNS Injury and Repair…………………………………………………..1 Astrocytes and their normal physiological functions………………………………...... 1 Glial scar formation……………………………………………………………………….4 1.3 Exosomes and Axon Regeneration…………………………………………………….10 1.4 The role of Immune System in CNS Injury…………………………………………..14 1.5 Earlier Studies on CNS Recovery……………………………………………………..16 1.6 Role of Macrophages in CNS Injury…………………………………………………..17 Physiological and Pathological functions of CNS Macrophages and Microglia………..17 M1 vs. M2 phenotype……………………………………………………………………20 1.7 Role of Histamine in Neuroinflammation…………………………………………….23 Histamine synthesis and physiological function……………………………………...... 23 Histamine receptors and their expression………………………………………………..23 Effect of Histamine on Astrocyte function………………………………………………27 1.8 Rationale………………………………………………………………………………..31 Studies showing regenerative effects of Macrophages in Lens Injury or ONC model….31 Studies arguing against the involvement of Oncomodulin or Activated Macrophages in Axonal Regeneration……………………………………………………………………36 1.9 Hypothesis and Aims…………………………………………………………………..40

2 Chapter 2: Material and Methods……………………………………………………43

2.1 Isolation and Plating of Mixed Cortical cells…………………………………………....43 2.2 Obtaining an Enriched Astrocytes culture……………………………………………….45 2.3 Purification of Exosomes by Differential Ultracentrifugation...... 46 2.4 Quantification of Exosomes using ZETAVIEW Particle Meter………………………...46 2.5 Isolation of Peritoneal Macrophages…………………………………………………….49 2.6 Histamine H1 Receptor Knockout Animals……………………………………………..50 2.7 Cell Culture treatments…………………………………………………………………..50 2.8 Chick Retinal Explant Outgrowth Assay………………………………………………...51 2.9 Immunocytochemistry………………………………………………………………...…52

2.10 mRNA Sequencing…………………………………………………………………..53 2.11 Statement of Ethics for Animal Use…………………………………………………54 2.12 Microscopy…………………………………………………………………………..54 2.13 Statistical Analysis…………………………………………………………………...54

Chapter 3: Results………………………………………………………………………….55 Aim I: Role of Macrophages in promoting neurite outgrowth of Retinal Explants...... 55 3.1 Neurite outgrowth is promoted with the treatment of Macrophages Conditioned Media 3.2 Neurite growth promoting effects induced by Macrophages are dependent on glial cells 3.3 Activated Macrophages promote neurite outgrowth of Retinal Explants when cultured on Astrocytes

Aim II: Histamine is the Activated Macrophage-derived factor that is promoting neurite length………………………………………………………………………………64 3.4 Inhibiting histamine production inhibits neurite growth promoting effects of Activated Macrophages.. 3.5 Histamine treatment promotes neurite outgrowth of retinal explants..

Aim III: Histamine’s neurite growth promoting effects are mediated by its binding to H1 receptor…………………………………………………………………………………70 3.6 Treatment with H1 receptor agonist promotes neurite outgrowth of retinal explants.. 3.7 Histamine H1 Receptor Knockout Mice-derived astrocytes show decrease in the neurite outgrowth..

Aim IV: Exosomes are involved in mediating the neurite growth promoting effects….79 3.8 Differential gene expression analysis show H1-antagonist treatment results in downregulation of gene involved with exosome release 3.9 H1-agonist treatment promotes exosome secretion 3.10 inhibiting exosome secretion decreases neurite outgrowth..

Aim V: Astrocyte-derived exosomes promote neurite outgrowth both on Laminin and CSPG……………………………………………………………………………………….88 3.11 Astrocyte-derived exosomes treatment increases growth on Laminin.. 3.12 Astrocyte-derived exosomes treatment increases growth even on CSPG..

Aim VI: Astrocyte-derived exosomes promote neurite outgrowth via Wnt signaling pathway…………………………………………………………………………………....91 3.13 Blocking Wnt secretion decreases exosome-induced neurite growth promoting effects on Laminin…

3.14 Blocking Wnt secretion decreases exosome-induced neurite growth promoting effects even on CSPG

Chapter 4: Discussion (& Future Directions)..………………………………………….94 Chapter 5: Conclusion…………………………………………………………………...101 Chapter 6: Appendix…………………………………………………………………….102 Chapter 7: Bibliography…………………………………………………………………105

List of Figures

Figure 1-1. A schematic representation demonstrating astrocytes play an important role in supporting neurons in the CNS………………………………………………………………..3

Figure 1-2. Schematic (A) and photomicrograph (B & C) of a Spinal Cord Injury lesion showing the discrete tissue compartments ………………………………………………….5

Figure 1-3. Schematic representation of M1 and M2 polarization of macrophages……...... 20

Figure 1-4. Schematic representation of classical binding sites of histamine and the sequential signaling pathways that are activated on H1, H2, H3 or H4 receptor binding…...24

Figure 2-1. Zetaview Nanoparticle Tracking Analyzer …………………………………….46

Figure 3-1. Neurite outgrowth is promoted with treatment of Activated MCM……………57

Figure 3-2. The neurite growth promoting effects induced by Activated MCM treatment are dependent on the glial cells………………………………………………………………….60

Figure 3-3. Activated MCM promotes neurite outgrowth when retinal explants are cultured on just astrocytes…………………………………………………………………………….62

Figure 3-4. Blocking Histamine production inhibits the neurite growth promoting effects...65

Figure 3-5. Histamine treatment promotes neurite outgrowth of the retinal explants………68

Figure 3-6. Histamine H1 agonist treatment promotes neurite outgrowth of retinal explants cultured on non-activated astrocytes………………………………………………………...72

Figure 3-7. Histamine H1 agonist treatment promotes neurite outgrowth of retinal explants cultured on activated astrocytes…………………………………………………………..….74

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Figure 3-8. H1-KO astrocytes resulted in decreased neurite outgrowth…………………….77

Figure 3-9. H1-agonist treatment increases exosome secretion…………………………….82

Figure 3-10. GW4869 treatment decreases exosome secretion……………………………..84

Figure 3-11. Inhibiting exosome secretion decreases neurite outgrowth…………………..86

Figure 3-12. Astrocyte-derived exosomes promote neurite outgrowth…………………….89

Figure 3-13. Astrocyte-derived exosomes promote neurite outgrowth despite the presence of

CSPG………………………………………………………………………………………..90

Figure 3-14. IWP2 treatment decreases the exosome-induced neurite growth…………….92

Figure 3-15. IWP2 treatment decreases the exosome-induced neurite growth even on

CSPG……………………………………………………………………………………….93

Figure 6-1. The effects induced by GW4869, histamine, H1 agonist, H2 agonist and H3 agonist are dependent on the astrocytes…………………………………………………..102

Figure 6-2. The effects of IWP2 are dependent on the presence of astrocytes…………..104

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List of Tables

Table 3-8. Relative mRNA expression levels for H1-antagonist treated astrocytes………….80

List of Abbreviations

CNS Central Nervous System

PNS Peripheral Nervous System

GABA Gamma-amino butyric acid

ROS Reactive Oxygen Species

IL Interleukin

NO Nitric oxide

TNF Tumor Necrosis Factor

OPCs Oligodendrocyte Precursor cells

ASC Astrocyte Scar border

GFAP Glial Fibrillary acidic protein

ECM Extracellular matrix

HSPG Heparan Sulfate Proteoglycan

DSPG Dermatan Sulfate Proteoglycan

KSPG Keratan Sulfate Proteoglycan

CSPG Chondroitin Sulfate Proteoglycan

IFN Interferon

FGF Fibroblast growth factor

WT Wild type

EVs Extracellular vesicles

ILVs Intraluminal vesicles

MVBs Multivesicular bodies

ESCRT Endosomal Sorting Complex Required for

Transport

KO Knockout

PM Plasma membrane

MAG Myelin associated glycoprotein

PLP Proteolipid protein

CircRNAs Circular ribonucleic acids

FD Fibroblast-derived

RGC Retinal Ganglion Cells

BDNF Brain derived neurotrophic factor

CNTF Ciliary neurotrophic factor

GSK Glycogen synthase kinase

CS Casein kinase

APC Adenomatous polyposis coli

PTEN Phosphatase and tensin homolog

TSC Tuberous sclerosis protein mTOR Mammalian target of rapamycin

HEK Human embryonic kidney

NADPH Nicotinamide adenine dinucleotide

phosphate

NOX NADPH oxidase

Ig-SF Immunoglobulin superfamily

TLR Toll-like receptor

CCL C-chemokine ligand

NMDA N-methyl-d-aspartate

MMP Matrix metalloproteinase

CSF Colony stimulating factor

NGF Neuron growth factor

GDNF Glial cell derived neurotrophic factor

HDC Histidine decarboxylase

H1R Histamine-1 receptor

GPCR G-protein coupled receptor

APC Antigen presenting cells

PLC Phospholipase C

DAG Diacylglycerol

IP Inositol triphosphate

AC Adenylyl cyclase

PKA Protein kinase A

CRE Cyclic adenine monophosphate response

element

CREB CRE binding protein

MAPK Mitogen activated protein kinase

GLT Glutamate transporter

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NT Neurotrophin

NF Neurofilament

MCM Macrophage conditioned media

JAK Janus-kinase

ONC Optic nerve crush

LIF Leukemia inhibitory factor

LI Lens injury

PKC Protein kinase C

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Chapter 1: Introduction

1.1 Injury to the Central Nervous System

An injury to the Central Nervous System (CNS) in adult mammals is often considered irreversible, partly due to lack of the regenerative capacity of neurons (2,3). Over the past several years, extensive amount of research has been focused on exploring the different neurodegenerative diseases and CNS trauma in attempts to finding strategies to repair the

CNS. A complete recovery after an injury or trauma to the CNS entails several steps including promoting the ‘neurogenesis’ via triggering the individual-specific stem cells to reprogram and form new neurons, protecting the severed but still surviving neurons from undergoing further damage, inducing the spontaneous regrowth of the axons, providing signals to guide the new regenerating axons to their specific targets and finally the ability to form functional synapses (3,7). While there have been many studies on the topics of neurogenesis and neuroprotection, the scientific literature still falls short in the understanding of axonal regrowth and synapse restoration, both of which are just as crucial in establishing a functional neuronal circuit.

1.2 Astrocytes in CNS injury and repair

1.2.1 Astrocytes and their normal physiological functions

Neuroglia are known to be a highly heterogenous population of non-excitable cells that can be found in several different regions within the CNS (15). These comprise at least

50% of the cellular constituency and mainly include astrocytes, oligodendrocytes and microglia (1,2). Their main functions involve controlling, protecting and supporting the functions of neurons (12, 13). Astrocytes are amongst the largest and most abundant of these glial cells within the CNS. They received their name from their characteristic star-like appearance (12). Astrocytes play a variety of different physiological functions including regulating homeostasis, increasing synaptic plasticity and providing neuroprotection, therefore helping maintain a normal CNS function (14,15). It is known that astrocytes can also help in guiding migration of postnatal neuroblasts along with promoting functional coordination within the brain. They are seen particularly involved with the secretion and metabolism of amino-acid-based neurotransmitters like glutamic acid and gamma-amino butyric acid (GABA), thereby preventing the nearby neurons against their excitotoxicity (11,

12, 16). Additionally, these cells can influence the uptake and synthesis of several important neurotransmitters and neurotrophic factors.

Astrocytes also support neuronal metabolism by enhancing the release of lactate and pyruvate. These cells can take up glutamate and convert it to glutamine which can then be utilized by neurons (17,18). In addition to this, they are heavily involved with regulating the innate immune responses by regulating the inflammatory factors like, cytokines, chemokines, complement proteins and reactive oxygen species (ROS) (20). Amongst some of the pro- inflammatory mediators that are released by astrocytes under circumstances of a brain injury or an otherwise inflammatory insult include interleukin (IL) -1β, IL-6, IL-12, Nitric oxide

(NO) and Tumor necrosis factor (TNF)- α (19, 20, 22). Depending on the severity of the

2 injury and the reactive gliosis that follows, these reactive astrocytes can either maintain a neuroprotective phenotype or take on a rather neurotoxic phenotype.

Figure 1-1. A schematic representation demonstrating astrocytes play an important role in supporting neurons in the CNS. Here astrocytes are shown supporting neuronal metabolism, protecting them against glutamate build-up, and are seen secreting various pro- inflammatory mediators in response to an injury (16).

Due to the abundance of these cells and the large spectrum of their functions, they have been widely studied in CNS pathologies. Earlier studies on astrocytes reported that they

3 might be playing a role in the pathogenesis of different neurodegenerative diseases and that the dysfunction of astrocytes could result in widespread neuronal death leading to these pathologies (10).

1.2.2 Glial Scar formation

It was found that astrocytes respond to an injury to the CNS via a process called reactive astrogliosis, sometimes also known as gliosis (22, 24). This is a process whereby these astrocytes would become hypertrophic and undergo changes in their molecular expression and morphology. These injury-induced activated astrocytes are also referred to as

‘reactive astrocytes’ (20, 22). Reactive gliosis comprises of changes that occur both at the genetic level and at the cellular level regulated via inter- and intracellular signaling.

Depending on the type of signaling events, it can result in these reactive astrocytes gaining or losing functions. As more studies were conducted, it was found that many CNS injuries, especially ones that were severe enough to breach through the blood-brain barrier led to a secondary tissue damage. This would usually involve encapsulation of the lesion by reactive astrocytes which would form the so-called ‘glial scar’ (24, 25).

Figure 1-2. Schematic (A) and photomicrograph (B & C) of a Spinal Cord Injury lesion showing the discrete tissue compartments consisting of both neural and non-neural cells. It shows all the reactive glial cells, astrocytes, oligodendrocyte precursor cells (OPCs) and

5 microglia around the lesion; especially the reactive astrocytes stained in green (B) using

GFAP that are forming the astrocyte scar border (ASB). Pericytes and fibroblast-lineage cells in the lesion core are stained white using CD13, neurons are stained in blue with NeuN marker and cell nuclei in blue with DAPI. More importantly it shows the astrocyte response following the injury to the spinal cord (74).

For many years, this glial scar was considered an essential part of the wound healing process in the injured brain and spinal cord. This was mainly because this scar creates a physical barrier to separate the areas with severe injury and inflammation from spreading and damaging the surrounding tissues. However, with further studies in the axon regeneration field it was found that this scar maybe one of the biggest obstacles preventing the spontaneous regrowth of the injured axons (22).

Besides being a physical barrier, reactive astrocytes that mainly form this scar were later found releasing several inhibitory extracellular matrix (ECM) molecules called proteoglycans (22). The four classes of proteoglycans produced by astrocytes were identified to be heparan sulfate proteoglycan (HSPG), dermatan sulfate proteoglycan (DSPG), keratan sulfate proteoglycan (KSPG) and chondroitin sulfate proteoglycan (CSPG) (22,24). Soon after these were identified, studies conducted on spinal cord injury models reported that reactive astrocytes upregulate CSPG expression, which was already known to be inhibiting axon outgrowth (17,18).

