The Effects of Growth Hormone on Opioid-Induced Toxicity in Vitro

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 279

The effects of growth hormone on opioid-induced toxicity in vitro

ERIK NYLANDER

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6192 ISBN 978-91-513-0765-7 UPPSALA urn:nbn:se:uu:diva-393940 2019 Dissertation presented at Uppsala University to be publicly examined in B21, Biomedicinskt centrum (BMC), Husargatan 3, Uppsala, Friday, 22 November 2019 at 09:15 for the degree of Doctor of Philosophy (Faculty of Pharmacy). The examination will be conducted in English. Faculty examiner: Associate Professor Alexis Bailey (St. George’s University of London).

Abstract Nylander, E. 2019. The effects of growth hormone on opioid-induced toxicity in vitro. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 279. 60 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0765-7.

There is an ongoing opioid crisis in the United States that is portrayed by a large number of opioid-related deaths. Many of these cases involve commonly used prescription opioids, such as morphine, oxycodone, fentanyl, and methadone. This is concerning and highlights the problems associated with long-term opioid treatment. In addition to opioid-related deaths, long-term opioid use may impact higher brain functions, such as cognitive function. The cause of cognitive decline following opioid treatment may be associated with increased neuronal cell death, inhibited neurogenesis, and altered volumes of specific brain regions important for cognition. Growth hormone (GH), a pituitary hormone regulated by the hypothalamic somatotropic axis, may counteract several of these effects. The hormone, alongside with its mediator insulin- like growth factor-1 (IGF-1), is associated with pro-cognitive effects and display promising neuroprotective actions in the CNS. The main aim for this thesis was to examine the impact of opioids on cell viability and the potentially protective, restorative, and effects linked to pro-cognitive properties of GH in mixed neuronal cell cultures and cell lines. The results clearly display that specific opioids, such as methadone, decrease cell viability, possibly via negative effects on mitochondrial morphology. GH treatment alleviated the negative effects of methadone in cortical cell cultures as well as successfully restored mitochondrial and membrane integrity past injury. Moreover, GH treatment to primary hippocampal cell cultures increased the number of dendritic spines, which are linked to higher cognitive functions, indicating that the hormone act as a cognitive enhancer in the CNS. In conclusion, this thesis provides further evidence that opioids negatively impact cell viability, an effect that may underlie reduced cognitive function as seen in several patients consuming opioids-long term. GH was able to counteract these effects and also able to restore damaged cellular functions. This thesis further confirms the essential role of GH in acting as a cognitive enhancer in the CNS, highlighting the potential role of GH as a treatment for cognitive dysfunctions.

Keywords: Growth hormone, opioids, methadone, morphine, ketobemidone, fentanyl, oxycodone, hydromorphone, insulin-like growth factor, cell viability, NG108-15, SH-SY5Y, hippocampus, cortex

Erik Nylander, Department of Pharmaceutical Biosciences, Box 591, Uppsala University, SE-75124 Uppsala, Sweden.

ISSN 1651-6192 ISBN 978-91-513-0765-7 urn:nbn:se:uu:diva-393940 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-393940)

In loving memory of my grandfather

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Nylander, E., Katila, L., Zelleroth, S., Birgersson, J., Nyberg, F., Grönbladh, A., Hallberg, M. Mitochondrial function and membrane integrity: an in vitro comparison between six commonly used opioids. Manuscript.

II Nylander, E., Zelleroth, S., Nyberg, F., Grönbladh, A., Hallberg, M. The effects of morphine, methadone, and fentanyl on mitochondrial morphology: a live cell imaging study. Submitted manuscript.

III Nylander, E., Grönbladh, A., Zelleroth, S., Diwakarla, S., Nyberg, F., Hallberg, M. (2016) Growth hormone is protective against acute methadone-induced toxicity by modulating the NMDA receptor complex. Neuroscience, 339:538-547

IV Nylander, E., Zelleroth, S., Nyberg, F., Grönbladh, A., Hallberg, M. (2018) The protective and restorative effects of growth hormone and insulin-like growth factor-1 on methadone-induced toxicity in vitro. Int J Mol Sci, 19(11):3627

V Nylander, E., Zelleroth, S., Stam, F., Nyberg, F., Grönbladh, A., Hallberg, M. Growth hormone increases dendritic spine density in primary hippocampal cell cultures. Submitted manuscript.

Reprints were made with permission from the respective publishers.

List of Additional Papers

Diwakarla, S., Nylander, E., Grönbladh, A., Vanga, S.R., Khan, Y.S., Gutiérrez-de-Terán, H., Sävmarker, J., Lundbäck, T., Jenmalm-Jensen, A., Andersson, H., Rosenström, U., Sköld, C., Larhed, M., Åqvist, J., Chai, S., Hallberg, M. 2016. Binding to and inhibition of insulin-regulated aminopeptidase (IRAP) by macrocyclic disulfides enhance spine density. Mol Pharmcol., 89 (4), 413-424.

