Effect of Propionic Acid-Derivative Ibuprofen on Neural Stem Call Differentiation; a Potential Link to Autism Spectrum Disorder

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Honors Undergraduate Theses UCF Theses and Dissertations

2019

Effect of Propionic Acid-derivative Ibuprofen on Neural Stem Call Differentiation; A Potential Link to Autism Spectrum Disorder

Aseelia Samsam University of Central Florida

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Recommended Citation Samsam, Aseelia, "Effect of Propionic Acid-derivative Ibuprofen on Neural Stem Call Differentiation; A Potential Link to Autism Spectrum Disorder" (2019). Honors Undergraduate Theses. 579. https://stars.library.ucf.edu/honorstheses/579

EFFECT OF PROPIONIC ACID-DERIVATIVE IBUPROFEN ON NEURAL STEM CELL DIFFERENTIATION; A POTENTIAL LINK TO AUTISM SPECTRUM DISORDER

ASEELA (Aseelia) SAMSAM

A thesis submitted in partial fulfillment of the requirements for the Honors in the Major Program in Biomedical Sciences in the College of Medicine at the University of Central Florida Orlando, Florida

Summer Term 2019

Thesis Chair: Saleh A. Naser, PhD

ABSTRACT

Propionic acid (PPA) is a short chain fatty acid that is produced by the human gut microbiome.

Propionate, butyrate and acetates are the end products of the fermentation of the complex carbohydrates by human gut friendly microbiome and are being used as sources of energy in our body. PPA is used as a food preservative against molds in various daily products and has been implicated in the pathogenesis of autism. In a recent study we showed that PPA in human neuronal stem cell (NSC) culture increases the astrocyte population and decreases the neuronal number and increases the inflammatory cytokines. In this study, we investigated the potential effects of a propionic acid-derivative, Ibuprofen, a member of the non-steroidal anti-inflammatory drugs

(NSAIDs) on neural stem cells proliferation and differentiation in vitro. Ibuprofen is an over counter drug that is used for alleviating pain, headache, and fever. To examine the effect of ibuprofen on developing brain we used human NSC in vitro, exposed them to increasing concentrations of ibuprofen, and investigated neural proliferation and differentiation. Here we show that NSAIDs, not at therapeutic, but very high concentrations cause an imbalance in NSC differentiation towards glial cells, therefore causing astrogliosis seen in some cases of autism spectrum disorder (ASD). Furthermore, upon removal of Ibuprofen, inflammatory cytokines;

TNF-alpha, IL-6 and IL-10, significantly increase (p<0.05) in cells previously exposed to NSAIDs compared to control. Therefore, we are speculating that if such drugs were to be taken in the circumstances of a developing child during the early trimesters of pregnancy, this could result in increased glial:neuron ratio leading to lifelong impediments. Based on the current study our

iii recommendation is to avoid high doses of propionic acid derivatives such as ibuprofen during pregnancy.

ACKNOWLEDGEMENTS

I would like to express my deepest gratitude to my committee chair, Dr. Saleh A. Naser, and my lab supervisor, Dr. Latifa S. Abdelli for their guidance, supervision and continuous support throughout my academic career as an undergraduate student and assistant researcher in Dr. Naser’s lab. I also would like to thank my thesis committee members, Dr. Dinender K. Singla and Dr. Shibu Yooseph, for providing additional advice to further my success in my endeavors. Many thanks to my family and friends who have continuously motivated me in my endeavors. Finally, I would like to thank and appreciate all my laboratory members for their helpful support.

TABLE OF CONTENTS

CHAPTER 1. INTRODUCTION ...... 1 CHAPTER 2. BACKGROUND ...... 4 2. 1 Autism Spectrum Disorder Prevalence and Causes ...... 4 2. 2 Glial versus Neurons Imbalance in Autism ...... 5 2. 3 Propionic Acid (PPA) ...... 6 2. 4 Non-Steroid Anti-Inflammatory Drugs (NSAIDs) ...... 8 2. 5 Structure of Ibuprofen ...... 8 2. 6 Mechanism of Action of NSAIDs...... 8 2. 7 Absorption of Ibuprofen ...... 10 CHAPTER 3. RESEARCH METHODOLOGY ...... 13 3. 1 Proliferation and Differentiation of Human Neural Stem Cells (hNSCs) ...... 13 3. 2 Cells Plating and Treatment with NSAIDS ...... 13 3. 3 Neurosphere Measurements ...... 14 3. 4 Immunostaining Detection of Glia versus Neuronal Cells ...... 14 CHAPTER 4. RESULTS ...... 16 4. 1 Ibuprofen Enhances Neurosphere Proliferation in vitro ...... 16 4. 2 Ibuprofen Enhances Glial Cell Differentiation versus Neurons in vitro: ...... 17 4. 3 Ibuprofen Modulates Inflammatory Response in hNSC...... 18 CHAPTER 5. DISCUSSION ...... 19 CHAPTER 6. CONCLUSIONS AND FUTURE DIRECTIONS ...... 21 REFERENCES ...... 22