On further exploration, it was revealed that the astroglial component of this scar undergoes five critical processes. The very first process which happens within the days of the injury or trauma, is the migration of astrocytes from the lesion epicentre to the outermost

6 edges (22,23). The second process is the proliferation of the thin layer of these astrocytes which by now, have underwent activation (gliosis) and are residing at the lesion margin.

Followed by this is the accumulation of the intermediate filaments, especially glial fibrillary acidic protein (GFAP), vimentin and nestin that further lead to cellular hypertrophy of these cells (22, 23, 24). The fourth process involves the restructuring of the gliotic layer from the radial longitudinal orientation to more of a mesh-like envelope which starts to become highly obstructive to any growing axons nearby (23). By this point, the glial scar has fully formed, and the last process involves the production of the growth inhibitory molecules, especially the CSPGs that offer a non-permissive atmosphere for the recovering severed axons, further contributing to their regeneration failure.

Astrocytes are vastly heterogeneous which is partly attributed to the variations in their response to an injury or a trauma (15, 16). This response varies with the severity of the injury and influences both morphological changes and changes in the activity of these astrocytes. In mild-to moderate injury, astrocytes occupy non-overlapping domains almost like a non- injured tissue; whereas in the cases of a more severe injury, these astrocytes occupy overlapping domains and result in a glial scar formation (23). Most of the structural changes that occur following a more extensive injury persist for longer periods and result in axon regeneration failures. Amongst the factors that can further induce this scar formation include

TGF-β1, TGF- β2 and IL-1 which are particularly released in response to recruitment and activation of macrophages (15, 16, 17). Other cytokines that are known to be involved are interferon (IFN) and fibroblast growth factor 2 (FGF)-2, which by promoting astrocyte proliferation leads to glial scarring.

While most studies on the glial scar have been painting reactive astrocytes as one of the main players contributing to the failed regeneration outcomes in CNS injury models, there have been few studies that reported the beneficial effects of these reactive astrocytes

(15). These studies suggested that reactive astrocytes may also have a permissive role in the functional recovery of these axons, considering they demonstrate the ability to regulate and minimize the inflammation cascade following the injury. Also, astrocytes have been reported to protect the CNS against excitotoxicity by glutamate, and against the oxidative stress or ammonium ions or amyloid β particles (13, 15). Some studies also noted that ablation of proliferation scar-forming astrocytes led to more aggravated inflammatory response and greater tissue damage. They also reported greater degree of demyelination and blood-brain barrier permeability (12, 13). These few studies speculated that perhaps this varying response of astrocytes on the functional outcomes of axotomized neurons have to do with the degree of their activation status or with the type and expression levels of molecules (pro- inflammatory or neurotrophic) they produce in response to the insult.

A more recent 2016 study by Sofroniew and his group in Nature was amongst the only in-depth studies so far that challenged this widespread view of astrocyte scar being the biggest barrier in CNS axon regrowth (9). Despite the several reports correlating the failed axon regeneration with the presence of reactive astrocytes and glial scar, and the evidence that these produce the growth inhibitory CSPGs, they decided to test whether there was a more causation relation between the glial scar and the failed regeneration outcome. To test this, they used multiple transgenic loss-of function mouse models where they either completely removed these scar-forming astrocytes or significantly weakened their activity in

8 the injured spinal cord (9). Their results indicated no spontaneous axonal regrowth with the prevention of scar formation, which was contrary to previous speculations.

To understand the molecular mechanism underlying this response, they decided to measure the total CSPG levels. Considering that these animals had fewer reactive astrocytes, it was expected that their CSPG levels would also be relatively lower compared to their wild- type counterparts. However, this was not the case. It seemed that the total CSPG levels were in fact not significantly reduced with the ablation of the astrocyte scars 9). This means that

CSPGs may be contributed from other non-astrocyte sources and suppressing scar formation had little to no implications on the axonal functional recovery. Considering that variety of different molecules some with growth supportive properties, while others with growth suppressive properties are produced in these injury lesions.

To further analyze these different molecules, this group also decided to perform a genome-wide RNA sequencing of both astrocyte-specific RNA and non-precipitated RNA derived from other non-astrocyte cells (9). The results from this experiment was one of their more important breakthroughs. They reported that of the 59 differentially expressed genes that were identified from the wild type (WT) animal mice, 28 were growth inhibitors while

31 were growth supporters (9). However, interestingly both these groups of molecules included specific CSPGs. Of the five growth-inhibitory CSPGs, WT-scar forming astrocytes had increased only versican, while they significantly decreased neurocan and phosphocan. In addition, the two axon growth supportive CSPGs, Cspg 4 and Cspg 5 were upregulated by these astrocytes (9).

These results indicated that both astrocytes and non-astrocyte cells expressed wide variety of growth promoting and inhibiting molecules. Especially CSPGs seemed to be contributed largely by these non-astrocyte cells. Also, scar-forming astrocytes upregulate and largely express the axon growth promoting CSPGs, which were seen decreased once these scars were prevented from forming. This was further supported when they showed axon regrowth despite the scar formation (9). In fact, weakening these astrocytes also removed the robust regrowth which was seen. These findings were contrary to the prevailing dogma since they not only disproved that this scar is detrimental to the axon recovery but instead, they showed that its formation helps with the successful axon regeneration in spinal cord injury models (9). This study called for more extensive research to further explore the characteristic responses of astrocytes to find better CNS repair strategies.

1.3 Exosomes and Axon Regeneration

Extracellular vesicles (EVs) were first observed almost 50 years ago in the plasma and since then virtually all biological fluids that have been tested seem to contain these vesicles (29, 30). There are three main types of EVs that vary based on their size and their mechanism of release from their cells of origin. These include, exosomes (less than 150nm in diameter), microvesicles (more than 100nm in diameter) and apoptotic bodies (also more than 100nm in diameter) (30). Exosomes are the smallest amongst these membrane bound

EVs and are virtually released by all cell types. In terms of their biogenesis, these are generated from inward budding of the membrane in the endosomes, forming intraluminal vesicles (ILVs) into multivesicular bodies (MVBs) which then eventually fuse into the

10 plasma membrane (PM) to finally release these exosomes into the extracellular space (34).

The Endosomal Sorting Complex Required for Transport (ESCRT), a complex multi- molecular machinery regulates the formation of these ILVs, therefore is responsible for the exosome secretion (34, 35). After ILVs get encapsulated into MVBs, these can either get transferred to lysosomes for degradation or they can fuse with the PM to release the enclosing exosomes (34).

Some of the earliest reports suggested that exosomes were essentially just another mechanism whereby cells discarded their garbage until some of the more recent studies that showed these serve as mediators for intercellular communication. This was supported by the evidence that they deliver variety of different molecules between cells that include proteins, lipids, nucleic acids and many other components (29). The cargo that exosomes carry is very specific to the cell type that they are generated from, which also determines their specific function. However, some studies have found that exosomes contain proteins like actin and β- tubulin that are required for axonal growth. It was also reported that myelin proteins like myelin-associated glycoprotein (MAG) and proteolipid protein (PLP) are also found within the exosomes which are critical for nerve remyelination process. In fact, these are also enriched in circular ribonucleic acids (circRNAs) specifically involved with glutamatergic synapse and cyclin guanosine monophosphate-protein kinase G signaling pathways and can be playing a role in improving functional recovery in some CNS injury cases (30, 38, 39).

Besides this, exosomes also contain many other molecules that seem to indicate their involvement in the axon regeneration process.

Amongst the more specific studies showing the influence of exosomes in the CNS injury model is the study from our own lab (29). Our lab was amongst the very first groups of researchers to show that exosomes can promote neurite outgrowth despite the presence of inhibitory myelin and CSPGs known to inhibit axon regrowth following CNS injuries. Our

2017 study showed that injecting fibroblast-derived (FD) exosomes into the vitreous of the eye promotes axon regeneration after optic nerve injury, another commonly used model to study CNS injuries. Furthermore, this pro-regenerative effect was abolished when the same exosomes were injected into Wnt 10b knockout animals (29). This proved that the exosome effects are mediated by the Wnt signaling.

Wnt proteins are involved with a variety of cellular and physiological functions including cell proliferation, differentiation and migration. These have classically been divided into canonical (β-catenin -dependent) and noncanonical (β-catenin-independent) signaling pathways (77). In the canonical Wnt signaling, β-catenin is actively degraded with a complex comprising of Axin, glycogen-synthase kinase-3 (GSK3), casein kinase 1(CS1) and adenomatous polyposis coli (APC) (29,77). In a classical pathway, binding of the Wnt protein to its receptors would disrupt the function of this degradation complex, hence stabilizing the levels of β-catenin which would then be able to enter the nucleus and activate the genes. Moreover, studies have demonstrated that these changes in the β-catenin levels can also occur as a result of extracellular ligands, for instance members of Dickkopf families, that can bind to Wnt receptors or ligands and can modulate their effects (77). Since more studies have reveled their implications in many pathologies, the knowledge on Wnt signaling

12 has been used in developing drugs that can manipulate the Wnt signalling, one of which is

IWP2 (29). It is a potent porcupine inhibitor that can potentially block Wnt secretion.

It was already known that the regeneration can be achieved using the genetic deletion of phosphatase and tensin homolog (PTEN) or tuberous sclerosis protein 1 (TSC1) which are known to downregulate mammalian target of rapamycin (mTOR) pathway (29, 30). Our lab further demonstrated that FD exosomes can activate the Wnt10b autocrine signaling pathway which further activates mTOR in neurons and helps elicit the regenerative effects (29).

Similar experiments were conducted to see the effects of exosomes derived from other cell lines like transformed human embryonic kidney (HEK)293 cells and African green monkey fibroblast cells or COS-7 cells. However, the growth promoting effects on neurites were not observed for these cell lines, which indicates that exosomes from different sources may have different effects.

Another more recent study reported that reactive oxygen species (ROS) which are produced via NADPH oxidases (NOX) which have been only known for contributing to tissue damage and neurodegeneration, can also promote axon regeneration following the injury (30). They proposed that macrophages that are recruited to the injury site as part of the inflammation process releases exosomes containing these NOX complexes. These exosomes are then incorporated into the severed axons from where the NOX complex is transported to their cell body through the dynein-dependent mechanism (30). Here the endosomal NOX oxidizes PTEN thereby causing its inactivation which has been known to promote regeneration. While this study raises many concerns about the newfound role of ROS in

13 promoting regeneration, it also prompts further studies to be conducted to better understand the role of exosomes.

One of the ways through which the functional implications of exosomes have been further explored is via the inhibition of neutral sphingomyelinases. Sphingomyelinases are enzymes known to cleave sphingomyelin to produce ceramide and phosphocholine.

Ceramide mediates the inward budding of the MVBs further helping with the release of mature exosomes. A very established neutral sphingomyelinase inhibitor called GW4869 is commonly used to inhibit exosome secretion and to study its effects in different animal models (30). Previously, knockdown of dynein, a microtubule associated protein which is an active part of endosome trafficking system has shown similar reduced exosome release from neuroblastoma cells. However, GW4869 is a more widely used agent for blocking exosome release since its effects have been confirmed in many different cell types.

1.4 The role of the Immune System in CNS injury

The CNS is often viewed as part of the body that is ‘immune privileged’, mainly because the local immune responses within the CNS are relatively restricted. Unlike most peripheral tissues CNS functions through a network of post-mitotic cells called neurons that are incapable of regeneration (53). Considering that these neurons are essential for survival and are unable to recover spontaneously post injury or a trauma, they are in an even more special need of protection from pathogens and other insults. However, its been shown that

CNS is more vulnerable to damage by the very means that the immune system uses to defend the rest of the peripheral tissues. This is the reason why this so-called immune privilege in the CNS is viewed as an evolutionary adaptation to protect the CNS from damage by its own immune system (43, 53).

An early description of the term ‘immune privilege’ in this context was based on the very assumption that the immune system ignores the CNS (53). This in fact was supported by the tendency of the CNS to not reject the allografts or inability of the immune cells to enter the CNS under normal physiological conditions. This is also why any leukocyte entry used to be viewed as evidence of pathology. Since then, many observations had challenged this definition including, CNS antigens can escape and induce immune responses within the host and that the activated T cells can enter the CNS under normal conditions (1,2). Soon, leukocyte recruitment into the CNS was seen beneficial against several CNS pathologies without developing any long-term bystander effects (1,2, 3). These findings indicated that

CNS is accessible to the body’s immune system, however there maybe mechanisms specifically within the CNS to limit these immune responses for the functioning of the healthy CNS. Nonetheless, the immune system is an essential requirement for both the protection and maintenance of the damaged CNS. Like the misconception around the interaction between the CNS and immune system, is another misconception that- since the activated immune cells are seen infiltrating the injured lesions within the CNS, these must be responsible in the degeneration process (1,2, 4). While this conclusion has now been recognized as an oversimplification, there still seems to be few gaps in our understanding of

15 the role of these different immune cells in both the degeneration and regeneration processes of the CNS.

1.5 Earlier studies on CNS Recovery

One of the first studies on recovery after CNS injury were focussed on the assumption that CNS is a unique tissue whose post-injury behaviour is different from the rest of the peripheral tissues at the time of the injury. Further studies during the 1900s showed that although CNS axons fail to regenerate post injury in their own degenerative environment, they can grow into the transplanted peripheral nerve bridges (1, 53, 41). This opened the idea that at least some of these CNS axons possessed the ability to regenerate and that their incapability to regrow stems is due to the very nature of their environment, which if changed can help achieve their structural recovery.

As more research began in this field, more evidence supported the concept that the

CNS regenerative failure in part may be due to the inability of the cellular components around the injured axons to create a growth supporting environment (44). Amongst the factors that were later found to be hostile to this re-growth in the adult CNS came to be myelin-associated proteins and extracellular matrix proteins such as chondroitin sulfate proteoglycans (CSPGs) (48, 45). Thus, it was concluded that for regeneration to successfully occur, these inhibitors must be masked or neutralized. These discoveries prompted more studies on different strategies for the treatment of the severed CNS axons such as transplantation of intercostal nerve segments into completely transected spinal cords of adult

16 rats, and introduction of monoclonal antibodies directed specifically against the myelin- associated proteins, where partial regeneration was seen (44, 48). Similarly, regrowth was also promoted with the treatment of proteases that degrade CSPGs, or with the modification of the axonal environment via the local transplantation of embryonic tissue, or Schwann cells or cells that can provide a source of trophic factors to increase the survivability of the cell bodies (48, 50).

1.6 Role of Macrophages in CNS injury

On further research, a comparative study of the local inflammatory response in injured peripheral nervous system (PNS) and CNS axons demonstrated that in both systems the degeneration and regeneration processes were seemed to be controlled with involvement of macrophages (47). Since most studies since then had been viewing the role of immune cells as detrimental to the spontaneous regeneration process, this controversial finding prompted more studies in this area.