Diwakarla, S., Nylander, E., Grönbladh, A., Gutierrez, H., Sävmarker, J., Lundbäck, T., Jenmalm-Jensen, A., Andersson, H., Rosenström, U., Sköld, C., Larhed, M., Åqvist, J., Chai, S., Hallberg, M. 2016. Aryl sulfonamide in- hibitors of insulin-regulated aminopeptidase enhance spine density in primary hippocampal neurons cultures. ACS Chem. neurosci., 7 (10), 1383-1392.

Grönbladh, A., Nylander, E., Hallberg, M., 2016. The neurobiology and addiction potential of anabolic androgenic steroids and the effects of growth hormone. Brain Res Bull., 126 (PT1), 127-137.

Contents

Introduction ...... 11 Growth hormone ...... 11 The endogenous somatotropic system ...... 12 GH receptors and the signaling pathway of GH ...... 14 Protective and restorative effects of GH ...... 15 Opioids ...... 16 The opioid crisis ...... 16 Opioid receptors ...... 17 Opioids and cell viability ...... 18 Cognition ...... 19 NMDA receptors ...... 19 Dendritic spines ...... 21 GH and cognition ...... 22 GH and the NMDA receptor – a potential mechanism of action ...... 22 Opioids and cognition ...... 23 Aims ...... 24 Methods ...... 25 Animals ...... 25 Cell cultures ...... 25 Primary cortical and hippocampal cell cultures ...... 25 NG108-15 cells...... 25 SH-SY5Y cells ...... 26 Cell viability assays ...... 27 Mitochondrial function ...... 27 Membrane integrity ...... 27 Apoptosis ...... 27 Immunocytochemistry ...... 28 Gene expression ...... 28 High-throughput screening assays ...... 29 ImageXpress ...... 29 Mitochondrial morphology ...... 29 Dendritic spines ...... 29 Image analysis ...... 29 Statistical analyses ...... 30

Results and discussion ...... 31 Opioids reduces cell viability ...... 31 Opioids disrupt mitochondrial morphology ...... 33 The protective effects of GH ...... 36 The restorative effects of GH and IGF-1 ...... 40 GH increases dendritic spines in cell cultures ...... 42 Conclusions ...... 46 Populärvetenskaplig sammanfattning ...... 47 Acknowledgement ...... 50 References ...... 52

Abbreviations

AC Adenylyl cyclase AMPA α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid ANOVA Analysis of variance BDNF Brain-derived neurotrophic factor CNS Central nervous system CaMKII Ca2+/calmodulin-dependent protein kinase II cAMP Cyclic AMP dbcAMP Dibutyryl-cAMP DAPI 4′,6-diamidino-2-phenylindole DIV Day(s) in vitro DOP Delta-opioid peptide FDA Food and Drug Administration GFAP Glial fibrillary acidic protein GH Growth hormone GHD Growth hormone deficiency GHR Growth hormone receptor GHRH Growth hormone releasing hormone HTS High-throughput screening IGF-1 Insulin-like growth factor-1 JAK Janus kinase KOP Kappa-opioid peptide LD50 Lethal dose 50% LDH Lactate dehydrogenase LTP Long-term potentiation MAP2 Microtubule-associated protein-2 MOP Mu-opioid peptide MTT 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide NMDA N-methyl-D-aspartate PKA Protein kinase A PKC Protein kinase C qPCR Quantitative polymerase chain reaction

rhGH Recombinant human growth hormone STAT Signal transducer and activator of transcription

Introduction

In 1905, Professor Ernest Starling first used the word ‘hormone’, which orig- inate from the Greek meaning ‘to arouse or excite’, in one of his lectures. This was the start of new scientific era of advancement in the field of biology where many new essential hormones, such as insulin, thyroxine, testosterone, pro- lactin, and many more were discovered. Hormones are named after their role and the hormone that is important for growth is named ‘growth hormone’ (Tata, 2005).