LIST OF FIGURES Figure 1: The 2D structure of Ibuprofen ...... 8 Figure 2: Neurospheres in the human neural stem cell cultures ...... 16 Figure 3: GFAP versus Tubulin-IIIB concentrations in ascending concentrations of ibuprofen . 17 Figure 4: Ibuprofen Modulation of Inflammatory Response in hNSC ...... 18

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LIST OF ACRONYMS/ ABBREVIATIONS ADHD: Attention deficit hyperactivity disorder ASD: Autism spectrum disorder AUC: Area under the curve BBB: blood brain barrier bFGF: basic Fibroblast growth factor CDC: Center for Disease Control CGG: Cytosine Guanidine Guanidine Co-A: Coenzyme A EGF: Epithelial-derived growth factor ELISA: Enzyme-Linked Immunosorbent Assay FMR1: Fragile X mental retardation Ib: Ibuprofen FDA: Food and drug administration GI: Gastrointestinal GPR41 (43): G-protein-coupled receptor IL: interleukin- IUPAC: International Union of Pure and Applied Chemistry NSAIDs: Non-steroid anti-inflammatory drugs NSC: Neuronal stem cell PA: Propionic acidemia PG: Prostaglandins PPA: Propionic acid PTEN/AKT: Phosphate and tensin homologue detected on chromosome 10 SCFA: Short chain fatty acid TNF-α: Tumor necrosis factor-α 2D: Two dimension

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CHAPTER 1. INTRODUCTION

1. 1 Introduction:

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder leading to lifelong psychological health problems1. ASD is characterized by repetitive behaviors and abnormal communication1. Recent reports indicate that 1 in 59 children across the United States have been diagnosed with autism2. There are known genetic causes for autism that are mostly affecting the synaptogenesis3. However, other causes such as environmental factors, gastrointestinal infections and inflammation have been implicated in the pathogenesis of ASD4. For instance, propionic acidemia (PA), a genetic disorder where the concentration of propionic acid is significantly increased in the blood, has been shown to cause autistic symptoms5,6,7. Propionic acid (PPA) is a short chain fatty acid (SCFA) that is a byproduct of microbiome fermentation of dietary fibers.

Many foods that contain fibers are rich in complex carbohydrates that are the source of energy are being fermented and digested by human gut microbiome. This process is necessary for the production of SCFAs such as acetate, propionate and butyrate that are being used as sources of energy for the intestinal cells and gluconeogenesis and release of glucagon-like peptide-1 hormone by acting through G-protein-coupled receptors such as GPR41 and GPR438. PPA is also a well- known food preservative protecting against molds. PPA was also shown to be upregulated in autistic stools compared to healthy counterparts; this is of particular interest considering that patients with autism have characteristic GI issues such as irritable or inflammatory bowel syndrome9. Additionally, autism is widely regarded as a disorder of glial cells as multiple reports

1 indicated an imbalance in neural versus glial cell populations in the autistic human brain with significantly elevated numbers of glia cells10. In a recent study, we discovered the molecular mechanism of propionic acid activity on GPR41 receptor in human neural stem cell (hNSCs) culture. Our results show PPA induces gliosis and neuro-inflammation through modulation of

PTEN/AKT signaling pathway11. Also, a single intracerebroventricular infusion of PPA (4μl of

0.26 molar solution) in rats caused autistic-like behaviors and seizure, and increased inflammatory and oxidative stress biomarkers in the brain homogenates12. A recent study also shows subcutaneous injection of 500mg/kg PPA (sodium propionate) in rats once a day for 5 days caused significant reduction in neuronal diameter and thickness of the brain granular cell layer13. These compelling reports stipulate a very plausible link between PPA, glial cell over-proliferation and the development of ASD

Besides its use in food industry, PPA is also heavily present in over the counter pharmaceutical drugs, such as Ibuprofen. This non-steroid anti-inflammatory drugs (NSAIDs) is known as a PPA derivative since it contains PPA in its molecular structure. Of interest, NSAIDs are currently considered safe to use during the 1st and 2nd trimester of pregnancy according to the food and drug administration (FDA)14. Therefore, and in light of our previously established PPA role in NSC plasticity, it appears of great importance that the role of NSAIDs on NSCs proliferation and differentiation be tested, since most of embryonic neural development occurs during the 1st trimester of pregnancy.