1.6.1 Physiological and Pathological Functions of CNS Macrophages and Microglia

Macrophages are myeloid cells that have both homeostatic and immune roles. They ingest and degrade dead cells, cell debris along with potentially hazardous pathogens, similar to microglia cells (44, 47). This way they constitute the body’s first line of defense against the pathogens by regulating both innate and adaptive immune responses. Many earlier studies referred to microglia as resident macrophages considering the similarities in

17 their functions (46). For the longest time, their functional heterogeneity was largely ignored but as more research was conducted into the CNS injury models it became clearer that the myeloid-derived infiltrating macrophages are quite functionally distinct from the resident microglia. Since then migratory macrophages were named monocyte-derived macrophages

(mo-Mφ) (45, 46, 47). This led to better understanding of their functional roles with respect to the resident microglial cells within the CNS. While microglial cells are already known to perform both protective and maintenance roles, under certain circumstances such as after an injury or a trauma these cells can no longer provide the needed protection to the damaged

CNS. This is when the macrophages are recruited to the damaged region, to not act as the microglial replacements but to assist the resident microglia in preventing more damage (45,

47, 50). Considering that these cells are crucial to inducing the inflammatory response to an injury to CNS, it is important to further explore their development and their role with respect to one another, both during normal physiological and pathological states. Perhaps they can open more gates to finding newer, more improved therapeutic treatments to axonal degeneration. Moreover, there is still quite a bit of debate with regards to their functional phenotypes and under what circumstances these are induced.

In terms of their developmental origin, microglia are known to originate from early yolk sac myeloid progenitors, while macrophages are known to originate from peripheral blood monocytes. Microglia are regarded as self-renewing resident cells of the CNS that play a wide variety of functions like synaptic pruning, facilitating learning, removal of the dead cells etc. (45, 50). The wide spectrum of their functions during a CNS infection or an injury become largely dependent on the nature of their activation stimuli. While moderate CNS

18 damage can evoke protection via these microglia, an intensive activation of these cells for instance after the spinal cord injury or after an optic nerve crush can render these cells rather neurotoxic, hence detrimental to the neuronal activity or regeneration. On the other hand, the recruitment and activation of the peripheral macrophages to the injured site which is another important part of this inflammatory response can have differential effects depending on the specific phenotype that is induced (44, 48, 49). While there are past several studies that have been highlighting these cell responses as detrimental to the neuronal recovery, due to the variability in macrophages responses’ it seems more research is needed in this field.

There are times when in order to meet the increasing demand for the control of infection or tissue injury, the functional activity of these cells is increased in response to the specific stimulus they are exposed to (42). The nature of the stimulus then can determine the distinct morphology and the movement of these cells to help better perform their functions.

Although this enhanced activity of the cells allows them to better respond to the changes in their surroundings in attempts to re-establish the homeostasis, it also increases the risk of hyperactivation and sequential tissue damage (42, 50). To counterbalance this activation program, these cells are subjected to silencing programs that set tissue-specific thresholds for their activation (42). The intensity and duration of activation or inhibition is balanced through activating or inhibitory receptors they express. For instance, immunoglobulin superfamily (Ig-SF) molecules deliver either activating or inhibitory signals via protein tyrosine kinase and phosphatase pathways, respectively ((42, 44). Therefore, the imbalance between these activating and inhibitory signals that regulate both microglial and macrophage activity, can lead to tissue pathologies including CNS autoimmunity or tumorigenesis.

1.6.2 M1 vs. M2 phenotype

Moreover, in response to these diverse stimuli, microglial cells and macrophages can undergo either classical activation which leads to induction of their M1 phenotype, or alternative activation in which case there is induction of their M2 phenotype (42, 44, 53).

Depending of the phenotype they take on, their roles would often change. The induction of these phenotypes is normally controlled by intrinsic (epigenetic factors) or extrinsic (ex.

Inflammatory cytokines) regulatory factors. The nomenclature M1/M2 phenotypes that is often applied to CNS infiltrating microglial and macrophages has mostly been studied in the context of CNS inflammation or tumor (53).

Figure 1-3. Schematic representation of M1 and M2 polarization of macrophages(75) .

M1 phenotype is usually characterized as the pro-inflammatory and often neurotoxic state. It is typically induced by simultaneous triggering of the Toll-like receptors (TLRs) and interferon (IFN)-  signalling pathways which are further associated with immunity to bacteria and similar intracellular pathogens (53). The M1 macrophages produce pro- inflammatory cytokines and chemokines such as Tumor necrosis factor (TNF) -α, interleukin

(IL)-6, IL-1β, IL-12 and C-C chemokine ligand-2 (CCL2). Moreover, M1 macrophages express the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which in turn generated superoxide and reactive oxygen species (ROS) (42, 44, 53). Nitric oxide production increases, which then leads to increasing the toxic effect of glutamate, thereby potentiating N-methyl-d-aspartate (NMDA) receptor-mediated neurotoxicity. It also produces inflammatory mediator called matrix metalloproteinase (MMP)-12 (41, 48, 53).

Some studies have found that these macrophages also express high amounts of MHC class I or II molecules, Fc receptors and integrins, all of which can induce inflammation and neurotoxicity (53).

On the other hand, M2 phenotype is often characterized as anti-inflammatory and potentiating tissue remodelling often seen in cases of asthma or allergy. M2 phenotype can be induced by IL-4, IL-10, IL-13, ligation of Fc receptors by immunocomplexes and sometimes from presence of apoptotic cells (40, 41, 53). M2 activation has been found to promote the release of prosurvival factor progranulin and many other anti-inflammatory cytokines such as IL-10, TGF-β. These cells also secrete growth factors such as insulin growth factor (IGF)-I, fibroblast growth factor (FGF) and colony stimulating factor (CSF)-1

(46, 47, 53). Furthermore, neurotrophic factors such as nerve growth factor (NGF), brain- derived neurotrophic factor (BDNF), neurotrophins 4/5 and glial cell-derived neurotrophic factor (GDNF) are also released (46, 47, 53). While the M1 and M2 phenotypes are often used to conceptualize the different macrophages activities in vitro, it is still difficult to clearly characterize the two phenotypes for in vivo models, owing to our limited understanding of the M1/M2 paradigm.

Macrophages are a source of both inflammatory cytokines and growth factors that can act directly or indirectly to induce axonal regrowth. One of the many evidence supporting this theory is the regeneration of the optic nerves in lower vertebrates and peripheral nerves in mammals with the upregulation of the macrophage-derived apolipoproteins, known to recycle the lipids needed for membrane rebuilding (41, 52). Similarly, synthesis of nerve growth factors and cytokines such as interleukin-1(IL-1) and tumor necrosis factor-α (TNF-

α), secreted by macrophages can induce the recovery. On the other hand, some literature reports suggest that the microglia in CNS is in its downregulated form as its activation is relatively restricted compared to the invading macrophages. To further prove that the macrophages can be beneficial to the repair of damaged CNS, some studies showed that implantation of homologous macrophages in the injured optic nerves of adult rats induced regrowth of the injured axons (42, 52, 53). This process was also accompanied with the efficient clearance of dead cells and other debris from the already damaged axons and large numbers of activated macrophages were found at the injury sites. In terms of the functional recovery aspect, these animals showed partial recovery of their motor function and electrophysiological activity.

1.7 Role of Histamine in Neuro-inflammation

1.7.1 Histamine synthesis and physiological function

These inflammatory conditions have long been thought to be also mediated by the histaminergic receptor signaling. Histamine is an important neurotransmitter or neuromodulator of the CNS (54, 55). In the brain histamine is not transported via the plasma due to its inability to penetrate the blood-brain barrier, but it is formed from L-histidine using a special enzyme called Histidine decarboxylase (HDC) (55). This enzyme, as its name suggests catalyzes the decarboxylation of the amino acid L-histidine to synthesize histamine.

There are two major pathways involving its metabolism that include ring methylation and oxidative deamination by diamine oxidase. Inside the brain, most of the histamine is catalyzed by histamine-N-methyltransferase to form tele-methylhistamine which is further converted with the help of monoamine oxidase B in order to form tele-methylimidazole acid

(55, 57).

1.7.2 Histamine receptors and their expression

The different effects of histamine are mediated via its four histamine receptors, H1R,

H2R, H3R and H4R, all of which are G-protein coupled receptors (GPCRs) (55, 57). Their active and inactive conformations are maintained during the equilibrium, while their agonists

23 can stabilize their active conformations, their antagonists would act by stabilizing their inactive conformations.

Figure 1-4. Schematic representation of classical binding sites of histamine and the sequential signalling pathways that are activated on H1, H2, H3 or H4 receptor binding

(76).

H1 receptors are found expressed in multitude of different cell types that include but are not limited to neurons, endothelial cells, muscle cells, hepatocytes, monocytes, T cells and B cells. Their signaling can result in synthesis of prostacyclins, activation of platelet

24 factor, synthesis of nitric oxide and contraction of smooth muscle cells (54, 57).

Additionally, their activation can also increase the chemotaxis of eosinophils and neutrophils especially at the site of inflammation along with the increased functional capacity of antigen- presenting cells (APCs) (55, 56). In terms of signaling, binding to H1R mediates excitatory actions on the neurons by recruiting Gq/11 and phospholipase C (PLC) which further leads to the formation of two secondary messengers namely, diacylglycerol (DAG) and inositol1,4,5- triphosphate 3 (IP3) and results in calcium release from the internal stores (63). By activating H1R, histamine results in contraction of the smooth muscles of respiratory tract, increases vascular permeability and induces production of prostacyclin and platelet activating factor. Furthermore, since its activation can be observed in almost all hypersensitivity reactions, use of H1R antagonists generally helps with ameliorating symptoms of allergies and asthma.

The H2Rs on the other hand, couple to GS following which the adenylyl cyclase

(AC) and protein kinase A (PKA) phosphorylates proteins and activates transcription factor cyclic-AMP-response element (CRE)-binding protein (CREB). H2R is often seen inducing relaxation of the airways, uterus and the smooth muscle cells of the blood vessels (63). In addition, H2R signalling is also associated with the induction of the immune responses such as, Th1 cytokine production, reduction of basophil degranulation, T-cell proliferation and antibody synthesis (55, 56).

Furthermore, H3Rs are found expressed at histaminergic and other cell somata, dendrites and axons. Binding of the histamine to H3R usually act as a negative feedback since it restricts further histamine synthesis and release. In fact, it also provides negative

25 feedback on the release of other neurotransmitters like glutamate, acetylcholine and noradrenaline. Unlike H2Rs, H3Rs couple to Gi/o and inhibit the high-voltage activated calcium channels, which is commonly what regulates the exocytosis process. Studies suggested that H3R knockout led to increased severity of many neuro-inflammatory conditions and some found its closely associated with the blood-brain barrier function (56,

63). Overall, while H1Rs and H2Rs are mostly seen eliciting excitatory actions on neurons,

H3Rs on the contrary, exhibits inhibitory actions on the neurons and the neurotransmitter release. While the physiological roles of these three metabotropic histamine receptors, H1R,

H2R and H3R have been clearly established, information on the fourth histamine receptor

H4R and on its functional expression in the CNS is still limited and controversial. Few studies have reported that stimulation of H4R can reduce forskolin-induced cyclic AMP formation, further leading to mitogen activated protein kinase (MAPK) activation and increased calcium release (55, 63).

Many studies on brain injuries and other neuro-inflammatory conditions have reported increasing circulating levels of histamine both in the blood and in the cerebrospinal fluid (CSF), along with its association with increasing blood-brain barrier permeability.

Therefore, it is believed that histamine maybe responsible for modulating the expression of several inflammatory mediators released by macrophages, monocytes and other T and B lymphocytes 54, 63). Histamine is thought to be playing a dual role with regards to the inflammatory response mediated by macrophages. For instance, histamine was found to induce interleukin-6 (IL-6) release by macrophages via its interaction with H1R, while it inhibits chemotaxis, phagocytosis and production of tumor necrosis factor (TNF)-α, IL-12

26 and superoxide anion via H2R activation (55, 56, 63). Therefore, when it comes to its effects on the inflammatory processes its effects can be either stimulatory or inhibitory depending on the type and abundance of the involved receptor subtype.

Some studies also reported the effects of histamine on the microglial activity, such that it can induce NO release by the microglial cells and subsequently result in dopaminergic neuronal death. Not only microglial cells but its receptors are also expressed on the astrocytes (57, 63). H1R subtype was particularly found connected to several of the astrocytic functions such as increase in intracellular Ca2+ levels; glucose allocation and histamine-induced glycogen breakdown; upregulation of the glutamate transporter-1 (GLT-1) expression and glutamate clearance by the astrocytes which normally help with neuroprotection against glutamate excitotoxicity (59, 61). H1R activation on astrocytes also enhance neurotrophic activity via stimulation of release of several neurotrophic factors like

NGF, neurotrophin-3 (NT-3) and FGF-1 (57, 63). Similar enhanced neurotrophic effects had also been observed with H2R and H3R activation in the cultured astrocytes. In fact, some reports have shown evidence of histamine production by the macrophages. This was not very surprising since histamine effects had been reported in many neuro-inflammation studies.

1.7.3 Effect of Histamine on astrocyte functions

More recent studies on the role of histamine in CNS injury have also looked at its effects on the astrocyte functions. Especially since it was known that histamine upregulated the expression of astrocytic glutamine synthetase and glutamate transporter 1, it was clear that histamine also plays a rather protective role via astrocytic functions ((58, 58, 60).

Further SCI animal models too showed elevated histamine levels at the injury site as early as

2 hours following the injury. This led to many researchers speculating whether histamine was contributing to the functional recovery after injuries to the CNS. Some researchers hypothesized that histamine may be influencing the glial scar formation, since this had been known to be the physical obstacle in the regeneration process. One specific study (5, 60) reported that exogenous delivery of histamine to the lesion site after spinal cord injury both reduced the lesion size and improved the motor functions in the animals. Moreover, the exogenous application of histamine had shown increased preservation of myelin, and decreased number of GFAP-labelled areas (5, 60, 61). This indicated that histamine treatment had decreased the number of reactive astrocytes. Further the thickness of the astrocyte scar seemed to have reduced in the treatment group, all of which indicated that histamine may have had some effect in weakening the reactive astrogliosis following the spinal cord injury.

This group also reported that the inhibitory CSPGs, that are also the major contributors to regenerative failures were downregulated with the histamine treatment.

Histamine was in fact shown to promote axonal growth since the neurofilament (NF-200)- labelled axons which were lost at the time of injury had started to grow beyond the scar (59,

60 61). To ensure that these neuroprotective effects were in fact mediated through histamine, they also used HDC knockout animals which were unable to produce histamine. These animals showed relatively more aggravating reactive astrogliosis, all of which proved that due to endogenous absence of histamine, the glial scar formation was stimulated and functional recovery in these animals was found to be relatively poor compared to the wild

28 types (599, 60, 61). In addition to this, they showed that the treatment with the H1R antagonist diphenhydramine successfully reversed the inhibitory effects induced by histamine on astrogliosis. This showed that perhaps the neuroprotective effects of histamine on reactive astrogliosis is mediated by activation of H1R.