Growth hormone Growth hormone (GH) is secreted from the anterior pituitary and was first isolated in 1944 from bovine pituitaries and confirmed to increase body weights in female rats (Li and Evans, 1944). The hormone is essential for reg- ulating growth, body composition, and metabolic effects, but also for stimulating the secretion of other hormones, such as insulin-like growth factor-1 (IGF-1), which mediates many of GH’s effects. Both GH and IGF-1 reach peak circulatory levels at puberty and thereafter slowly declines throughout adulthood until reaching low basal levels in the elderly population (Corpas et al., 1993; Iranmanesh et al., 1991; Zadik et al., 1985). The primary function of GH is growth, and the first medical therapy of GH was reported in 1958 where pituitary GH treatment successfully enhanced growth in a GH-deficient dwarf (Raben, 1958). The success from this study accelerated the use of pituitary GH as a replacement therapy to children suffering from GH-deficiency (GHD). But later in 1985, reports of children treated with pituitary GH displayed symptoms that was characterized as Creutzfeldt Jakob Disease (Koch et al., 1985; Underwood et al., 1985), a fatal degenerative brain disorder. These severe side effects associated with the use of pituitary GH caused the American Food and Drug Department (FDA) to end the use of pituitary GH as a replacement therapy. This FDA decision shifted the focus from pituitary GH to recombinant GH, which had been successfully manufactured in the 80’s by a small company called Genentech Inc. (Cronin, 1997). The cheaper recombinant GH enabled mass production that was scaled up within the years to come. Today, recombinant GH is widely used in the treatment of childhood- onset GHD (Wit, 2002) but also for other diseases affecting growth (Kirk,

11 2012). Scientists are now also exploring the unknown and exciting positive effects that GH may mediate in the central nervous system (CNS).

The endogenous somatotropic system The pulsatile secretion of the 22 kDa polypeptide GH from somatotropic cells in the pituitary gland is regulated by the somatotropic axis. Several life factors impact GH secretion. For instance, sleep, stress, starvation, and exercise all promote the secretion of GH while overeating and obesity inhibits the secretion (Bartke et al., 2013). There are many factors and hormones promoting or inhibiting the secretion of GH, but two hypothalamic hormones are mainly involved: growth hormone releasing hormone (GHRH) and somatostatin. The stimulating GHRH promotes activation of the adenylyl cyclase (AC) pathway that in turn increase the levels of cyclic AMP (cAMP) and protein kinase A (PKA). This results in depolarization of somatotropic cells and open- ing of voltage-gated calcium channels. The increase of calcium in the cytosol activates the secretion of GH into the bloodstream. The effects of GHRH on GH secretion is very effective as a rapid increase of GH is observed following GHRH administration in humans (Thorner et al., 1983). On the contrary, a hypothalamic polypeptide was confirmed in 1973 to inhibit the secretion of GH (Brazeau et al., 1973), a peptide that was later named somatostatin. The inhibitory somatostatin is part of the negative feedback loop of GH secretion. When secreted, somatostatin stimulates a specific pertussis toxin-sensitive Gi- protein that inhibits the AC pathway. This leads to a somatostatin-induced inhibitory effect on cAMP production and calcium release, which in turn will suppress the release of GH by hyperpolarizing the somatotropic cell. The relationship between GHRH and somatostatin is important as the hormones regulate the pulsatile nature of GH secretion (Sato et al., 1988; Tannenbaum and Ling, 1984; Vance et al., 1985). When GHRH is administered in intervals, the peaks of GHRH-induced secretion of GH declines over time, which may be linked to depletion of the GH pool, or to desensitized GHRH receptors in somatotropic cells. However, when GHRH is co-administered with somatostatin, no peak decline of GH secretion is observed (Sato, et al., 1988). Thus, the link between GHRH and somatostatin is essential to maintain as the pulsatile secretion of GH is important for physiological growth and development. Continuous release of GH has been shown to be less effective in promoting growth compared to frequent pulses (Clark et al., 1985). Apart from the effects that GHRH and somatostatin have on GH secretion, other important factors that regulates GH secretion is GH itself and IGF-1 that is secreted from the liver following release of GH. When exogenous GH is administered, both the transcription of GHRH mRNA and secretion of GH are effectively inhibited (Abe et al., 1983; Bertherat et al., 1993; Katakami et al., 1986; Rosenthal et al., 1986). This effect is likely caused by an increase of somatostatin secretion from the hypothalamus (Chihara et al., 1981; Patel,

12 1979). In hypophysectomized animals, where the pituitary gland is removed or damaged, the mRNA levels of somatostatin are usually low. However, when exogenous GH is administered, the levels are normalized (Patel, 1979). Similar to exogenous GH administration, IGF-1 attenuates the secretion of GH, GHRH, and reduces GH mRNA when administered to somatotropic cells, an effect that is caused by increased secretion of somatostatin (Abe, et al., 1983; Berelowitz et al., 1981; Goodyer et al., 1984; Yamashita and Melmed, 1986). These studies provide evidence that both GH and IGF-1 regulate their own secretion. A simplified overview of the feedback mechanism of the somatotropic system is illustrated in Figure 1.