1. 2 Hypothesis:

The prediction in our study is that Ibuprofen, a propionic acid derivative, will induce over- proliferation and differentiation of glial cells over neurons leading to gliosis and inflammation, as commonly described in Autism.

1. 3 Specific Aims:

To address our hypothesis, we designed two specific aims;

Aim 1: Demonstrate that Ibuprofen will induce glia versus neuronal proliferation and differentiation in vitro:

1. hNSCs will be exposed to either PPA or ascending concentrations of Ibuprofen and neurosphere expansion and proliferation will be evaluated.

2. Previously obtained neurospheres will be plated for differentiation and ratio of neural versus glial cells will be quantified using glia and neuron specific cell surface markers.

Aim 2: Demonstrate the Effect of NSAIDs on NSC Inflammatory Response in vitro:

1. Cytokine release by fully differentiated glial cells will be measured on supernatants obtained from Ibuprofen differentiated hNSCs and cells differentiated in presence of Ibuprofen than allowed to further proliferate following differentiation without the drug.

CHAPTER 2. BACKGROUND

2. 1 Autism Spectrum Disorder Prevalence and Causes

ASD has become more prevalent by the years. According to a recent report by the Center for

Disease Control (CDC), 1 in 59 children in the United States have been diagnosed with ASD in

2014; demographic values show that ASD is more prevalent in boys than girls, by about 4 times15.

More striking is a 2012 CDC report shows the prevalence of ASD had increased by 78% in the US only during 2002-200816. It is also present across all socioeconomic and ethnical groups; however, a recent comprehensive study in seven states in the US shows community-level differences, diagnostic services and the accessibility of data resources may contribute to differences in the prevalence of ASD among different states and may affect the age of identification of ASD since cognitive problems was found to be higher among children aged 4 compared to 8 years old children17. ASD is believed to results from a combination of genetic, environmental, and gastrointestinal factors18. Some related genetic causes for autism include fragile X syndrome: individuals have multiple nucleotide repeats of CGG in the FMR1 (fragile X mental retardation) gene, and this mutation can hinder expression or even lead to the gene being lost; symptoms are prevalent in the behavioral side, such as ADHD or ASD19. Another possible genetic cause of ASD is propionic acidemia (PA), where an enzyme called propionyl Co-A is not functioning properly

(mutation on its respective gene) and there is efficient breakdown of macromolecules; due to the gene defect, there is a buildup of propionic acid (converted from propionyl Co-A) in the bloodstream and that can be detrimental to the patient; it has been reported that individuals with

PA show autistic symptoms5,6. Other factors can be stemmed from the environment. Various exposures to factors such as toxins, drugs, organisms causing infections and valproic acid (a

4 commonly used medication for seizures) are possible causes for ASD20,21. Signs and symptoms of

ASD include repetitive behavior, aloofness, self-injurious behavior, tics, and impaired social skills22. Because of the many studies and conditions that indicate a possible link between PPA and

ASD, we decided to investigate the effect of this on differentiation of neural cells, which are vital for the developing brain.

2. 2 Glial versus Neurons Imbalance in Autism

Through our hypothesis, we predicted that PPA could possibly induce more glial cell differentiation than neural. Although the ratio of glial to neural is normally 1:1 to 3:1, it potentially can shift more towards glial in certain neurological disorders including autism23. Stem cells in the nervous system begin to proliferate at 12 to 14 weeks of the fetus, followed by migration of these cells to different areas of the nervous system and eventual commitment to differentiate into one of two type of nerve cells: glial or neural cells24. Neural cells, or neurons, have an important role in signaling other cells and transmitting messages through chemicals called neurotransmitters; however, glia cells are more of the helper cells, assisting the neurons through maintaining a proper environment for them, whether it be monitoring the release of neurotransmitters or cleaning any excess electrolytes or waste around the cells. There are four types of glial cells in the central nervous system; the first is astrocytes. Astrocytes are the cells that help maintain the surroundings of the neurons; microglial cell, ependymal cells and oligodendrocytes that myelinate the axons of the central neurons (while Schwann cells myelinate the peripheral nervous system). Of note, myelin is crucial for the electrical signals, known as action potential. Axons with thicker myelin sheath have a higher conduction velocity and carry the nerve impulses much faster. The thickest fibers are the motor fibers that conduct the electrical impulses to the skeletal muscles for

5 contraction. Microglial cells are like the cleaners of the CNS, where they remove any particles and debris lurking around and are involved in the immune mechanisms in the CNS23. In the case of autism, there have been reports of increased levels of glial cells (specifically astrocytes and microglia cells), based on not only at the cellular level, but the analysis of ASD patients’ brain matter including functional studies; it was shown in post mortem samples of the brain a presence of gliosis and inflammation on the brain matter (due to accumulation of glial cells and release of proinflammatory cytokines such as interleukin 6 or TNF-α)25. The cause of such an imbalance in glial cells versus neurons is not clear yet, but this study will focus on finding a possible link to propionic acid.