Another study looked at the neuroprotective effects of histamine on both early and late stages of cerebral ischemia. Since glial scar formation has been known to be largely responsible for impeding neurogenesis and functional recovery following the ischemia, suppression of this scar is thought to be beneficial. Studies showed that histamine seem to be protecting astrocytes from injuries induced by oxygen or glucose deprivation (57, 59). Also, that the treatment with its precursor, histidine at a high dose during the early stage and low dose during the late stage can elicit the same neuroprotective effects. This was seen when intraperitoneal treatment with histidine showed a significant reduction in the glial scar area after the cerebral ischemia. It was later shown that histidine was promoting the migration of reactive astrocytes towards the infarct area which was attributing to the formation of thinner scar barrier (59, 60). Further H2 agonist amthamine showed similar effects as histamine on astrocyte migration which led many to believe that these neuroprotective effects may have been elicited via the involvement of histamine H2 receptor (57, 59).

Similar more recent study on the role of histamine and astrocytes found that primary astrocytes selectively express H1, H2 and H3 histamine receptors. More specifically, the mRNA expression comparisons indicated that H2 and H3 receptor mRNA levels were 0.7 and 0.1 times the levels of H1 receptor mRNA (57,60, 63). Furthermore, histamine treatment induced astrocyte activation and upregulated the H1, H2 and H3 expression in these

29 astrocytes. More importantly, it was seen that histamine dose-dependently decreased both the

TNF-α and IL-1β secretion from the primary astrocytes. Since injury induced activated astrocytes do take part in secreting these pro-inflammatory mediators, with this finding, it was suggested perhaps that histamine could modulate the immune functions of the astrocytes. It was also found that histamine stimulated the secretion of an important neurotrophic factor GDNF, by the astrocytes (57, 59, 61). This factor is already seen playing a major role in neuronal survival and can also inhibit microglial activation along with neuroinflammation. Moreover, they found that these effects of histamine were reversed with the treatment of H1 and H3 antagonist, indicating that they may be mediated via these receptors. Since astrocytes and their activation following injury is known to induce diverse effects from developing protective immune responses to inducing progression of damaging inflammation. It seems that histamine can modulate its functions and facilitate its neuroprotective effects which could be mediated by one or more of the three specific receptors.

In fact, it was also discovered that stimulation of specifically histamine H1 receptor could induce increase in the exosome secretion which was measured as the frequency of fusion of multi-vesicular bodies (MVB) with the plasma membrane (PM) (62). This was found by detecting the real-time visualization of MVB-PM fusion using live total internal reflection fluorescence and dynamic correlative light-electron microscopy. This was one of the very few studies that discovered an agent that could stimulate the secretion of exosomes, therefore can help understand the dynamics of MVB exocytosis (62).

1.8 Rationale

1.8.1 Studies showing regenerative effects of macrophages in a Lens Injury or an Optic Nerve Crush (ONC) model

The role of the macrophages was specifically explored in greater depths using the optic nerve as a model to understand the axonal regeneration failure in the CNS. As it is previously known that under normal circumstances, Retinal Ganglion cells (RGCs), like

CNS neurons, fail to regrow their injured axons distal to the site of the optic nerve injury. In fact, if the injury to these axons occurs within the orbit, more than 95% of the RGCs undergo cell death within 2 weeks of the injury (64, 67). Owing to this failure in the axonal recovery and rapid RGC apoptosis within days of the optic nerve injury, for the longest time it was believed that regeneration failure within the Optic nerve was inevitable until the 1991 study by DeFelipe and Jones (64, 66, 67). This study was amongst the earliest studies to show that mature RGCs can still show axonal regrowth through a peripheral nerve (PN) graft sutured on to the Optic Nerve stump (injured site) (64). This discovery led to further studies that attempted to discover specific factors that can support or inhibit this axonal regeneration process.

Later it was reported that there are several polypeptide trophic factors such as brain- derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), fibroblast growth factor-1 (FGF-1) that can promote the RGC survival following the axotomy, but neither had been reported to promote axonal regeneration (64, 66). Some studies showed the overexpression of anti-apoptotic protein Bcl-2 also led to RGC survival but no effects were

31 seen in the context of axon regeneration. Then some studies showed that inducing an injury to the lens or implanting a fragment of the peripheral nerve into the vitreous body of the eye stimulated the RGCs to switch to a rather regenerative state and allowed them to grow axons through the injury site into the distal optic nerve, which had not been previously shown (64,

66, 67). In fact, few reported that if the optic nerve were to simply cut and then sutured back, this lens injury would stimulate the RGCs to spontaneously regrow their severed axons back to the superior colliculus. Similar modest levels of this optic nerve regeneration had also been seen with the use of angiotensin II, or with the antibodies targeting the axon-growth inhibitory myelin proteins.

As more studies came on, it was noted that increased regenerative capacity of the

PNS axons in comparison to CNS axons including optic nerve is also paralleled with the recruitment of larger numbers of macrophages to the degenerating peripheral tissues. It seems that the phagocytic functions of the macrophages which are of great use in the axon regrowth are inhibited by the exposure to the optic nerve segments (CNS), in comparison to the sciatic nerve segments (PNS) (65, 66, 67). This difference in macrophages activity in the

CNS injured regions can be due to presence of specific inhibitors or due to the absence of certain chemoattractants.

Since like most CNS or PNS injuries, lens injury leads to macrophages recruitment and activation, Benowitz and their group decided to look into whether these activated macrophages secreted any specific factor(s) that could be mediating the regenerative effects of the induced lens injury on the optic nerve (64, 67). To better understand the macrophages involvement they developed two animal models, first the optic nerve crush model that

32 allowed to study the axon regeneration in the native environment of the Optic Nerve, and then by grafting a segment of the peripheral nerve on to the cut end of the optic nerve. Along with the Optic Nerve Injury, they also performed intravitreal injections of a compound called zymosan A which is a yeast cell wall suspension commonly used as a potent macrophage activator. They speculated that axon regeneration in the optic nerve is sensitive to the time of zymosan administration. In order to test this, they injected zymosan on the same day of the injury, 7 days or 3 days before the injury or 3 days or 7 days after the injury (66, 67). While they suspected that by activating macrophages earlier on before the optic nerve crush might make more macrophage-derived factors available for the RGC axon regeneration, the results were contrary. Introducing zymosan 7 days prior to the injury failed to stimulate any regeneration whatsoever, while injecting it 3 days prior to the injury resulted in some regeneration but no higher than seen in the animals that received it on the same day of the injury. Surprisingly, the greatest levels of regeneration were seen when zymosan was injected 3 days after the injury (66, 67). It was also noted that the number of intravitreal macrophages was relatively higher than the controls during the first 7 days after the stimulation and remained high up to 2 weeks of the injury. Animals with the Lens injury and zymosan injection showed upregulation od GAP-43 positive axons extending beyond the injured site and numerous ED-1 positive macrophages lining both the vitreous and the inner retinal surface. Moreover, the animals that had been given the zymosan after 3 days of the injury showed a 9-fold increase compared to their control counterparts, and 1.6-fold increase compared to animals that received zymosan injection on the same day as the injury (65, 66,

67).

The PN animal model results confirmed that lens injury does enhance the axon regeneration into the PN graft. However, to further test if the macrophages activation is the principal mediator of this lens-injury induced axon regeneration, zymosan injections were given in the similar manner to the optic nerve crush model. As expected, zymosan injection resulted in more than 5700 RGCs extending their axons to the distal end of the PN graft, which is twice in number than control group of animals that received saline injections (65,

66, 67). Additionally, delaying zymosan injection by 3 days similarly increased this number further, with even more RGCs regenerating their axons by the factor of 1.5-fold in relative to the group that received immediate injections (67).

To further confirm whether macrophages-derived factors are causing the RGCs to grow axons, an in vitro primary cell culture model was also used. They showed that a previously discovered vitreous-derived factor called AF-1 along with forskolin that elevates intracellular cAMP levels did a small growth promoting effect on the rat dissociated RGCs in culture. However, this effect was enhanced when the zymosan-activated macrophage- conditioned media (MCM) was added (64, 66). Further fractionation of this MCM revealed that these zymosan-activated macrophages secreted both cytotoxic and axon-promoting effects. This was supported when the components of MCM were separated by ultrafiltration with the use of 30kDa molecular weight cut-off membrane. The fraction containing proteins that were larger than 30kDa seemed to be detrimental to both the axon outgrowth and RGC survival. While fraction containing proteins smaller than 30kDa showed no toxicity and in fact enhanced the axon outgrowth by 1.8-fold compared to cells exposed to AF-1 and forskolin (66, 67). This discovery gave to the idea that maybe these axon regenerating effects

34 are mediated by the macrophage-derived factors of molecular weight lower than 30kDa. This led to testing of the different trophic factors that included neurotrophins like BDNF and

NGF, and cytokines like, CNTF, IL-6, GDNF, FGF-2 and many more. Although none of the factors showed any growth promoting effects except CNTF that showed increased axon outgrowth 1.4-fold above levels seen for AF-1 and forskolin treated RGC (65, 66, 67).

Benowitz and his group revealed another macrophage-derived factor called oncomodulin which is claimed to be a potent axon-growth promoting factor for RGCs. It was shown that the macrophages that entered the vitreous within 24 hours of the lens injury showed elevated levels of oncomodulin mRNA and protein. This finding was further supported when this Ca2+ binding protein almost nearly doubled the axon outgrowth of the in-culture RGCs when treated with mannose and forskolin that had already been known for their moderate regenerating effects (64, 66, 67). In fact, later it was found that the effects of oncomodulin were equivalent to the effects seen with treatment of MCM prepared from zymosan stimulation. On comparing its effects with the other trophic factors, it was shown that while CNTF did show axon growth promoting effects, its effects were not nearly as significant as oncomodulin’s (66, 67). Moreover, oncomodulin also exhibited high-affinity binding to RGCs which was cAMP-dependent. Later in order to show that oncomodulin is the principal factor that is stimulating the RGCs to regenerate their axons following the lens injury and intravitreal inflammation, the Benowitz group used the optic nerve (ON) model.

The ON was transected, and an autologous peroneal nerve was sutured onto the ON stump.

Further to prevent the macrophage recruitment into the eye, they had injected the animals with the clodronate liposomes that is known to cause apoptotic death to monocytes and

35 macrophages. They also performed intraocular injections of peptides blocking the action of oncomodulin. By both depleting the macrophages and inhibiting the effects of oncomodulin, they showed that the beneficial effects of lens injury were inhibited (66, 67). While this discovery had many researchers convinced, there was another group that claimed that it is not in fact the oncomodulin or the activated macrophages that are mediating these regenerative effects of the Lens injury.

1.8.2 Studies arguing against the involvement of Oncomodulin or Activated

Macrophages in Axonal Regeneration

While the studies that followed the Benowitz’s discoveries confirmed the beneficial effects of inducing a lens injury or intravitreal injections of the pro-inflammatory zymosan on the regeneration of the RGC axons. Some of these studies especially one conducted by

Fischer and his group, seemed to have contradicting results with regards to the involvement of macrophages in mediating these regenerative effects. In fact, they were amongst the first groups that directly challenged the role of oncomodulin as the active macrophage-derived factor that promotes RGCs axon regeneration (68). It is true that the inflammatory reactions that follow a lens injury affects variety of different cells around the lesion site. While many research studies had been focused on exploring the role of macrophages and their secreted factors, other cells like Muller cells and astrocytes are also heavily influenced during this process. These cells respond by increasing the expression of glia fibrillary acidic protein

(GFAP) which is already known as a common marker for inflammation (68, 69).

The study by Fisher shed light on the previously reported findings where intravitreal injections of activated primary macrophages did not promote axonal regeneration, nor did they improve the expression of the growth-associates proteins like GAP-43 in RGCs. Fischer and his group conducted a study where they showed that primary macrophages did not express oncomodulin in significant amounts, even on their activation (68). Moreover, they showed that neither lens injury, nor administration of zymosan significantly increased oncomodulin levels in the eye that could result in any substantial effects. In fact, on depleting macrophages from the eye, the neuroprotective and axon growth promoting effects of the

Lens injury seem to persist. More importantly their clodrolip treatment to deplete macrophages showed an increase in the number of regenerating axons growing at least

0.5mm distally to the lesion site by more than 3-fold compared to the group that received control liposomes (68, 699, 70).

This group’s results did not question the role of oncomodulin in promoting axon growth, however they concluded it cannot be the ‘principal factor’ mediating these regenerative effects of intraocular inflammation. This was supported by the findings that beneficial effects of oncomodulin in vivo were dependent on the co-administration of drugs that elevate cAMP levels, and that this treatment was still way less potent than inducing the

Lens injury or administrating zymosan alone (68, 69). In terms of the role of macrophages in mediating the axon regeneration in CNS injury models, this topic is still controversial. While

Fischer and his group did not negate the possibility of the pro-regenerative effects of macrophages or their derived factors, their results emphasized that in terms of the major effects induced by the Lens injury or zymosan on the injured RGC axons, there may be

37 another alternative mechanism involved, perhaps one that is independent of the macrophages.

Later they showed that lens injury or zymosan alone increased the levels of the cytokine Ciliary Neurotrophic Factor (CNTF) in retinal astrocytes (72). Further by culturing the naïve untreated retinas with increasing concentrations of lens proteins or zymosan and showing the increase in CNTF in a dose-dependent manner, they showed that this induction in the CNTF expression was happening independent of the macrophages. Intravitreal injection of CNTF showed the strongest outgrowth, while these effects were seen diminished with the treatment of anti-CNTF antibody or Janus-kinase (JAK) inhibitor, while the injections of anti-oncomodulin did not remove these effects at all. However, another glial- derived factor called Leukemia inhibitory factor (LIF) was found to have similar neuroprotective and axon regenerative activity (71, 72). This was discovered when the retinal

LIF expression was found correlated with CNTF expression after the Lens injury. It was confirmed when the CNTF/LIF knockout mice that underwent lens injury or zymosan administration did not show beneficial effects of regeneration. Additionally, they showed similar results through their in-vitro RGC culture that both CNTF and LIF significantly improved the neurite outgrowth (71, 72). However, their data on the role of CNTF as a mediator in exerting strong neuroprotective and regenerative effects on the axotomized

RGCs was questioned by several groups of researchers. Amongst the various arguments that were raised included the fact that the treatment with anti-CNTF antibody failed to significantly abolish the lens injury-induced regenerative effects (71, 72).

Since the discovery of another mediator LIF and owing to the ongoing controversy on the involvement of macrophages, it is certain that there is still a lot that needs to be explored (70, 71, 72). Perhaps there is some other factor that has not yet been found, which is responsible for mediating these beneficial effects. It can also be true that there is no single factor responsible for eliciting these protective and regenerative effects. Overall, these new discoveries, although contrary reflect on the need to further conduct more in-depth studies.