Figure 1. A simplified overview of the somatotropic system in the brain. The two hypothalamic hormones that regulate the secretion of growth hormone (GH) are growth hormone releasing hormone (GHRH) and somatostatin. GHRH act stimulating on the secretion of GH while somatostatin acts inhibitory. Furthermore, both GH and its mediator, insulin-like growth factor-1 (IGF-1) regulate their own secretion by a negative feedback mechanism. Illustration by Erik Nylander.

13 GH receptors and the signaling pathway of GH The GH receptor (GHR) was successfully cloned in 1987 (Leung et al., 1987) and the transmembrane receptor was later characterized to belong to the cytokine class 1 family (Cosman et al., 1990). It is abundantly expressed in various tissues, including specific brain regions such as hippocampus and cortex (Lai Z. N. et al., 1991). Just like GH itself, the expression of the GHR diminishes with ageing (Lai Z. et al., 1993). An overview of the GHR and the signaling mechanism of GH are illustrated in Figure 2.

Figure 2. An overview of the growth hormone receptor (GHR) and the signaling mechanism of growth hormone (GH). (1) Binding of a single GH molecule to the GHR; (2) Formation of the GH-GHR complex; (3) Phosphorylation of Janus kinase 2 (JAK2) and the intracellular SH2 domain of the GHR that enables docking of a protein belonging to the ‘signal transducer and activator of transcription’ (STAT) family; (4) Phosphorylation of docked STATs by JAK2, which subsequently dissociate the STATs from the GHR; (5) Dissociated homodimers of STAT migrates to cell nucleus; (6) Transcription or gene expression of proteins that mediates the effects of GH. Illustration by Erik Nylander.

When available GH binds to the GHR it forms a GH-GHR homodimer complex (Leonard and O'Shea, 1998). At this state the receptor complex is considered to be active. The GH-GHR complex enables a further link with a tyrosine kinase, namely Janus kinase 2 (JAK2), which is activated and phosphorylated upon binding of GH. In contrast, without GH binding to GHR, no link between GHR and JAK2 is found (Argetsinger et al., 1993). However,

14 when the GH-GHR complex is linked with JAK2, both JAK2 and the intracellular part of GHR are phosphorylated. After these events, the molecular signaling follows the traditional JAK downstream pathway. The phosphorylated GHR enables docking of a specific protein belonging to the family ‘signal transducers and activators of transcription’ (STAT) that binds to the intracellular SH2 domain of the GHR. These STATs are phosphorylated by activated JAK2 and subsequently dissociate from the receptor-complex as homodimers and migrate to the nucleus and activate gene transcription of a wide array of different proteins that mediates the effects of GH.

Protective and restorative effects of GH There are accumulating evidence suggesting that GH act neuroprotective in the CNS. In rats subjected to hypoxia-induced ischemic injury, the mRNA levels of both IGF-1 and GHR increased in the damaged part of the brain (Gustafson et al., 1999) indicating that the somatotropic axis may have an important role in neuroprotection. This observation was later confirmed, as GH treatment was capable of maintaining the integrity of the CNS by acting protective against ischemic insults (Alba-Betancourt et al., 2013; Gustafson, et al., 1999; Scheepens et al., 2001). The role of GH in protecting against hypoxia-induced injuries has been suggested to be related to the anti-apoptotic effects of GH (Shin et al., 2004). Further reports suggest that GH protects hippocampal cell cultures from morphine-induced apoptosis (Svensson et al., 2008), against excitotoxicity in neuroretinal cells (Martinez-Moreno et al., 2018), and that the hormone inhibits the executioner apoptosis enzyme caspase-3 in lymphocytes (Mitsunaka et al., 2001). These observations indicate that GH acts protective in the CNS, although further research is required to fully confirm these results. The restorative effects of GH have so far primarily been reported in patients suffering from various brain trauma. For instance, a 10-year old child suffering from severe brain trauma caused by asphyxia during delivery had her memory, intelligence, and general cognitive abilities improved following GH treatment (Devesa et al., 2016). The observed results from this particular case support earlier studies, where GH-therapy displayed improved rehabilitating effects in patients suffering from various traumatic brain injuries, such as high-speed motor- and skiing accidents. Although the effects varied, these patients all displayed improved cognitive functions when subjected to GH-therapy (Devesa et al., 2013). The positive effects were independent whether or not a GHD was present (Devesa, et al., 2016; Devesa, et al., 2013). In cases when patients are suffering from brain trauma and display a pronounced GHD, severe cognitive dysfunction has been observed. Treatment of GH in these specific cases improved their cognitive capabilities (Moreau et al., 2013). Moreover, studies using animal models reveal similar results. In cases where a leisure in the brain have been induced, GH treatment improves various brain

15 functions (Heredia et al., 2013; Ong et al., 2018), effects that likely are associated with the effects GH and IGF-1 exert on neurogenesis (Aberg D., 2010; Heredia, et al., 2013).