2. 3 Propionic Acid (PPA)

Propionic acid (PPA) is a short chain fatty acid (SCFA) and a bacterial end-product of the GI tract.

It is also commonly used as an anti-fungal food preservative. In addition, it is known to be a

26,27 category derivative of NSAIDs such as ibuprofen or naproxen . Microbiota over-populating

ASD gut include clostridia and disulfovibrio among others28. Interestingly, all of these bacteria have PPA as their fermentation byproduct, which is a normal process by which our body can use the complex carbohydrates that otherwise, could not digest and use as a source of energy.

Additionally, elevated concentrations of PPA producing bacteria such as various clostridia taxa29, and a decrease in bifidobacteria30 has been reported in autistic stools compared to healthy siblings

(controls). Consistent with this, increased levels of propionate and butyrate (due to increased number of their corresponding microbiota) and significantly higher branched-chain fatty acids have been found in the feces of Rett syndrome patients, an often co-occurring condition with autism31 . Indeed, a recent study in Chinese autistic children found decreased butyrate producing

6 bacteria and increased valeric acid producing bacteria in their feces, indicating such imbalance as a cause of constipation in autistic children32. Furthermore, Propionic Acidemia (PA), a rare metabolic disease resulting from a dysfunction in propionyl-CoA carboxylase enzyme, induces accumulation of PPA in the bloodstream33. This enzyme is responsible for fatty and amino acids metabolism by converting propionyl-CoA to methylmalonyl-CoA; however, if mutated,

Propionyl-CoA is converted instead to PPA which builds up in the bloodstream leading to a range of life-threatening symptoms noticeable at birth, including poor feeding, vomiting, hypotonia, lethargy, and seizures. If untreated, PA may lead to coma and death33. Interestingly, lack of development, self-absorbed behavior, limited to absent speech development, and various autistic- like social impairments are also noticeable in PA, however, whether PPA is responsible for these symptoms has yet to be clarified. Other studies have since established a correlation between elevated PPA levels and autistic behavior. For instance, a recent study has showed that intra- cerebrovascular injection of PPA in rats (in vivo) caused autistic symptoms13,34. Furthermore, fecal microbiota transplant from healthy counterparts in children with autism reduced autistic behavior, suggesting that replacing misbalanced microbiota with healthier population may play a role in alleviating autistic symptoms due to increased number of various microbiota taxa including

Bifidobacterium, Provotella, and Desulfovibrio35. With all the aforementioned literature taken together, we concluded that elevated PPA levels may be responsible for some autistic cases. In a recent study, we investigated the direct effect of various concentrations of propionic acid in neural stem cells culture. Our observations strongly suggest that PPA favors glial cell differentiation over neurons. Therefore, it is worth asking whether PPA derivative drugs such as NSAIDs may show similar results.

2. 4 Non-Steroid Anti-Inflammatory Drugs (NSAIDs)

Non-steroid anti-inflammatory drugs are some of the commonly used medications in the treatment of pain and inflammation. These include a number of medications such as aspirin, acetaminophen, naproxen, ibuprofen and others.

2. 5 Structure of Ibuprofen

Ibuprofen (nomenclature name by the International Union of Pure and Applied Chemistry or

IUPAC): 2-[4-(2 methylpropyl)phenyl] propionic acid), with the molecular formula of C13H18O2, is a water soluble compound (21mg/L at 25oC) that has the following 2D structure36:

Figure 1: The 2D structure of Ibuprofen36

Ibuprofen structure has propionic acid in its chemical structure. Ibuprofen comes in two forms: R and S isomers; these are essentially mirrored molecules that are different in how they are oriented.

The pharmaceutically active form in the body is the S isomer, while the inactive is the R isomer37.

2. 6 Mechanism of Action of NSAIDs

NSAIDs are known for their ability to inhibit the cyclooxygenase (COX) enzymes that mediate the production of prostaglandins from the cell membrane arachidonic acid. Mechanism of action of NSAIDs such as ibuprofen is through the inhibition of COX-1 and COX-2 enzymes that leads to decreased production of prostaglandins and thromboxanes from arachidonic acid.