Due to these controversies around the role of macrophages in the CNS injury models, we through our present study wanted to further investigate if macrophages were involved in mediating these axon regenerative effects following an injury or a trauma. Moreover, considering the sparking interest in identifying a specific factor(s) responsible for these effects, we wanted to test whether histamine was that specific macrophage-derived factor that is responsible for promoting the axon outgrowth. In order to test this, we designed an invitro model using three different primary cell cultures namely, peritoneal macrophages, cortical astrocytes and retinal explants. The reason behind using primary astrocytes was to further address the more recent discoveries on the supportive role of glial scar and reactive astrocytes in the context of regeneration. Further the use of retinal explants allowed to see the effects of these cells on the neurite outgrowths from these explants. In addition, these explants have been proven to be a good in vitro model to test the regeneration outcomes.

1.9 Hypothesis and Aims

We hypothesized that Histamine is the specific macrophage-derived factor that is playing a critical role in promoting the neurite outgrowth of the in-culture RGC explants. To test this hypothesis, we completed the following aims:

Aim I. Role of Macrophages in promoting neurite outgrowth of Retinal Explants

1. Test the effect of Macrophage Conditioned Media (MCM) on the neurite outgrowth

of retinal explants, while explants are cultured on all glial cell layers.

2. Test whether the effects observed in 1. Are dependent on glial cells.

3. Test the effects of MCM on just Astrocytes

Aim II. Histamine is the Activated Macrophage-derived factor that is promoting neurite outgrowth in Retinal Explant cultures.

1. Test if the effects observed with the treatments of MCM are influenced with the

inhibition of histamine production

2. Test the effects of histamine directly on the retinal explant cultures

Aim III. Histamine’s neurite growth promoting effects are mediated by it binding to the H1 receptor.

1. Test the effects of all three histamine receptor agonists- H1, H2 and H3 on the neurite

outgrowth of retinal explants.

2. Compare the neurite outgrowth from the retinal explants when cultured on histamine

H1 receptor knockout animal-derived astrocytes versus wild type animal-derived

astrocytes.

Aim IV: Exosomes are involved in mediating the neurite growth promoting effects

1. Perform mRNA sequencing on the astrocytes treated with H1 receptor antagonist to

identify any differentially expressed genes

2. Isolate and quantify exosomes using ZETAVIEW Particle Meter for H1, H2 and H3-

agonist treated astrocytes

3. Test the effects of inhibiting exosome secretion on the neurite outgrowth of retinal

explants

Aim V: Astrocyte-derived exosomes promote neurite outgrowth on both growth supporting and inhibitory substrates

1. Test the effects of astrocyte-derived exosome treatments on the neurite outgrowth

while explants are added on coverslips coated with Laminin

2. Repeat the experiment in 1., except the explants are added on coverslips coated with

Laminin and CSPG

Aim VI: Astrocyte-derived exosomes promote neurite outgrowth via Wnt signaling pathway

1. Test the effects of blocking Wnt secretion on the neurite outgrowth of retinal explants

plated on just Laminin

2. Repeat the experiment in 1., except the explants are added on coverslips coated with

Laminin and CSPG.

Chapter 2: Materials and Methods

2.1 Isolation and Plating of Mixed Cortical Cells

Mixed cortical cell isolation for astrocyte cultures was performed using post-natal P1 to P4 C56BL/6 mouse pups. In order to achieve proper astrocyte density at least 3 pups were used per T75 tissue culture flask. Before the dissection procedure, astrocyte culture media

(DMEM, high glucose + 10% heat-inactivated Fetal bovine serum +1% penicillin/streptomycin, Medstore) was prewarmed to 37 °C. Coat one T75 flask with 20ml of poly-D-lysine (PDL, Sigma) at a concentration of 50ug/ml in cell culture grade water for at least one hour at 37 °C. For the dissection procedure, the necessary reagents and materials used included, surgical scissors, smooth fine forceps, flat tip forceps, 70% ethanol, 2 dissecting dishes on ice filled with 2ml Hank’s Balanced Salt Solution (HBSS, Medstore) each. The animal was gently held, and its head and neck were sprayed with 70% ethanol. The animal was sacrificed by decapitation using the scissors. A midline incision was performed, posterior to anterior along the scalp to reveal the skull. The cranium was carefully cut from the neck to the nose. Two additional cuts were performed to further allow access to the brain- the first cut was made anterior of the olfactory bulbs, the other one inferior to the cerebellum to disconnect the cranium from the skull base. Using the flat tip forceps, the cranial flaps were gently flipped to the side and the brain was taken out and placed in the dissecting dish filled with HBSS, which was always placed on ice.

The remainder of the dissection procedure was performed using the stereomicroscope.

First the olfactory bulbs and the cerebellum were removed using the fine dissecting forceps.

Then in order to retrieve the cortices, the posterior end of the brain was held in place using fine forceps while a midline incision between the hemispheres. During this using the second set of forceps inserted into the created groove, the plate-like structure of the cortex from the brain was extracted. After this, the meninges from the cortex hemispheres were removed carefully to avoid any contamination of the final astrocyte culture by the meningeal cells and fibroblasts. The prepared cortex hemispheres were then transferred into the second dissecting dish filled with HBSS and returned to the ice. The steps were repeated for all the pups.

Finally, each cortex hemisphere was cut into further smaller pieces using sharp forceps.

Under sterile conditions, these cortex pieces were transferred into one 50ml falcon tube with HBSS brought to the total volume of 22.5ml. To this additional 2.5ml of 2.5% trypsin was added, mixed and incubated in 37°C tissue culture incubator. Mixture was occasionally shaken every 3-5 minutes, while the incubation was performed for no more than

15 minutes. After this the mixture was centrifuged for 5 minutes at 300g to pellet the cortex tissue pieces. The supernatant was carefully removed. The tissue pellet was resuspended in

5ml fresh astrocyte media until the tissue pieces had dissociated into single cell suspension.

This was then passed through a 100um cell strainer after which the total volume was adjusted to 20ml astrocyte media. The initially added PDL was aspirated from the T75 flask and was replaced with the 20ml dissociated cell suspension which was incubated at 37°C in the CO2 tissue culture incubator.

2.2 Obtaining an Enriched Astrocyte Culture

After the plating of mixed cortical cells, the media was changed every 3 days thereafter. After 7-8 days when cells would reach about 85%-90% confluency, the T75 flasks were shaken at 180rpm for 30 minutes on an orbital shaker to remove the overlying microglia. The supernatant containing the microglia was discarded and fresh 20ml astrocyte media was added. Then shaking was continued at 240rpm for 6 hours to remove the oligodendrocyte precursor cells (OPCs) that had also detached from the bottom astrocyte layer. To ensure all the OPCs were removed, the flask was again vigorously shaken by hand for 1 minute to prevent any OPC contamination. The supernatant was similarly discarded, and the remaining confluent astrocyte layer was washed with PBS twice. After aspirating the

PBS, 5ml of trypsin-EDTA was added and the cells were incubated in the cell culture incubator at 37°C. The cell detachment was checked every 5 minutes and to enforce further detachment, the flask was hit against the palm 2-3 times. After the astrocytes had completely detached 5ml of astrocyte media was added to neutralize the trypsin, and the cells were spin at 180g for at least 5 minutes. The supernatant was carefully aspirated, and the pellet was resuspended in 5ml of the fresh astrocyte media by pipetting multiple times. Then the total media volume was brought to 40ml and 20ml of cell suspension media was distributed in each T75 flask. One T75 flask would normally yield around 1X 106 cells after this first cell split. The cells in the two T75 flasks were left to grow in 37°C tissue culture incubator for the future experiments.

2.3 Purification of Exosomes by Differential Ultracentrifugation

After the astrocytes reach about 85%-90% confluency, their culture media was collected and centrifuged at 300g for 10 minutes to eliminate any large dead cells or cell debris. The supernatant was collected and centrifuged again at 2000g for 10 minutes at 4°C.

From hereon after, all the steps were carried out at 4°C, so the samples were always on ice.

The pellet was discarded while the retained supernatant was centrifuged using the ultracentrifuge at speed 10,000g for 30minutes. The supernatant was collected and ultracentrifuged at 100,000g for 70 minutes to pellet the small vesicles that corresponded to exosomes. This pellet was then washed in a large volume of PBS to eliminate any contaminating proteins. In order to this, the pellet was resuspended in cold PBS and ultracentrifuged again at 100,000g for 70 minutes. The final pellet at this step was collected and resuspended in 100ul of PBS and was aliquoted and stored in -80°C freezer for future experiments (29).

2.4 Quantification of exosomes using ZETAVIEW Particle Meter

Figure 2-1. Zetaview Nanoparticle Tracking Analyzer (73)

After the purification of exosomes using the method in section 2.3, the samples were brought to Mount Sinai Hospital. Here the exosomes wee quantified using their shared

Zetaview Particle Meter. The program was opened and the instructions on the screen for an automated implementation of the Startup procedure were first followed. Boxes for both cell quality check and auto-alignment were selected, although either steps could be repeated separately as needed. The inlet and outlet port were opened and 10ml of sterile water was injected by the syringe into the cell channel through the inlet port. A beaker was placed under the outlet port to collect the waste solution. At all times, the measurement cell needed to be devoid of any air bubbles. The cell quality check was performed. The software would display the results within few seconds. If any particles are visualized in the connected live- view screen of the software, or if the results from the cell quality check were only good or poor, the previous steps were repeated until the measurement cell was fully free of any particles or bubbles. After this the control suspension was prepared that contained uniform

200nm sized polystyrene particles to be used to align the foci of the laser and microscope.

One drop of this suspension concentrates, provided by the instrument manufacturer, was added to 500ml of distilled water to obtain the required concentration, such that 600±100 particles were displayed per screen in the live view. This alignment suspension was then injected, and the auto-alignment was started, which is an automated routine through which the system itself finds the optimal position between the two foci.

Once the prompt appeared on the screen stating that the system was now ready for experiments, the actual exosome measurement was started. Before measuring the first sample, the cell channel was washed once again with distilled water after which the exosome suspension in PBS was injected. The main parameters of the software that included, sensitivity, minimum brightness and minimum and maximum size were adjusted. In order to find the optimal sensitivity range, an area on the curve for measured particles per screen for different sensitivity levels was chosen, preferably before the maximum slope of the curve. A higher sensitivity level would allow for visualization of more small particles but would also increase the background noise. In terms of brightness, the starting brightness of 20 for exosome measurement was chosen. The parameter could be regulated up to blank out the strong light-scattering particles, and down to amplify the weakly scattering particles. The digital filter was set to eliminate pixel noise and unwanted scatter from oversized particles or clumps by regulating the minimum and maximum size. The range of 10 to 500 pixels

(maximum particle diameter cut-off was 150 nm) for measurement of exosomes was chosen.

The period that the camera was open for was adjusted to 1/300 second. The number of particles counted in the field of vision was noted from the live display as well. Once the measurement began, before each acquisition the movement of the particles was checked to check for the particle drift test. If the particle drift was found to be higher than 20

µm/second, the measurement was paused until the sample would slow down for the final acquisition. Camera sensitivity for all samples was adjusted to be at 75, shutter at 100, scattering intensity to be detected automatically and the cell temperature around 25°C. For each measurement, three cycles were performed by scanning 11 cell positions each and capturing 30 frames (73).

2.5 Isolation of Peritoneal Macrophages

5-8 weeks old C57BL/6 mouse were used to collect the peritoneal macrophages. The mice were euthanized by rapid cervical dislocation. Then the skin was sprayed with 70% ethanol and the mouse was mounted on the Styrofoam block. Using the scissors and forceps a small incision was made in the outer skin of the peritoneum and the skin was gently pulled back to expose the inner lining the peritoneal cavity. 5ml of ice cold 30% sucrose in PBS was injected into the peritoneal cavity using a 27-gauge (g) needle, avoiding puncturing the intestines or any leakage. After the injection, the area was gently massaged for few minutes to dislodge any attached cells into the solutions. A 25g needle attached to a 5ml syringe was then inserted to collect the fluid while moving the tip of the needle gently to avoid clogging by any fatty tissues or other organs. After this, the fluid was centrifuged at 1500g for 5 minutes. The supernatant was discarded and the pellet that supposedly contained macrophages was retained. The cells were resuspended in the Macrophage media- DMEM- high glucose with 10% FBS and 1% penicillin-streptomycin and counted using the hemocytometer. Normally one mouse yielded close to 1 X 106 cells/ml. The cells would be plated in a 12-well plate. The cells were washed 2 hours with PBS to remove any B-cells or

T-cells. The macrophages that were attached were maintained in the culture for 2-3 days before some of them were activated to induce the M1-phenotype.

2.6 Histamine H1 Receptor Knockout (H1-KO) animals

H1-KO adult animals of the strain ‘B6.129P2-Hrh1/BrenJ’ (Jackson

Laboratories) were bred to start a colony which was maintained. The pups of age P1-P4 were used to establish astrocyte cultures that were later used for experiments.

2.7 Cell Culture Treatments

While establishing enriched astrocyte cell cultures, to ensure more efficient removal of microglia cells, the cells were treated with 25 mmol/L of L-leucine methyl ester (LME,

Sigma-Aldrich) for 90 minutes. Then the media was discarded, and the cells were washed thoroughly with PBS 2-3 times, after which fresh astrocyte media was added and the cells were maintained. Astrocytes and Macrophages were activated using 1ug/ml LPS (Sigma) and

5ng/ml IFNϒ (Sigma) for 48 hours after which the conditioned media was collected for future experiments. The astrocytes were also treated with 10µM GW4869 (Sigma), or 100

µM Histamine (Tocris) or 100µM 2-pyrilethylamine dihydrochloride (H1 agonist, Tocris) or

100µM Dimaprit dihydrochloride (H2 agonist, Tocris) or 100µM R-)-(-)-α-methylhistamine dihydrobromide (or H3 agonist, Tocris) or 50µM Diphenhydramine hydrochloride (H1 antagonist, Cedarlane) or 50 µM Pinocembrin (5,7-dihydroxyflavonone, Histamine

Decarboxylase enzyme inhibitor, Sigma). In some experiments, explants were added along with 20µM IWP2 (Sigma) (29).

2.8 Chick Retinal Explant Outgrowth Assay

Coverslips were autoclaved and treated with 10 µg/ml poly-L-lysine prepared in sterile water for 24 hours and stored at 4°C. The next day these coverslips were washed with sterile water and allowed to dry for 20 minutes before coating with 10 µg/ml laminin prepared in PBS, with or without 10 µg/ml CSPG, for 2 hours in a tissue culture incubator at

37°C. Except for few experiments where prepared retinal explants were directly added on the coverslips, most experiments involved a monolayer of astrocytes growing underneath on top of which the explants were then added. Astrocytes would be maintained in their specific media until the explants were added. On reaching confluency, either the astrocytes were activated using LPS and IFN ϒ or would be left as non-activated.

To prepare the retinal explants, the fertilized eggs from Leghorn chicken were purchased and incubated in a 37.5 °C and 60% humidified egg incubator. The eggs were removed from the incubator at the embryonic age E7 day (Note: The age of chicken embryo is determined as the time of incubation and is denoted as the embryonic day). The eggs were placed in the laminar flow hood. The eggshells were wiped with 70% ethanol to avoid contamination of the embryo, and all the procedures were conducted under aseptic conditions using sterile solutions.