Opioids Opioids are widely used in the clinics as a potent treatment for pain due to their strong antinociceptive effect. The first opioid discovered was the natural opioid morphine, which was isolated and extracted from the opium poppy (Papaver somniferum) by Friedrich Wilhelm Adam Serturner in the beginning of 1800 (Krishnamurti and Rao, 2016). Since then, many new substances, both natural, synthetic, and semisynthetic opioid drugs have been discovered and approved for the treatment of pain. They are pharmacologically classified after their potency, whereas the more potent opioids, such as methadone, fentanyl, and oxycodone are used in the treatment of severe cancer pain while less potent opioids, such as morphine, codeine, and hydromorphone are used as a treatment for moderate acute pain when NSAIDs (non-steroid anti-inflammatory drug) or paracetamol is not enough. The use of opioid drugs is associated with various side-effects, ranging from the mild effects such as obstipation and sleepiness to the more severe or fatal side-effects such as addiction and respiratory failure.

The opioid crisis Since the 90’s, there have been an increasing number of overdose deaths involving opioids in the United States. These statistics raised massive concerns and are referred to as an opioid epidemic or the ‘opioid crisis’. The first wave of opioid overdose deaths that occurred in the United States was primarily associated with the introduction of OxyContin®, containing an extended release of oxycodone, to the market. At that time, it was generally considered safe to prescribe opioids for long-term treatment of non-cancer pain. The ag- gressive marketing from pharmaceutical companies, the excessive prescription of opioids and increased addiction, as well as failed efforts to address these problems, led to the first wave of opioid-related deaths and the start of the opioid crisis (Kolodny et al., 2015). In 2010, a second wave of opioid overdose deaths was reported. This time, the rise in deaths was associated with the use of heroin, an illicit synthetic opioid. The use of prescription opioids had at this time declined, albeit it still remained high (Rudd et al., 2014) indicating that previous users of prescription opioids had made their switch to il- legal non-medical opioids instead. The third wave of opioid overdose deaths started in 2013, and was characterized by the illicit use of a new class of potent synthetic opioid, fentanyl and its derivates. In the United States during 2013- 2014, law enforcement reported a large increase (up to 426%) in cases where

16 obtained drugs were tested positive for fentanyl. The same study also reported that the number of overdose deaths involving synthetic opioids increased by 79%, even though the prescription rates remained the same (Gladden et al., 2016). The illicit use of fentanyl or synthetic opioids continued and in 2016 during July-December, fentanyl was involved in 56 % of all opioid overdose deaths while fentanyl analogs were involved in up to 14% of all death cases (O'Donnell et al., 2017). During the same year, fentanyl, methadone, morphine, and oxycodone were all ranked within the top 10 drugs involved in overdose deaths (Hedegaard et al., 2018). A total number of 42,249 overdose deaths involving opioids was reported, an increase by 28% from 2015. This represent 66% of all drug overdose deaths (63,632) during 2016 (Seth et al., 2018). The opioid prescription rates declined during the period 2010-2015 but as the number of opioid-related overdose deaths still remains high (Guy et al., 2017), this indicate that there are ongoing issues associated with illicit use of opioids as previously observed. In Sweden, there is no epidemic in comparison to the opioid crisis in the United States. However, a worrying trend in the use of the more potent opioids, such as oxycodone, has been observed. The number of patients with pre- scribed opioids declined from 2006-2015 but data indicate that an increase in the prescription of the more potent opioids occurred (Backryd et al., 2017). According to the Swedish National Board of Health and Welfare (Socialstyrel- sen), oxycodone prescription has increased from approximately 8 to 32 patients per 1000 inhabitants during the last 10 years (Swedish National Board of Health and Welfare, 2019), which is worrying given that oxycodone partly initiated the opioid crisis in the United States. However, as the Swedish laws regarding marketing and prescription of pharmaceuticals are rather strict alongside with the knowledge that the opioid crisis is an ongoing problem in the United States, it seems unlikely that an opioid epidemic to similar extent will occur in Sweden.