Prostaglandins are unsaturated fatty acid derivatives that possess a twenty-carbon structure that includes a cyclic ring that are often called the eicosanoids due to the twenty carbon atoms.

Arachidonic acid, the precursor of prostaglandins is also a twenty-carbon fatty acid that is a component of the phospholipid of the cell membrane38. Prostaglandins (PG) including the PGD2,

PGE2, PGF2, and PGI2 (prostacyclin) are synthesized from arachidonic acid by prostaglandin synthase. These prostaglandins are responsible for multiple symptoms such as pain, fever, and inflammation. In addition, Ibuprofen causes a decrease in the synthesis of thromboxane-A2 by thromboxane synthase, inhibiting platelet aggregation36. However, prostaglandins are involved in a number of activities in human body including the relaxation of smooth muscle, induction of labor, decreasing intraocular pressure, hormone regulation, calcium regulation, cell growth, filtration rate of kidney, and inhibiting the acid secretion of the stomach among others39.

COX inhibitors prevent the combination of arachidonic acid with its substrate-enzyme thus preventing the formation of prostaglandins, eicosanoids, and thromboxane36.

COX inhibitors are classified into four categories due to their mode of action: 1- time-independent competitive inhibition like that of ibuprofen, 2- time-dependent, tight binding inhibition such as that of the indomethacin, 3- time-dependent, weak binding inhibition such as that of the naproxen,

4- the covalent inhibition of COX such as that of the aspirin40,41. Time-independent inhibitors bind to COX and lead to competitive inhibition through a rapid reversible fashion40,41. However, the

9 time-dependent, tight binding inhibitors first bind to the active sites of the COX in a rapid and reversible fashion but then transit into a time-dependent complex where the inhibitor slowly dissociates from the COX40,41. Ibuprofen is a reversible non-selective inhibitor of both COX-1 and

COX-236. Ibuprofen is believed to be in the time-independent category, binding rapidly and competitively to COX-2 prevent oxygenation of arachidonic acid40,41,42. The COX-1 activity leads to preferential oxygenation of arachidonic acid while the COX-2 oxygenates several fatty acids including the arachidonic acid, linoleic acid and the eicosapentaenoic acid more efficiently41,43,44,45.

Ibuprofen is used for patent ductus arteriosus, cystic fibrosis, rheumatoid arteritis, osteoarthritis, various pains and headache, toothache, fever, and often in neurodegenerative diseases with known inflammatory pathophysiology among other conditions46,47.

2. 7 Absorption of Ibuprofen

Ibuprofen is available in 200-800mg tablets or other forms, is easily absorbed through oral administration, and reaches a peak concentration in the blood within 1-2 hours46,47.

Ibuprofen’s average concentration ©max, time (T)max and area under the curve (AUC, drug plasma concentration-time curve) includes 20 μg/ml, 2 h and 70 μg.h/ml, although they may change due to the rout of the delivery48. After entry into the blood, it is bound to plasma proteins such as albumin, reaching the liver and is being metabolized into hydroxylated and carboxylated components47. After absorption its R-enantiomer converts (up to 65%) to S-enantiomer to form an active compound46,49. The metabolism occurs in two phases: in phase I hydroxylation of isobutyl chains results in -hydroxy metabolites and continues to the phase II that is the oxidation into 2-

10 carboxy-ibuprofen and p-carboxy-2-propionate mediated by cytochrome P450 system46,48. The oxidative products will be conjugated to glucuronide that facilitates ibuprofen excretion through urine48.

A newer study looking into 17 trials shows that following a 400mg dose of ibuprofen its plasma concentration reaches 8.4μg/ml in 20 minutes50. An earlier study shows that following a 600mg dose the plasma concentration of ibuprofen was at 39.4-63.7μg/ml within one hour and remained at similar range following 2 or 3 doses daily51. The plasma concentration of ibuprofen at the therapeutic dose is at 3mg/dl52, which is approximately 30μg/ml.

The passage of the short chain fatty acids such as PPA through the blood brain barrier (BBB) into the central nervous system is mediated by monocarboxylate transporters53.

It is not known how the metabolites of the ibuprofen enter the cell from the blood stream; however, the same monocarboxylate transporters might mediate this process. Nevertheless, most of the

NSAIDs are weak organic acids and in the gastric environment they are non-ionized and can diffuse through the cell membrane of the gastric epithelial cells into the cytoplasm due to being lipid soluble54,55. Although with this process, the NSAIDs can hurt the gastric mucosa by inhibiting the protecting effect of prostaglandins there through mitochondrial damage54,55, this might be a possible way the ibuprofen enters the cells elsewhere to act on COX enzymes.