The side of the egg was tapped and cut using a scissor to empty its contents into a dish. Then the embryo was transferred to a petri dish containing pre-warmed HBSS to prevent the tissue from drying. Using the dissecting microscope in the laminar flow hood, the embryo was decapitated by pinching its neck with the fine forceps and the body is discarded.

Using fine forceps, an incision was made through the mouth, ventral to the eye. The tissue

51 around the eye was removed while the optic nerve was pinched off and the eye was removed from the eye socket. Then the eyes were transferred into a new 35mm petri dish containing pre-warmed HBSS to prepare retinal explants. Using fine forceps all the remaining tissue around the eye was removed including the scleral anlage. At this point all that remained was the retina on the vitreous body along with the lens and the pigment epithelium (Fard, S.S.,

2015).

Using forceps, a small incision was made into the pigment epithelium to tear it open and remove it. Afterwards, the lens and the vitreous body was removed too. Then the retina was cut into smaller pieces using a tissue chopper. After that these retinal explants were ready to be used for experiments where they were maintained in a specific culture medium containing 1:1 DMEM: F12 nutrient mix supplemented with 10% FBS, 2% Chick serum and

1% penicillin/streptomycin. Retinal explants were then cultured for at least 18hr with or without the different treatments like GW4869, histamine, or one of the three agonists. The next day they were fixed with 4% paraformaldehyde (PFA) and stained (29).

2.9 Immunocytochemistry

After leaving the retinal explants in culture for at least 18 hours, the explants were fixed with 4% Paraformaldehyde (PFA) prepared in PBS and incubator for at least an hour in

4°C. This was followed by the rinsing with 0.1M PBS/ 0.1% triton for 3-5times for 5minutes each. The explants were blocked for 1 hour in 5% FBS in PBS/0.1% Triton, after which the primary antibody GFAP (1:300) produced in mouse along with beta-III tubulin (1:1000)

52 produced in rabbit was added in the same blocking buffer for overnight at 4°C on an orbital shaker. The next day the explants were washed and the secondary antibodies (1:1000) were added in PBS/0.1% Triton and incubated for 1hr at room temperature. Dapi was also added for 10 min. Then after couple washes, the coverslips were mounted on to the microscope slides for imaging.

2.10 mRNA sequencing

The astrocytes were grown to 90% confluency and were then treated with the 50 µM

Diphenhydramine hydrochloride (H1 antagonist) for 24 hours while half of the wells were left untreated. Then these cells were used for mRNA sequencing. The kit that used was

Illumina TrueSeq Stranded mRNA (Thermo Fischer) and the protocol was also obtained from TrueSeq Stranded mRNA Reference Guide. The protocol involved series of steps starting with capturing and purifying of the poly-A mRNA using the magnetic beads with poly-T oligos. mRNA was then fragmented using divalent cations under elevated temperature. The purification was conducted by the postdoctoral fellow in the lab, while they were sent for bioanalysis at Dr. Lively’s lab. The workflow involved addition of primers which were bound to RNA fragments in preparation for synthesis of first cDNA strand. Then

3’ ends of cDNA were adenylated to prevent cDNA fragments from ligating to one another during the following adapter ligation reaction. After this cDNA was amplified and enriched.

The library generation was conducted with the help from the common facility at Toronto

Western Hospital. The library cleanup was conducted to remove any cDNA fragments that

53 were too small. Then the results were sent to Mount Sinai Hospital to Dr. Wrana’s lab for analysis.

2.11 Statement of Ethics for Animal Use

All animals were handled and sacrificed in accordance with the guidelines of the

Canadian Council of Animal Care and approved by Toronto Western Hospital animal care committee.

2.12 Microscopy

All images were obtained using the Standard Fluorescence Microscope, and the

Software used for quantification was CellSens.

2.13 Statistical Analysis

Data was analyzed using GraphPad Prism software. Student t-tests were used for direct comparison between two groups. One-way Anova with Bonferroni post-hoc correction for comparisons between multiple groups was utilized. Statistical Significance was set at p<0.05 (*), p<0.01 (**) and p<0.001(***). For every figure, mean and standard error of the mean (SEM) are presented.

Chapter 3: Results

Aim I: Role of Macrophages in promoting neurite outgrowth of Retinal explants

3.1 Neurite outgrowth is promoted with the treatment of Activated Macrophage

Conditioned Media

Since an injury to the CNS is usually followed by a cascade of inflammatory reactions including infiltration of the macrophages to the injury site. Considering the controversies around the role of macrophages in axonal regeneration, we hypothesized that the pro-inflammatory macrophages are beneficial to the regeneration of the severed axons.

To test this hypothesis, we developed an in-culture model using three primary cell lines namely, peritoneal macrophages, cortical glial cells (astrocytes, oligodendrocytes, microglia) and retinal ganglion explants. The peritoneal macrophages were extracted from C57BL/J mice and were treated with LPS and IFN  for 48 hours to induce the M1-phenotype.

Afterwards, fresh media was added, and the cells remained in culture for up to 3 days to allow the macrophage-derived factors to be released into the media. Then the supernatants from the non-activated and activated macrophages were collected to be used as cell-culture treatments. These will be referred to as Non-activated Macrophages Conditioned Media

(MCM) or Activated Macrophages Conditioned Media (MCM), respectively. The primary glial cells were extracted from the cortices of the P1-P4 C57BL/J mouse pups and were maintained in culture for 7 days after which they were split in order to be used for experiments. The retinal explants were prepared using the chick embryos of age E7. The

55 prepared explants were added on to the glial layers already plated on the coverslips, and were treated with the non-activated or activated MCM in the ratio of 1:1 of MCM to supplemented

DMEM media, while the control group received no macrophages’ supernatant and just had media. The retinal explants were allowed to grow neurites for 24 hours after which the explants along with the cells were fixed for immunostaining. The glial cells and explants were visualized using GFAP and β-III tubulin under a fluorescence microscope. Up to 10 longest neurites were measured for each explant from at least 10 different explants for each condition. The neurite lengths were quantified using the CellSens software and plotted as mean lengths ± SEM (error bars) for n=3 independent experiments. For the control group, the neurite length was measured to be 400 µm while the treatment of Non-activated MCM increased the neurite outgrowth to the mean length 457.2 ± 16.93 µm and Activated MCM increased it to 574.4 ±22.92 µm, in comparison to control untreated group that had a mean length of 347.9 ± 15.25 µm. We found that the explants that had been treated with the activated MCM showed the longest neurites of the three groups.

beta-III tubulin GFAP DAPI Merge

con

MCM

act

+Act +Act

MCM

Neurite outgrowth Mean ± SEM

800

600

400

200 Neurite length (um) length Neurite

con

+Act MCM

+Non-act MCM Groups

Figure 3-1. Neurite outgrowth is promoted with treatment of Activated MCM. A) Representative images of embryonic retinal explants cultured on the glial cells (astrocytes, oligodendrocytes and microglia) treated with media (Con), or Non-activated MCM or Activated MCM, Scale bars, 100 µm, n=2. B) . B) Quantification of the neurite outgrowth for the groups plotted as mean lengths ± SEM (error bars)

3.2 The neurite outgrowth promoting effects induced by macrophages are dependent on glial cells

To further investigate whether the glial cells are an essential component in mediating the observed effects on the neurite outgrowth of the retinal explants, a control experiment was conducted. Here, the prepared retinal explants were cultured directly on the coverslips that had been coated with laminin. The following steps were similar as the previous experiment. The control explants were left untreated and were cultured in the media that had been supplemented with the chick serum. The other two groups had either Non-activated

MCM or Activated MCM added to them. Here the neurite outgrowth length in the control group was quantified to be 289 µm, while the non-activated MCM and activated MCM treated groups showed neurite lengths of 231 µm and 245 µm, respectively. The treatments with Non-activated or activated MCM did not show any significant difference in the neurite lengths from the control group. Since the neurite growth promoting effects seen previously were not seen here, we speculated that the glial cells may be playing some role in mediating the effects of the M1-macrophages.

Con +Non-act MCM + Act MCM

B Neurite outgrowth

Mean ± SEM

300

200

100

Neurite length (um) length Neurite

Con

+Act MCM

+Non-act MCM Groups

Figure 3-2. The neurite growth promoting effects induced by Activated MCM treatment are dependent on the glial cells. A) Representative images of embryonic retinal explants cultured in the absence of the glial cells, treated with media (Con), or Non-activated MCM or Activated MCM, Scale bars, 100 µm. B) Quantification of the neurite outgrowth for the groups plotted as mean lengths ± SEM (error bars) for n=3 independent experiments

3.3 Activated Macrophages promote neurite outgrowth of the retinal explants when cultured just on astrocytes

Since one of the reasons why the injured CNS axons are unable to spontaneously regenerate past the lesion site is due to glial scar formation, which is formed by astrocytes undergoing reactive gliosis, these glial cells are also particularly studied in the context of axon regeneration failure. Further since the 2016 Nature study by Anderson, M. which reported that these scar-forming astrocytes are not the principal causes behind the regeneration failure but are in fact necessary for the axonal regrowth, makes it even more necessary to further study these cells. Therefore, considering the functional heterogeneity of astrocytes and the effects of their cell responses following the injury, we decided to replicate the previously described in-vitro model but this time using just the primary astrocytes.

The cortical cells were extracted as described earlier however after the cells remained in culture for 7 days which is how long it takes for all the three glial cell layers (astrocytes, oligodendrocyte precursor cells and microglia) to start maturing, the astrocytes were isolated.

In order to create an enriched primary astrocyte cell line, cells were shaken at a high speed to sequentially isolate and remove the overlaying microglia and then the oligodendrocyte precursor cells. These astrocytes were then plated on the pre-coated coverslips and the retinal explants were then added once the cells had reached 90% confluency. The results from this experiment were consistent with the previous results where we had all three glial cell layers.

The longest neurites were seen from the explants that were treated with the M1-macrophage supernatant, meaning the activated MCM. The untreated explants cultured on primary astrocytes showed the mean neurite length of 354.3 ± 9.25 µm, which was quite similar to

the measured neurite length of control from the experiment with all three glial layers. The

explants treated with Non-activated MCM showed the mean neurite length of 463.1 ± 9.03

µm, while the explants treated with the Activated MCM were even longer with the mean

lengths of 622.6 ± 8.87 µm. Both these treatments showed a significant increase in

comparison to the control (***p<0.001). The Activated MCM treated explants consistently

resulted in significantly longer neurites compared to their control counterparts. This further

proves that the pro-inflammatory macrophages were responsible for promoting the neurite

outgrowth of these explants.

beta-III tubulin GFAP DAPI Merge

Con

+Non -

act MCM

+Act MCM

Neurite outgrowth Mean ± SEM

800 *** 600 ***

400

200 Neurite length (um) length Neurite

con

+Act MCM

+Non-act MCM Groups

Figure 3-3. Activated MCM promotes neurite outgrowth when retinal explants are cultured on just astrocytes. A) Representative images of embryonic retinal explants cultured on just astrocytes treated with media (Con), or Non-activated MCM or Activated MCM, Scale bars, 100 µm. B) Quantification of the neurite outgrowth for the groups plotted as mean lengths ± SEM (error bars) for n=3 independent experiments

Aim II: Histamine is the activated macrophage-derived factor that is promoting neurite outgrowth in the Retinal Explant cultures

3.4 Inhibiting Histamine production inhibits the neurite promoting effects of

Activated Macrophages

We further hypothesized that the pro-regenerative effects observed with treatments of retinal explants with activated macrophages conditioned media are mediated by histamine. In order to test this, a pan inhibitor called Pinocembrin was used. This inhibitor targets the histidine decarboxylase (HDC) enzyme responsible for producing histamine. After the macrophages were activated using LPS and IFN , to some of the cells this inhibitor was added after which all the cells remained in culture for another 3 days. The rest of the procedure was followed as described earlier. The neurite length was found to have decreased to 345.4 ± 9.25µm on treatment with the pan inhibitor, which is a significant decrease from

622.6 ± 8.87 µm (*** p<0.001) mean neurite length seen with the treatment of activated

MCM. This suggested that histamine may be playing a crucial role in mediating those regenerative effects.

beta-III tubulin GFAP DAPI Merge +Act MCM w/

inhibitor

B Con +Act MCM

+Act MCM w/ inhibitor

C Neurite outgrowth Mean ± SEM

***

800

600

400

200 Neurite length (um) length Neurite

con

+Act MCM

+Act MCM w/ Inhibitor Groups

Figure 3-4. Blocking Histamine production inhibits the neurite growth promoting effects. A) and B) Representative images of embryonic retinal explants cultured on astrocytes treated with Activated MCM containing Pan Inhibitor Pinocembrin (top row), Comparison between the enlarged representative images of untreated retinal explants (con), with explants treated with Activated MCM and with explants treated with Activated MCM and the inhibitor. Scale bars, 100 µm. C) Quantification of the neurite outgrowth for the groups plotted as mean lengths ± SEM (error bars) for n=3 independent experiments

3.5 Histamine treatment promotes neurite outgrowth of the retinal explants

To confirm the result findings from the previous experiment, we decided to test the effects of histamine (100 µM) added directly on to the retinal explants cultured while they were on primary astrocytes. To see if the different activation status of the primary astrocytes can change its induced effects, histamine was tested on both the non-activated astrocytes, along with the activated astrocytes. In order to activate the astrocytes, LPS and IFN  was added and the cells were incubated for 48 hours. After this the media was replaced and the cells were used for the experiment. The neurite length of the explants treated with histamine on non-active astrocytes was found to be 576 ± 39.81µm compared to the control with the mean neurite length of 417.9 ± 29.08µm (* p<0.05). Further, when the retinal explants were cultured on the activated astrocytes and treated with histamine, the neurite length was even longer and was quantified to be 735.3 ± 38.97µm, compared to control explants on non- treated activated astrocytes with neurite length 385.8 ± 29.85µm (*** p<0.001). However, there was no significant difference in the neurite outgrowth between untreated explants cultured on non-activated and activated astrocytes group. Histamine treatment significantly increased the neurite length for both groups of astrocytes, but its pro-regenerative effect was more pronounced on the activated astrocytes given there was a greater increase in the neurite length for this group. We speculated that the histamine must be binding to its receptor on these astrocytes to mediate its growth promoting effects on the cultured explants on top.

beta-III tubulin GFAP DAPI Merge

Non - act act astro

Non -

act astro + Hist

Act astro

Act astro + Hist

Neurite outgrowth Mean ± SEM

* ***

1000

800

600

400

200

(um) length Neurite 0

Act astro Non-act astro Act astro + Hist Non-act astro+ Hist Groups

Figure 3-5. Histamine treatment promotes neurite outgrowth of the retinal explants. A) Representative images of embryonic retinal explants cultured on non-activated astrocytes with no treatment (con), or non-activated astrocytes treated with histamine (non-act + hist), or activated astrocytes treated with histamine (act astro + hist). Scale bars, 100 µm. B) Quantification of the neurite outgrowth for the groups plotted as mean lengths ± SEM (error bars) for n=3 independent experiments