Opioid receptors In the beginning of the 70’s, opioid peptides were demonstrated to bind to specific receptors (Pert and Snyder, 1973; Simon et al., 1973; Terenius, 1973). Later in 1976, opioids were suggested to bind to three distinct opioid receptors subtypes (Gilbert and Martin, 1976; Martin et al., 1976). These receptors were later characterized to belong to the family of G-protein coupled receptors and are classified into the following subtypes; the mu-opioid peptide (MOP) receptor, delta-opioid peptide (DOP) receptor, and kappa-opioid peptide (KOP) receptor (extensively reviewed in Harrison et al., 1998). These different subtypes all mediate specific effects in the CNS. For instance, stimulation of the MOP receptor is associated with most of the clinical effects as seen from opioids, such as nociception, euphoria, reward, and withdrawal symptoms

17 (Kieffer, 1999). The DOP receptor does exert specific nociception but is also involved in the regulation of anxiety and depression (Chu Sin Chung and Kieffer, 2013). Finally, the KOP receptor is associated with aversive and dys- phoric effects (Wee and Koob, 2010) that primarily have been observed from animal models mimicking stress using dynorphin, a KOP receptor agonist (Knoll and Carlezon, 2010). The clinical effects as seen from morphine treatment, such as most of the analgesia, reward, withdrawal, respiratory depression, and obstipation, among others, are effectively inhibited in animal MOP receptor knock-out models when compared to wild-type animals (Kieffer, 1999). The opioid system is important for maintaining reward and controlling pain sensation. Although studies of the opioid system have not been a major part of the investigations in the present thesis, it is important to consider the effects of the opioid system in the brain. When endogenous opioids, such as endor- phins or enkephalins, binds to their corresponding receptor, a cascade of signaling events occurs resulting in stimulation or inhibition of second messengers (e.g. AC, calcium, potassium, PKC, mitogen-activated protein kinase) that ultimately reduces neuronal activity giving rise to euphoria and reduced pain transmission (Williams et al., 2001). The same principle applies for opioid drugs, although at a much higher potency. This is a problem in chronic opioid users, as they will develop a tolerance to the drug and effectively inhibit the endogenous opioid system. In the 80’s, animal experiments using opioids revealed that the function of the opioid receptors is reduced in tolerant animals (Chavkin and Goldstein, 1984; Christie et al., 1987). Later studies have revealed that development of tolerance is a rather complex phenomenon and that chronic opioid treatment induces neuronal adaptations associated with receptor tolerance, opioid-sensitive cell tolerance, and changes in synaptic strength (Christie, 2008).

Opioids and cell viability The impact of opioids on cell viability is rather inconclusive. There are several studies providing proof indicating that opioids induce toxic effects in cells leading to reduced cell viability. This reduction is observed in various cell types or cell lines from different tissues, for example in the oral squamous carcinoma HSC-3 cells (Nishiwada et al., 2019), in the reproductive Sertoli cells (Soltanineghad et al., 2019), in the gastric SGC-7901 cell line (Wu et al., 2014), in the human neuroblastoma SH-SY5Y cell line (Faria et al., 2016; Kokki et al., 2016), in human fibroblasts (Aguirre et al., 2016; Ficklscherer et al., 2013), in hippocampal cell cultures (Svensson, et al., 2008), and in mouse spinal cord neurons (Hauser et al., 2001). The main cause for the observed negative effects in most of these studies are related to opioid-induced apoptosis, which may serve as the main catalyst to reduced cell viability. However, there are studies demonstrating that opioids may in fact act protective in

18 various cell cultures. For instance, opioids are shown to act anti-inflammatory in oral epithelial cells (Wang Y. et al., 2015), protective against oxidative stress in the glioma C6 cells (Almeida et al., 2014), protective in SH-SY5Y cells (Wang B. et al., 2018), and protective against ischemic toxicity in isolated adult cardiomyocytes and HL-1 cells (He et al., 2016). The inconsistency of the above-mentioned studies highlights the need for additional studies examining the effects of opioids on cell viability, although most of the studies do indeed provide evidence that opioid treatment negatively impacts cellular health. In addition, morphine is more commonly studied whereas less is known about other clinical opioids and few comparative studies exist.

Cognition Cognition (Latin cognosco, meaning ‘I know’) is a complex physiological phenomenon involving higher brain functions, such as learning and memory, perception, and problem solving. As stated in (Purves, 2008), cognition can be described as “the process by which we come to know the world”. Different brain regions are associated with specific functions, but the two areas of interest for this particular thesis is prefrontal cortex and hippocampus. A large meta-study by Cabeza and Nyberg (Cabeza and Nyberg, 2000), who investigated 275 PET (positron emission tomography) and fMRI (functional mag- netic resonance imaging) studies, concluded that the prefrontal cortex is associated with most of the cognitive related tasks, and was most dominantly activated during working memory and memory retrieval. The hippocampal areas, which were classified as the medial temporal lobe, were associated with episodic memory encoding as well as nonverbal episodic memory retrieval (Cabeza and Nyberg, 2000). Both of these regions are of interest to study when investigating the ‘learning and memory’ aspect of cognition.