As ibuprofen is one of the most utilized NSAIDs, it is known to be used by even pregnant women, when they need coping with pain. The food and drug administration (FDA) classifies ibuprofen and naproxen as categories B during the first two trimesters of pregnancy, and D during the last trimester in their pregnancy risk classification by trimester table56. This table is used to classify

11 the safety use of drugs during the three trimesters of pregnancy (Class/Category A, B, C, D, X). It ranges from Class A, which is known as not detrimental to the health of the developing child, B for which the studies have shown no risk on the developing fetus, to class X which is known to lead to congenital abnormalities or worse for the child57. Based on the categories, ibuprofen and naproxen are considered safe during the first two trimesters, while unsafe during the third trimester of pregnancy, due to potential congenital malformation in the heart (pre-closure of the ductus arteriosus; which usually closes right after birth)58. Although the categories suggest that these drugs are deemed safe in early pregnancy, some latest studies challenge that thought. For instance, ex-vivo exposure of ovarian germ cells of the developing fetus (about 7 weeks) to ibuprofen resulted in cell death59; however, it remains to be clarified how ibuprofen may cause germ cell death or affect overall stem cell development.

Nevertheless, some general side effects of NSAIDs (chronic use) include peptic ulcer disease due to the damage to the GI mucosa60, abdominal pain, nausea, vomiting, and vertigo, drowsiness, loss of consciousness, kidney complications, and coma among others46. The toxic effects of ibuprofen is seen with consumption of 99 mg/kg and higher46.

CHAPTER 3. RESEARCH METHODOLOGY

3. 1 Proliferation and Differentiation of Human Neural Stem Cells (hNSCs)

StemPro Neural Stem Cells (Cryopreserved human fetal-derived neural stem cells (hNSCs);

Thermo Fisher Scientific, A15654), were grown on T-25 suspension flask. The proliferating media were prepared following the list provided by GIBCO; contents include Knockout D-MEM/F12 media base, growth factors EGF and bFGF, StemPro Neural Supplement and antibiotics based on the standards of the stock concentration. At this point, clusters of progenitor cells known as neurospheres appeared and were seeded for differentiation on Geltrex (1:100) pre-coated 24 well plates supplemented with differentiation media. Differentiation media consists of complete

StemPro media with exclusion of the growth factors.

3. 2 Cells Plating and Treatment with NSAIDS

To study the effect of NSAIDs of hNSCs proliferation, NSCs were equally distributed in 24 well plates and exposed to media containing ascending concentrations of Ibuprofen (0.1, 0.2, 2, 4, and

6mM) (Thermo-Fisher) diluted in methanol. Control cells were exposed to either methanol, pure sodium propionate (2mM; Sigma Aldrich), or cell culture media alone. Treatment went on for about 10 days. Meanwhile, neurosphere diameter was monitored and measured. For differentiation studies, previously treated neurospheres were plated on Geltrex pre-coated (1:100) 8 well chambers (for immunostaining analysis) or 24 well plates (for molecular analysis) and supplemented with differentiation media for an additional 7 days. Cells treated with Ibuprofen were allowed to differentiate in presence of the drug for 7 days followed by an additional 7 days without the drug.

3. 3 Neurosphere Measurements

Neurospheres were imaged using a digital camera mounted on an inverted tissue culture microscope (Amscope, Japan) at 10x magnification every other day for 3 time points post plating

(Day 4, Day 8, and Day 10). Neurospheres with a diameter superior to 25μm were measured using

Amscope software version x64.

3. 4 Immunostaining Detection of Glia versus Neuronal Cells

Cells were allowed to differentiate for 7 to 10 days and then washed and fixed with 10% formalin and 3% Triton-X for 10 min before incubation in 10% normal goat serum (NGS; Vector

Laboratories) for 1hr. Next, cells were double immune-stained for GFAP (Glial cell marker) and

Tubulin-IIIβ (Neural cell marker) markers. Briefly, slides were incubated at 4oC over night with mouse anti-human-GFAP antibody (1:20 in 10% NGS; Sigma; Cat# SAB4100002) or rabbit anti- human Tubulin-IIIβ antibody (1:50; in 10% NGS Sigma; Cat#T5076). Cells were next washed and incubated for 1hr with goat anti-rabbit FITC (Tubulin-IIIβ) or goat anti-mouse TRITC (GFAP);

Sigma] diluted in 1:50 in PBS. Antifade Vectashield medium containing 4’,6-diamino-2- phenylindole (DAPI; Vector Laboratories) was used to co-stain nuclei and slides were imaged using Amscope IN480TC-FL-MF603 Fluorescent Microscope.