Aim III: Histamine’s neurite growth promoting effects are mediated by its binding to H1 receptor

3.6 Treatment with Histamine H1 receptor agonist promotes neurite outgrowth of retinal explants

Many studies have reported that histamine has a strong influence on many of the activities of astrocytes, including ion homeostasis, energy metabolism, neurotransmitter clearance, neurotrophic activity and immune response. These responses are mediated through the binding of the histamine to one or more of its three receptor subtypes, H1, H2 and H3 that are expressed on the astrocyte surface. To find which of these three receptors it is binding to in order to promote the neurite outgrowth, we decided to test the effects of H1 agonist 2- pyrilethylamine dihydrochloride (100 µM), H2 agonist 100µM Dimaprit dihydrochloride (100 µM) and H3 agonist R-)-(-)-α-methylhistamine dihydrobromide (100

µM) on the neurite outgrowth of retinal explants. H1, H2 and H3 agonists were added on to the retinal explants which were cultured on either the non-activated astrocytes or the activated astrocytes. For the non-activated astrocyte cultures, the mean neurite length for the control or untreated explants was found to be 417.9 ± 8.87 µm. In comparison, H1, H2 and

H3 agonist treatments resulted in the mean neurite length of 680.1 ± 41.54 µm, 443.4 ± 18.26

µm and 435.6 ± 31.48 µm, respectively. The neurite lengths were the longest for the H1 agonist treated explants, when compared to the H2 and H3 agonist treated groups. In fact, the

H1 agonist treatment promoted the neurite growth significantly in relative to the control untreated explants (*** p<0.001), while there was no significant difference between the other

70 two agonists and control. On the other hand, for the activated astrocytes the explants that remained untreated had the mean neurite length of 358.8 ± 29.85 µm. While on treatment with H1, H2 and H3 agonists they showed neurite lengths of 771.0 ± 35.93 µm, 307.8 ±

21.32 µm and 275.4 ± 29.66 µm, respectively. The effect of H1 agonist was far more pronounced when the explants were cultured on the activated astrocytes instead of non- activated. Since after the injury to CNS, the immediate response of astrocytes nearby is to undergo activation, these results can be used to extrapolate that the activated astrocytes may become beneficial for the regeneration process following the injury.

beta-III tubulin GFAP DAPI Merge

in Non

- act act astro

+H1

+H2

+H3

Neurite outgrowth Mean ± SEM

***

800

600

400

200

Neurite length (um) length Neurite

+H1 +H2 +H3

Non-act astro Groups

Figure 3-6. Histamine H1 agonist treatment promotes neurite outgrowth of retinal explants cultured on non-activated astrocytes. A) Representative images of embryonic retinal explants cultured on non-activated astrocytes with no treatment (con), or non- activated astrocytes treated with H1 agonist, or H2 agonist or H3 agonist. Scale bars, 100 µm. B) Quantification of the neurite outgrowth for the groups plotted as mean lengths ± SEM (error bars) for n=3 independent experiments

beta-III tubulin GFAP DAPI Merge l in in in in

Act astro

+H1

+H2

+H3

Neurite outgrowth Mean ± SEM

***

1000

800

600

400

200 (um) length Neurite 0 +H1 +H2 +H3 Act astro Groups

Figure 3-7. Histamine H1 agonist treatment promotes neurite outgrowth of retinal explants cultured on activated astrocytes. A) Representative images of embryonic retinal explants cultured on activated astrocytes with no treatment (con), or activated astrocytes treated with H1 agonist, or H2 agonist or H3 agonist. Scale bars, 100 µm. B) Quantification of the neurite outgrowth for the groups plotted as mean lengths ± SEM (error bars) for n=3 independent experiments.

3.7 Histamine H1 receptor Knockout mice- derived astrocytes show decrease in the neurite outgrowth

The previous results showed that H1 agonist treatment showed the similar growth promoting effects seen with the histamine treatment, which were not observed with treatments of H2 or H3 agonist. We decided to further confirm this by using specific H1 receptor knockout (H1-KO) mice. The P1-P4 pups from the pregnant mice were used to extract cortical astrocytes in the similar manner as previously described. Presuming these astrocytes do not express the histamine H1 receptor it was expected that they will not show the similar growth stimulatory effects that were observed with the treatments of histamine, or

H1 agonist alone. As expected, we found that the H1-KO astrocytes resulted in much smaller neurites when compared to the wild-type astrocyte group. The mean neurite length for the

KO was found to be 234.3 ± 10.07 µm which was significantly smaller than the mean neurite length for the WT group which was 385.6 ± 9.54 µm (*** p<0.001). On treatment with histamine, the mean neurite length for the WT group was increased to 596.0 ± 12.75 µm, while the neurite length for KO group was found to be 236.3 ± 11.87 µm (*** p<0.001). This implies that perhaps the WT astrocytes (non-activated), alone without any treatments may be providing a growth permissive environment for these neurites. Furthermore, this neurite length is improved for the WT non-activated astrocytes group that were treated with histamine, but it does not improve for the KO group. The neurite length for both the untreated H1-KO astrocytes and the ones that were treated with histamine were found to be significantly smaller than the WT group. This confirmed that histamine must be promoting

the neurite outgrowth of these retinal explants through binding to its H1 receptor found on

the astrocytes.

beta-III tubulin GFAP DAPI Merge

l in in

WT astroWT in

astro

+ hist +

H1 - KO as KO

tro

- KO astro + Hist

Neurite outgrowth

Mean ± SEM ***

800 ***

600

400

200

(um) length Neurite 0

H1-KO astro wild-type astro H1-KO astro + Hist wild-type astro + Hist

Groups

Figure 3-8.H1-KO astrocytes resulted in decreased neurite outgrowth. A) Representative images of embryonic retinal explants cultured on non-astrocytes extracted from the wild-type mice without (WT astro) or with histamine (WT astro + hist) , compared with the non- activated astrocytes extracted from the H1-KO mice without (H1-KO astro) or with histamine (HI-KO astro + hist). Scale bars, 100 µm. B) Quantification of the neurite outgrowth for the groups plotted as mean lengths ± SEM (error bars) for n=3 independent experiments

Aim IV. Exosomes are involved in mediating the neurite growth promoting effects

3.8 Differential gene expression analysis show that H1-receptor antagonist treatment results in downregulating gene involved with exosome release

To further investigate the specific factors that may be regulating this pro-regenerative effect of histamine at the genetic level, we decided to send these samples for mRNA sequencing. Primary astrocytes were grown and maintained in the similar manner as described before. When cells reached appropriate confluency, some of these cells were left untreated while remaining were treated with 50µM H1 receptor antagonist,

Diphenhydramine hydrochloride for 24 hours. After this, the mRNA from both these samples was isolated and purified using the TrueSeq mRNA Kit and was send for sequencing. The results from the sequencing were analyzed by Dr. Wrana’s lab at Mount Sinai Hospital. The method that was used to quantify and statistically infer the systematic changes between the two conditions was DESeq2 which uses negative binomial generalized linear models. This method produced 456 differentially expressed genes for the antagonist group (in relative to the control group). After adjusting the p-value cut-off to 0.05, this number was reduced to 10 differentially expressed genes which are arranged in the order of their increasing fold change.

Decreased fold change and log 2-fold change implies decrease in mRNA expression. It was found that amongst these 10 results, one specific gene (shown in highlight) that was downregulated with the treatment of H1 antagonist was ‘dnah6’ or dynein axonemal heavy chain 6 gene. This gene was found to be belonging to the dynein family whose members code for microtubule-associated motor proteins. Additionally, there are studies showing that

79 dynein may be involved in regulating MVB trafficking and exosome release. This was further supported when few reports showed that a dysfunction of dynein can result in significant disruption in exosome release. Seeing that dynein mRNA levels are significantly decreased with the histamine H1 receptor antagonist treatment of the primary astrocytes; it supports the speculation that histamine might be inducing these growth promoting effects through its influence on exosome secretion.

Table 3-8. Relative mRNA expression levels for H1-antagonist treated astrocytes

Symbol Fold change Log 2-fold change p-value

3.9 H1 receptor agonist treatment promotes exosome secretion

To further test whether treatments with histamine can influence exosome secretion, we decided to isolate and purify the exosomes from the conditioned media obtained after treating the primary astrocytes with H1, H2 or H3 agonist. This was repeated for both non- activated and activated astrocytes. The exosomes isolation and purification were performed using series of ultracentrifugation steps described in the Section 2.3. These exosome samples were then taken to the facility at Mount Sinai Hospital where they were quantified using the

Zetaview Particle Meter. The steps to quantify the exosome (microvesicles) concentration can be found in Section 2.4. The exosome concentration is normalized for all the groups against the control which in this case is the non-activated (untreated) group, to allow for systematic comparison between the groups. The results showed that the mean exosome concentration for the untreated non-activated astrocytes was 5.03 X 107 particles/cm2, while with the treatment of H1-agonist the concentration increased to 5.86 X 107 particles/cm2. On the other hand, the mean exosome concentration for the untreated activated astrocytes was quantified to be 4.0 X 107 particles/cm2, however with the treatment with H1-agonist the exosome levels were found to have significantly increased to 6.8 X 107 particles/cm2 (*** p<0.001). This was consistent with the results that we observed from the chick retinal explant outgrowth assay. The effect of H1-agonist was seen to be more pronounced for the activated astrocytes group, just like in the neurite outgrowth assay. Therefore, it indicates that H1-agonist may be resulting in longer neurite outgrowth through its promotion of exosome secretion. Furthermore, this effect may be particularly greater with the activation of astrocytes. On treatment with H2-agonist the exosomes level was found to be 4.63 X 107

81 particles/cm2 when cultured on non-activated astrocytes and 5.5 X 107 particles/cm2 when cultured on activated astrocytes. However, these levels were insignificant in relative to untreated non-activated and activated astrocyte groups, respectively. The similar trend was observed for the H3-agonist treatment. The exosomes levels were found to be 4.03 X 107 particles/cm2 for the non-activated group, while it was 3.75 X 107 particles/cm2 for the activated group. These levels were also statistically insignificant in relative to their untreated non-activated and activated groups. This was again consistent with the neurite outgrowth results observed with the treatments of H2- and H3- agonists for both non-activated along with activated astrocytes.

Microvesicle/exosome concentration Mean ± SEM

) 2

***

200

150

100

act

non-act act+H1 act+H2 act+H3 Microvesicle concentration (particles/cm concentration Microvesicle non-act+H1 non-act+H2 non-act+H3 Groups 82

Figure 3-9. H1-agonist treatment increases exosome secretion. Quantification of the exosome levels plotted as Microvesicle concentration ± SEM (error bars) for n=3 independent experiments. All groups are normalized to non-activated (untreated) group for easier comparison between the treatments. The astrocytes were either left non-activated or were activated. Following this, they were treated with H1, H2 or H3 agonist and the exosomes from this conditioned media were purified and quantified using Zetaview Particle Meter.

3.10. Inhibiting exosome secretion decreases neurite outgrowth

To further examine the role of exosomes secretion in promoting neurite outgrowth, we decided to test the effects of exosome secretion inhibitor GW4869. Before performing the retinal explant assay, to confirm that GW4869 can inhibit exosome secretion, the conditioned media from astrocytes treated with GW4869 was retained to collect the exosomes. The exosomes were quantified in the similar manner as before using Zetaview. The same process was repeated for histamine treatment. This allowed for direct comparison between their exosome levels. It was found that GW4869 treatment significantly decreased the exosome concentration for both non-activated and activated astrocytes (*** p<0.001). On the other hand, treatment with histamine did increase the exosome levels in relative to the untreated non-activated and activated groups, however these results were not statistically significant.

However, the exosome levels were significantly increased for the histamine treatments in relative to GW4869 for both non-activated and activated astrocyte groups.

Microvesicle/exosome concentration

Mean ± SEM )

2 *** *** 150

100

act

non-act

Microvesicle concentration (particles/cm concentration Microvesicle act+Hist

non-act+Hist act+GW4869 non-act+GW4869 Groups

Figure 3-10. GW4869 treatment decreases exosome secretion. Quantification of the exosome levels plotted as Microvesicle concentration ± SEM (error bars) for n=3 independent experiments. All groups are normalized to non-activated (untreated) group for easier comparison between the treatments. The astrocytes were either left non-activated or were activated. Following this, they were treated with GW4869 or histamine and the exosomes from this conditioned media were purified and quantified using Zetaview Particle Meter.

After confirming the effect of GW4869 on the astrocyte-derived exosome concentration, we decided to test its effects on the neurite outgrowth using the same astrocyte-chick retinal explant outgrowth assay. The treatments were performed as described in Section 2.7. Immunostaining results indicated that GW4859 treated astrocytes were showing significantly shorter neurite outgrowth. The mean neurite outgrowth for the untreated non-activated astrocytes was quantified to be 381.5 ± 15.82 µm, while this was decreased to 136.7 ± 10.51 µm on treatment with GW4869 (*** p<0.001). Furthermore, it was observed that this neurite outgrowth was not improved when histamine or H1-agonist was added in addition to GW4859. GW4869 is known to inhibit ceramide-mediated inward budding of the MVBs which directly results in inhibiting the formation of mature exosomes and by doing so, it indirectly results in inhibiting exosomes release from their cells. More importantly, these results were consistent with the results observed with its effects on exosome secretion. It further confirms that decreasing exosome secretion results in decreasing neurite outgrowth.

beta-III tubulin GFAP DAPI Merge

l in in

Con

+GW4869

+GW4869 + Hist

+GW4869 + H1

Neurite outgrowth

Mean ± SEM

500

400 300 *** 200

100 (um) length Neurite 0

con

+GW4869 Groups

Figure 3-11. Inhibiting exosome secretion decreases neurite outgrowth. A) Representative images of embryonic retinal explants cultured on non-astrocytes untreated (con) or treated with GW4869, or with GW4869 along with Histamine or H1 agonist. Scale bars, 100 µm. B) Quantification of the neurite outgrowth for the groups- con and +GW4869 plotted as mean lengths ± SEM (error bars) for n=3 independent experiments

AIM IV. Astrocyte-derived exosomes promote neurite outgrowth both on Laminin and CSPG

3.11. Astrocyte-derived exosomes treatment increases neurite outgrowth on Laminin

We decided to test whether these astrocyte-derived exosomes were promoting neurite outgrowth compared to untreated control retinal explants when plated on a known neurite-growth inhibitory substrate CSPG. First the experiment was conducted on laminin- coated coverslips to see the comparative effect of non-activated versus activated astrocyte- derived exosomes. This experiment showed that while both non-activated and activated astrocyte-derived exosomes were promoting neurite outgrowth compared to the control, this effect was specifically significant for the non-activated astrocytes (* p<0.05).