NMDA receptors The N-methyl-D-aspartate (NMDA) receptor is essential for maintaining normal function of various brain functions. The binding sites of the NMDA-receptor in the frontal cortex and cerebellum are significantly increased from fetus stage to birth (Court et al., 1993; Johnson et al., 1993). Blockade of the NMDA receptor during early-life development causes severe neurodegeneration in rats (Ikonomidou et al., 1999). On the contrary, mild head trauma to a young rat brain causes excitotoxicity of the NMDA receptor, which was associated with increased neuronal cell death. The observed effects were inhibited by MK801, a NMDA receptor antagonist (Ikonomidou et al., 1996). These observations suggest that caution should be taken when initiating treatment with NMDA receptor antagonists during early-life. The role of NMDA receptors during early prenatal life is essential for maintaining the integrity and

19 development of the brain. In the early adult brain, the NMDA receptor devel- opmental role is suggested to be less sensitive when compared to early prenatal life but altered function of the NMDA receptor may still impact certain cognitive-related behavior (Popke et al., 2001). In fact, alteration of the NMDA receptor structure and function has been associated with several neurodegenerative diseases affecting cognition, such as Alzheimer’s and Hun- tington’s disease (Fernandes and Raymond, 2009; Wang R. and Reddy, 2017). These studies provide further evidence highlighting the essential role of the NMDA receptor in the CNS. There is a strong link between the function of the NMDA receptor and cognitive function. The NMDA receptor, together with the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, play an important role in the underlying mechanism behind memory formation as they are associated with long-term potentiation (LTP) and synaptic strength (Frankiewicz et al., 1996; Matsuzaki et al., 2004; Tsien et al., 1996). This is a complex system involving many neurotransmitters, second messengers, receptors and will only briefly be touched in this thesis. Initiation of LTP is summarized in (Purves, 2008) and the molecular mechanism is illustrated in Figure 3. Briefly, glutamate bind to postsynaptic AMPA receptors, mediating the influx of sodium ions into the cell. At this stage, glutamate also binds to postsynaptic NMDA receptors although they are considered inactive as the channel pore is blocked by a magnesium ion. The repetitive activation of AMPA receptors and the subsequent influx of sodium will at a given threshold depolarize the cell, leading to removal of the magnesium ion blocking the NMDA receptor. This enables influx of calcium ions after glutamate binding to the NMDA receptor. While in the cell, calcium act as a second messenger and activates downstream factors, such as protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CaMKII). This cascade of events will eventually lead to substrate phosphorylation and the release of vesicles containing additional AMPA receptors. These receptors are transported and expressed on the cell surface and will increase the sensitivity of the postsynaptic dendrite. It has also been suggested that as a result of LTP initiation, AMPA receptors can also be expressed in synapses that normally do not contain any AMPA receptors that further strengthens the signal and maintains LTP (Purves, 2008). In hippocampus, LTP is inhibited in the presence of NMDA receptor antagonists such as MK801 (Engert and Bonhoeffer, 1999; Frankiewicz, et al., 1996; Matsuzaki, et al., 2004).

Figure 3. The basis of long-term potentiation (LTP) initiation and the role of the N- methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA) receptors. (1) Released glutamate from the presyn- aptic terminal binds to both the AMPA receptor and the NMDA receptor. However, due to the magnesium block of the NMDA receptor, the receptor is considered inactive; (2) Activated AMPA receptors mediate the influx of sodium ions in the postsynaptic cell leading to depolarization; (3) Depolarization of the postsynaptic cell releases the magnesium block from the NMDA receptor and effectively activates it; (4) Glutamate can now bind and activate the NMDA receptors leading to an influx of calcium ions into the postsynaptic cell; (5) Calcium acts as a second messenger and activates two main pathways, the PKC and CaMKII leading to substrate phosphorylation that ultimately results in the release of vesicles containing new AMPA receptors; (6) New AMPA receptors are transported and expressed at the cell-membrane leading to additional binding sites of glutamate that enables further signaling mechanism and the onset of LTP. Illustration by Erik Nylander.

Dendritic spines Dendritic spines are small protrusions in neurons where the synaptic activity occurs and they are suggested to be highly associated with learning and memory (Bourne and Harris, 2008; Kasai et al., 2010). There are different spine morphologies where mature spines are morphologically identified as spines with a large spine-head (e.g. mushroom-like spines). Immature spines are referred to as filopodium, which resembles thin-like spines. The mushroom-like spines are considered mature as they contain dense populations of NMDA and AMPA receptors mediating the effects of LTP and synaptic strength (Korobova and Svitkina, 2010; Matsuzaki, et al., 2004). Spine development occurs dynamically in the brain upon various stimulations. Long-term enhancement of synaptic strength is associated with lasting changes in the spine morphology, while short-term enhancement is not (Engert and Bonhoeffer, 1999). This may explain why humans remembers occasions associated with strong emotionally responses while only temporary remember

21 occasions associated with every-day life. In certain diseases, an increase in spine density may not always be optimal as an increase in spine density in the amygdala are associated with fearful memories (Gisabella et al., 2016).