3. 5 Evaluation of Protein and Cytokine Levels with Enzyme-Linked Immunosorbent Assay

(ELISA)

GFAP, Tubulin-IIIB proteins as well as TNF-a, IL-6, and IL-10 cytokine levels were determined using commercially available ELISA kits following the general protocol provided with each kit.

In general, we added about 50 µl of standard that was prepared and our sample of interest into the

14 well plate. After incubating at room temperature for 2 hours, we decanted the liquids and washed about 3 times. Adding the diluting detection antibody for one hour, followed by another decanting and washing 3 times. After, we added 100 µl of the substrate per well, we incubated the well plates in a dark room for half an hour at 37oC, followed by adding the same amount of the stop solution and read the results at the specified wavelength in nm (450 nm).

CHAPTER 4. RESULTS

4. 1 Ibuprofen Enhances Neurosphere Proliferation in vitro

Figure 2A, shows the neurospheres formation after 4 days of hNSCs plating as well as differentiation following adhesion to Gelatin coated plates starting at day 4 and reaching confluency at Day 7 to 10. Cultures were seen in phase contrast view using a light microscope

(25x magnification). The graph in 2B depicts neurosphere expansion (Day4, Day 8 and Day 10) under PPA (2mM) or Ibuprofen (2mM) treatments. *P<0.05. As the days progressed for proliferation, the neurospheres gradually expanded and became more distinguishable. As shown in the corresponding graph below, the progression of neurosphere diameter (in µm) for PPA and

Ibuprofen were quite similar in trend and they both increased neurosphere diameter significantly at day 10 when compared to untreated or methanol treated cells (PPA: 216.76um±44.97um),

(Ibuprofen: 170.11um±32.17um), against control (137.8um±53.48um) and Methanol

(127.74um±39.74um) (*p<0.05).

Figure 2: Ibuprofen Enhances Neurospheres Expansion in vitro

4. 2 Ibuprofen Enhances Glial Cell Differentiation versus Neurons in vitro

Figure 3A depicts positive immunostaining slide demonstrating hNSCs differentiate to equal ratio of glia versus neurons as evidenced by equivalent Tubulin-IIIβ and GFAP fluorescent staining

(Scale bar:25um). In Figure 3B, ELISA results indicate GFAP protein significantly increases in both PPA (2mM) and Ibuprofen (2mM) treated cells against controls. Of note the highest concentration for GFAP was achieved with PPA (2.66ng/ml±0.01ng/ml) followed by 2mM

Ibuprofen (2.54±0.02ng/ml) against controls. Of note, Ibuprofen concentrations beyond 2mM did not seem to correlate with higher GFAP concentration. As for the neuronal marker (Figure 3C),

Tubulin-IIIβ, both PPA and Ibuprofen treatments resulted in less concentration of the neuronal marker with the lowest concentrations achieved for PPA (3.94ng/ml±0.01ng/ml) and Ibuprofen

0.2mM (5.31ng/ml±0.01ng/ml).

Figure 3: GFAP versus Tubulin-IIIβ Concentrations in Ascending Concentrations of Ibuprofen

4. 3 Ibuprofen Modulates Inflammatory Response in hNSC

Figure 4 shows concentration of inflammatory cytokines TNF-α (A), interleukin (IL)-10 (B), and

IL-6 (C) in hNSC cultures treated with ibuprofen. Black bars (Phase 1) represent cells after 7 days of treatment with either methanol, PPA (2mM) or ascending concentrations of Ibuprofen (0.1mM to 6mM). After 7 days, the medium from Ibuprofen or other compound treated cells were removed and fresh medium without any compounds was added to the cells for an additional 7 days (Blue bar/Phase 2). Over all, pre-treating cells with Ibuprofen allowed for some increase in proinflammatory TNF-α (210.25pg/ml±11.81pg/ml) vs control (44,13pg/ml±1.12pg/ml) and anti- inflammatory IL-10 (43.51pg/ml±1.93pg/ml) vs control (21.44pg/ml±1.94pg/ml), however IL-6 levels (I-2mM: 1.15pg/ml±0.01pg/ml) were equivalent to control (1.21pg/ml±0.01pg/ml).