Neurite outgrowth

Mean ± SEM

Figure 3-12. Astrocyte-derived exosomes promote neurite outgrowth. Quantification of the neurite outgrowth for the untreated retinal explants (con), explants treated with non- activated-derived exosomes (non-act) and explants treated with activated astrocyte-derived exosomes (act) plotted as mean lengths ± SEM (error bars) for n=3 independent experiments

3.12. Astrocyte-derived exosomes treatment increases neurite outgrowth even on CSPG

This experiment was repeated on coverslips that were coated with laminin and CSPG.

It confirmed that CSPG has growth inhibitory effects, since the explants plated on CSPG showed much smaller neurites compared to the control group without CSPG. More importantly, when astrocyte-derived exosomes were added on to these explants, they were

89 able to overcome the inhibition by CSPG which was indicated by their relatively longer neurites. This effect was statistically significant (* p<0.05)for the non-activated group when compared with CSPG group.

Neurite outgrowth Mean ± SEM

Figure 3-13. Astrocyte-derived exosomes promote neurite outgrowth despite the presence of CSPG. Quantification of the neurite outgrowth for the untreated retinal explants plated on laminin in the absence of (con) or presence of CSPG (CSPG) with or without the treatments of non-activated-derived exosomes (non-act) and activated astrocyte-derived exosomes (act) plotted as mean lengths ± SEM (error bars) for n=3 independent experiments

AIM V. Astrocyte-derived exosomes promote neurite outgrowth via Wnt signaling pathway

3.13 Blocking Wnt Secretion decreases the exosome-induced neurite growth promoting effects on Laminin

Previously our lab has shown that FD exosomes promoted neurite outgrowth of the retinal explants and this effect was abolished with the treatment of IWP2, a known Wnt secretion inhibitor. Considering this, we decided to test whether the observed astrocyte- derived exosomes growth promoting effects were also influenced by the Wnt signaling. In order to test this, IWP2 was added alongside the retinal explants and the astrocyte-retinal explant assay was performed in the similar manner as before. This experiment was first conducted on laminin-coated coverslips. The results showed that explants that received non- activated and activated astrocyte-derived exosomes along with IWP2 resulted in shorter neurites. This indicates that Wnt signaling may be involved in mediating the growth promoting effects (* p<0.05).

Neurite outgrowth

Mean ± SEM

500

400

300

200

100 neurite length (µm) length neurite

con act

non-act act IWP2

non-act IWP2

Figure 3-14. IWP2 treatment decreases the exosome-induced neurite growth. Quantification of the neurite outgrowth for the untreated retinal explants (con), explants treated with non-activated-derived exosomes with or without IWP2 and explants treated with activated astrocyte-derived exosomes with or without IWP2 plotted as mean lengths ± SEM (error bars) for n=3 independent experiments

3.14 Blocking Wnt Secretion decreases the exosome-induced neurite growth promoting effects even on CSPG

The previous experiment was repeated on coverslips that were coated with laminin and CSPG. We wanted to test whether the results observed on Laminin were seen in the presence of CSPG. We found that when these explants were treated with non-activated or

92 activated astrocyte-derived exosomes in the presence of IWP2, they consistently showed shorter neurites (* p<0.05). This confirmed the involvement of the Wnt pathway.

Neurite outgrowth

Mean ± SEM

* *

400

300

200

100 neurite length (µm) length neurite

con CSPG act CSPG

non-act CSPG act IWP2 CSPG

non-act IWP2 CSPG

Figure 3-15. IWP2 treatment decreases the exosome-induced neurite growth even on CSPG. Quantification of the neurite outgrowth for the untreated retinal explants in the absence of(con) or presence of CSPG; explants treated with non-activated-derived exosomes with or without IWP2 and explants treated with activated astrocyte-derived exosomes with or without IWP2 while plated on coverslips coated with CSPG plotted as mean lengths ± SEM (error bars) for n=3 independent experiments

Chapter 4: Discussion (& Future Directions)

Since an injury to the CNS would usually be followed by a series of inflammation reactions including, recruitment of macrophages and their activation along with the activation of the surrounding astrocytes (46, 48). It is important to address whether these infiltrating macrophages are contributing to the recovery of the severed axons. Furthermore, because the spontaneous axon regeneration is difficult to achieve after an injury to the CNS, it will be further useful to explore this issue from different avenues. Our results indicate that peritoneal macrophages’ conditioned media has neurite growth promoting effects. More specifically, our results report that of these macrophages, the ones that are activated using

LPS and IFNγ seem to show a rather amplified effect on the neurite outgrowth. Since studies have reported that activating macrophages at the injury site usually take on a more inflammatory phenotype, we decided to induce this phenotype in these macrophages using

LPS and IFNγ (48, 49). This inflammatory or M1-macrophage phenotype is usually regarded as the neurotoxic one. However, with evidence from the Benowitz group studies and few others, it seems that these inflammatory macrophages may be mediating the pro-regenerative effects that have been observed in several lens injury and ONC models.

While these results may be consistent with theirs’, our study found that histamine is the specific macrophage-derived factor that is responsible for mediating these growth promoting effects. This, however, is different from what Benowitz group had reported. They reported that Oncomodulin is the axon growth promoting factor (67). Moreover, to further confirm whether this is in-fact the M1-phenotype polarization in these macrophages that is specifically eliciting the observed effects, it will be important to use specific M1-

94 macrophage, M2-macrophage and pan macrophage markers. This will allow us to further extrapolate the extent of the macrophage activation from the ratio of the macrophages stained using each of these markers. Perhaps, it might be moderate levels of activation that is able to produce these effects. Also, since most lens injury and ONC models have used zymosan as their potent macrophage activator, it will be useful to further conduct a similar experiment using astrocyte-retinal explant cultures, except the MCM is obtained from zymosan-activated macrophages. Moreover, our near future animal studies will also involve the use of zymosan injections into the vitreous humor of the animals to test its regenerative effects on the crushed Optic Nerve.

While our findings address the role of macrophages in axon regeneration, we did not examine whether the already proposed factors such as Oncomodulin by the Benowitz group or the astrocyte-derived CNTF by the Fischer group are contributing to increase in the neurite outgrowth (67, 72). Moreover, we decided to proceed with our experiments using just the astrocyte enriched cultures as opposed to the mixed glial layers because we wanted to address some of the recent controversial data on the role of reactive or activated astrocytes.

Nearly almost all studies since the mid 20th century have been viewing both activated astrocytes and the glial scar they form after the injury, as the biggest culprits responsible for the regeneration failure. However, the recent studies by the Sofroniew group uncovered that astrocyte scar is in fact needed for the spontaneous axon regrowth (9). This also highlighted the fact that the CSPGs which are produced by the reactive astrocytes are not all growth inhibitory by nature. There are many CSPG molecules that can exert growth promoting effects (9). Other studies also looked at the differential role of reactive astrocytes in this

95 context and found that despite their activation status, moderately activated astrocytes are also capable of producing several neurotrophic factors which facilitates the axonal recovery.

Therefore, with the use of the activated primary astrocyte culture, we wanted to somewhat mimic the injury-like setting in our in-vitro culture model. However, it would still be important to replicate these results using OPCs and microglia. Especially microglia- involving studies may be beneficial too, since there has been recent evidence on the crosstalk between the microglia and macrophages in the CNS injury models. Also, few studies reported that microglia can also activate astrocytes via the release of factors like IL-1α, TNF-

α and complement protein C1q (41). These activated astrocytes then take on an A1 phenotype which is shown to be neurotoxic.

Moreover, although the use of the Pan Histamine inhibitor in our study showed significant decrease in the neurite outgrowth indicating the involvement of histamine. It will also be crucial to perform an ultrafiltration experiment like the one conducted by the

Benowitz group with the molecular weight cut-off of 10KDa and 3KDa (60, 62). This will further confirm that histamine is the factor extracted from the MCM that is demonstrating these regenerative effects. Also, a control experiment with just the Pan inhibitor will be needed which can show that the inhibitor does not result in the same effects without the presence of astroglial cells. This will further imply that histamine must be interacting with its receptors on the astroglial cells to mediate its effects. Further we reported that H1 agonist treated cells resulted in the similar augmented neurite outgrowth like seen with the histamine treatment, while the H2- and H3- agonists did not seem to have that effect. These results are certainly consistent with lot of the studies that have reported that histamine’s suppressive

96 effects on the glial scar formation were reversed with the use of H1-receptor antagonist, but not as affected with the H2- or H3- antagonist. Also, histamine promotes the release of

GDNF from the astrocytes which was also seen to be mediated by the H1 receptor specifically in these studies (59, 60). Furthermore, the inhibitory effect of histamine on the release of some of the toxic pro-inflammatory cytokines was also reported to have reversed with the use of H1 receptor antagonist (59). However, it would also be important to look at the expression of these individual receptors on both untreated astrocytes and the ones that are treated with histamine. Since studies report that histamine’s treatment on astrocytes seems to upregulate these receptors expression, however they are increased to a varied degree (59, 60).

In addition, with regards to replicating these results using in vivo models, it may be difficult to inject histamine into the eyes of mice. Considering this has not yet done in the past studies, and the fact that it may stimulate an instant inflammatory reaction it may be a better option to use the H1-antagonist instead. Also, its effects can also be deduced if the HDC enzyme can be blocked. While to examine the role of macrophages, the macrophages can be depleted using the clodronate liposomes method that has been previously used.

Further our results also demonstrated that the H1-KO animal-derived astrocytes resulted in decreased neurite outgrowth compared to their wild type counterparts. This certainly confirmed the role of H1 receptor in mediating the regenerative effects. This was further supported because the control experiment where the retinal explants were treated with just H1 agonist did not show the similar effects which further indicate that these effects are mediated in the presence of astrocytes. Also, the neurite growth promoting effects were found to be quite pronounced for the activated astrocytes in relative to the non-activated

97 group. This indicates that perhaps the influence of histamine on the already activated astrocytes may be responsible for their rather more regenerative state. These results were consistent with their respective exosome levels. H1-agonist treated astrocytes reported to have the largest exosome concentration amongst all the three histamine agonist groups. In fact, they were found to be largely increased from the untreated cells. This difference from the control exosome levels was also specifically significant for the activated astrocytes group, also consistent with the neurite outgrowth results. The mRNA sequencing further reported the downregulation of the gene coding for dynein proteins, on treatment with the

H1-antagonist. This did in fact prove the involvement of exosomes release, however it will be important to also examine the samples tested with histamine. Furthermore, a confirmation study is needed to be performed using the H1-antagonist to test its effects on the retinal explant cultures.

In addition, our study only explored the aspect of exosome secretion and its influence on the axon regeneration. Perhaps there can be further studies on the composition of these exosomes. According to the literature, the content of these exosomes can vary depending on their source or the stimulus that the cells are exposed to. So, it will not be surprising to see that their composition varies between the non-activated astrocytes and activated astrocytes.

These can be conducted via mass spec of the isolated exosome samples, considering there are not many quantification tools available for vesicles as small as exosomes. Moreover, our results indicated that astrocyte-derived exosomes treated explants showed improved neurite growth compared to the untreated explants. However, the results were found to be significant for only the non-activated group. Similarly, these exosomes seemed to overcome the

98 inhibition by the CSPG substrate. However, when the Wnt was blocked using the IWP2 treatment, the neurite outgrowth for both non-activated and activated astrocytes had shown to decrease. This clearly indicated the involvement of the Wnt pathway which has already been shown in our 2017 study using the FD exosomes (29). Based on our findings we propose that the macrophages that infiltrate the injury site as part of the inflammatory response to a CNS insult, undergo activation, along with the astrocytes nearby. These activated macrophages take on a M1-phenotype and release histamine which then binds to the H1 receptors on the astrocytes. On interacting with the H1 receptor, it stimulates exosome secretion. Promoting exosome secretion then may lead to the axon regrowth past the lesion to the point that the neuronal circuit and its functions are re-established. Overall, these results certainly open several new possibilities in the context of improving the functional recovery of the severed axons. However further exploration of the histaminergic system and its role in the injured

CNS will have to be conducted and perhaps this system may open the path to more improved regenerative therapies for all kinds of CNS injuries and neurodegenerative pathologies.

Schematic Representation of the Proposed Model

Growth Cone

Injury

Activated Macrophages

Histamine Histamine binding to H1 Receptor

Astrocytes

Astrocytes-derived exosomes

CNS INJURY Recruitment & Release of Promotion of Axonal Activation of Histamine that Exosome secretion Regeneration Macrophages binds to H1 by nearby receptor astrocytes

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Chapter 5: Conclusion

Our work not only shows the involvement of macrophages in mediating the neurite growth promoting effects, but it also shows that histamine may be the active macrophage- derived factor responsible for this. In fact, our findings help propose a mechanism underlying these regenerative outcomes. Based on our findings, we propose that macrophages that recruit following the injury in the CNS, are undergoing activation which results in their M1- phenotype. On induction of this phenotype, these macrophages secrete histamine which binds to the H1 receptors on the surface of nearby astrocytes and promotes their exosome secretion. This promotion of exosome secretion can then have an effect in promoting axon regrowth at these injury sites. These findings help address the contradicting data on the involvement of macrophages in axon regeneration as well as the role of reactive (or activated) astrocytes in the injured CNS. They can even be extended and observed using different CNS injury models. Overall, it certainly calls for further research especially on the role of histaminergic system and its influence on the axon regeneration outcomes. This can help explore different therapeutic options and strategies to better repair the CNS and achieve a more efficient functional recovery following different CNS insults.

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Chapter 6. Appendix

To confirm that the effects of GW4869, Histamine, H1-agonist, H2-agonist and H3-

agonist are only mediated in the presence of astrocytes, a control experiment was conducted.

These reagents were added on the retinal explants which were cultured directly on the

laminin-coated coverslips. The quantification of the neurite outgrowth was conducted in the

similar manner. The results indicated that the mean neurite lengths for all these treatments

were insignificant compared to the control untreated retinal explants.

A Con

Con + GW4869 +Hist +H1

+H2 +H3

102

B Con

300

200

100 Neurite length (um) length Neurite

con +H1 +H2 +H3

+GW4869 +Histamine Groups

Figure 6-1. The effects induced by GW4869, histamine, H1 agonist, H2 agonist and H3 agonist are dependent on the astrocytes. A) Representative images of embryonic retinal explants cultured in the absence of astrocytes, treated with media (Con), or GW4869, histamine or one of the three histamine receptor agonists, Scale bars, 100 µm. B) Quantification of the neurite outgrowth for the groups plotted as mean lengths ± SEM (error bars) for n=3 independent experiments

103

To confirm that IWP2 alone on the retinal explants does not have the same growth inhibitory effect, we ran a control experiment. This time the retinal explants were plated on laminin and were treated with IWP2, however no other exosome treatments were added. The mean neurite length with the treatment with IWP2 was not so different from the control untreated retinal explants.

Neurite outgrowth

Mean ± SEM

Figure 6-2. The effects of IWP2 are dependent on the presence of astrocytes. Quantification of the neurite outgrowth for retinal explants left untreated or treated with IWP2 plotted as mean lengths ± SEM (error bars) for n=3 independent experiments

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