Interestingly, upon removal of Ibuprofen (Phase 1), previously treated cells started expressing significantly higher amounts of inflammatory cytokines (*p<0.05) regardless to the concentration of Ibuprofen (Fig. 4). Ibuprofen at therapeutic concentrations didn’t adversely affect neurogenesis.

Phase 1 Phase 1 Phase 1 Phase 2 Phase 2 Phase 2

Figure 4: Ibuprofen Modulates Inflammatory Response in hNSC

CHAPTER 5. DISCUSSION

In this study, we examined the effect of Ibuprofen, a PPA derivative, on neuronal stem cell differentiation to mimic our previous study suggesting PPA would shift NSC differentiation towards glial phenotype over neurons. Ibuprofen is one of the most commonly used NSAIDs for the treatment of pain, headache, fever and other conditions36.

It is a non-selective inhibitor of COX-1 and COX-2 enzymes in many human cells preventing the formation of prostaglandins, thereby alleviating the pain and inflammation36.

Being a PPA derivative, we postulated that Ibuprofen may mimic some of the biological side effects of PPA. For instance, PPA has been implicated in the pathogenesis of autism and propionic academia5,6, A genetic disease with autistic symptoms. Also, animal studies show that injection of PPA in rats can lead to signs and symptoms of Autism12,13.

Furthermore, our recent study shows that PPA increases astrocytic differentiation and proliferation while decreases the neuronal population in vitro. This was accompanied with increased inflammatory cytokine release, similar to what has been observed in the autistic brain11. Given the structural similarity between PPA and Ibuprofen, it appeared imperative that we figure out if Ibuprofen will affect NSC in a similar way, especially since its uptake is currently allowed during the first two semesters of pregnancy.

In the current study, we treated hNSCs with various concentrations of Ibuprofen. The neurosphere diameter in Ibuprofen treated cultures increased to approximately170μm

(about 25% increase in size) in 10 days compared to the control wells: 135μm (figure. 2).

PPA is a short chain fatty acid used as source of energy by cell8. This might be the reason

19 for the increase in the size of the neurospheres in PPA and Ibuprofen treated hNSC cultures in our study. However, excess or long time exposure might be detrimental as is the case of brain damage caused by acetate due to high alcohol consumption61.

Our result shows that at therapeutic concentrations (20, 50, and 100µg/ml) did not have any adverse effect on the neuronal and glial markers. However, at high concentrations of

Ibuprofen may interfere with the neuronal/glial differentiation and mimic the effect of PPA in vitro. Figure 3, shows that the astrocytic marker, GFAP, level increased significantly while neuronal cell marker Tubulin-IIIB significantly decreased in PPA and Ibuprofen treated hNSC. Higher Ibuprofen concentrations did not correlate with higher or lower

GFAP and Tubulin-IIIβ levels though. We believe that 2mM Ibuprofen saturates hNSCs.

Of note, PPA induces its effect via G-protein coupled receptor, GPR41. We therefore stipulate that Ibuprofen might be using the same route to influence hNSC differentiation fate; however, further studies are required in this area.

Our results further show that Ibuprofen increases inflammatory response regardless to the fact that it is an anti-inflammatory drug in nature. This is in accordance with some studies showing Ibuprofen to stimulate IL-6 production by human polymorphonuclear cells62, 63.

However, while Ibuprofen is present, the extent of inflammation is significantly lower to that registered upon removal of the drug. As far as IL-10 goes, Ibuprofen appears to increase its expression across the board. This data contradicts studies that show Ibuprofen decreases the IL-10 expression in both animal64 and humans62,65 .

CHAPTER 6. CONCLUSIONS AND FUTURE DIRECTIONS

Based on these preliminary data, it appears that similar to PPA, at very high concentrations,

Ibuprofen is capable of tampering with neural patterning and differentiation. Our result shows that at therapeutic concentrations, Ibuprofen did not have any adverse effect on the neuronal and glial markers. Since elevated concentrations of PPA was correlated with

ASD, it is safe assuming that high NSAIDs concentrations may resume the same condition.

It is therefore our recommendation to mothers in general or those with predisposing conditions that they avoid exposure to high concentration of PPA or PPA derivative drugs such as Ibuprofen during the earliest stages of their pregnancy (first trimester); which is most critical for brain development.

To further elucidate NSAIDs mediated glial over-proliferation, it is imperative that we figure out the mechanism by which the drug may influence the cell fate. Our guess is that

Ibuprofen may use GPR41 receptor, like PPA, to influence glial cell survival over neurons.

Another major point is to validate whether this effect is specific to Ibuprofen or if it encompasses all PPA derived NSAIDs.

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