GH and cognition Unlike the well-established role of GH during growth, the effects in the brain are still elusive and not fully known. Since the 90’s, research has aimed to discover the potential role of GH in the brain. These studies were particularly appealed by the knowledge that the GH receptor is expressed in various regions in the brain (Lai Z. N., et al., 1991), but also because GH may be able to pass the blood brain barrier (Pan et al., 2005). The outcome of these studies revealed that GH is essential in the CNS, as it is involved in many physiological processes such as sleep, appetite, immune system, memory, fear, neuroprotection, and cognition (Nyberg, 2009; Nyberg and Hallberg, 2013). One of the first evidences suggesting that GH improves cognitive function was observed in patients suffering from GHD. The inability of the pituitary gland to secrete GH does not only reduce growth, but also negatively impact cognitive function, which was improved in GHD-patients when subjected to GH replacement therapy (Elbornsson et al., 2017; Falleti et al., 2006). Recent studies demonstrate that GH treatment improve cognitive function in humans as assessed using various neurological tests (Dykens et al., 2017; Rhodin et al., 2014) as well as in older patients with low levels of circulating GH (Quik et al., 2012; Sonntag et al., 2005). In addition, several behavioral animal studies have reported improved memory following GH-treatment. In particular, studies examining the beneficial role of GH in improving spatial memory in rats (Enhamre-Brolin et al., 2013; Gronbladh et al., 2013; Le Greves et al., 2006; Ramis et al., 2013) or long-term memory in zebrafish (Studzinski et al., 2015) have been successful. Given the potential beneficial effects that GH exert on cognition, the hormone has been suggested to act as a ‘cognitive enhancer’ that may benefit patients suffering from various cognitive-related dysfunctions, but additional studies are needed to fully confirm these effects.

GH and the NMDA receptor – a potential mechanism of action There are still ambiguities as to how GH exerts its effects in the brain. One of the suggested mechanisms involves the interaction between GH and the NMDA receptor, which has been the main hypothesis in the present thesis. In several models where GH improves cognitive function, there is a link between GH and the NMDA receptor. In hypophysectimized rats, GH improves cognition and induces an increase in the transcription levels of the NMDA receptor subunits GluN1 and GluN2b (Le Greves, et al., 2006). In zebrafish, the GH-improved long-term memory was associated with an increase of NMDA receptor subunits (Studzinski, et al., 2015). In both mice and rats, a blockade

22 of the NMDA receptor also blocked the improved memory effects from GH treatment (Ramis, et al., 2013). Moreover, the GHR is closely associated with NMDA receptor subunits in the hippocampus where GHR levels correlates well with GluN2b mRNA levels (Le Greves et al., 2002). These observations indicate that GH may alter the structure or conformation of the NMDA receptor leading to improved memory or improved cognitive capabilities.

Opioids and cognition The main problem associated with opioids is addiction, which is acknowl- edged worldwide and is in many countries addressed pharmacologically in opioid maintenance programs using methadone or buprenorphine (Whelan and Remski, 2012). Patients who enlist at opioid maintenance programs are well controlled and many of these patients can, with proper treatment and mo- tivation, live a normal life. However, given the rise of the opioid crisis and the over-prescription of opioids, other long-term effects (excluding overdose deaths) associated with opioid use are not fully evaluated. It is likely that years of opioid use impact brain development and functions that has not yet been discovered. One of these negative effects may be cognitive dysfunction. As of date, there are limited and inconclusive evidence suggesting that opioids may negatively impact cognitive function. However, there are specific studies indicating that certain opioids, such as methadone and morphine, do in fact alter cognitive function. For instance, a man with chronic pain pre- scribed opioids for six years displayed certain cognitive-related symptoms such as concentration and memory difficulties, among other (Rhodin, et al., 2014). Furthermore, patients on long-term opioid treatment for chronic pain display reduced spatial memory and impaired performance in working memory when compared to patients without opioid treatment (Schiltenwolf et al., 2014; Sjogren et al., 2005). Impaired performance in cognitive-based tasks in comparison to non-drug users is also the case in patients actively using methadone (Zeng et al., 2016). Similar results have been observed in animal studies where opioid use impairs behavioral patterns associated with cognition, such as behavior linked to memory retrieval and working memory (Andersen et al., 2011; Cummins et al., 2012; Hepner et al., 2002; Tramullas et al., 2007). Opioid treatment in animals also demonstrate negative effects associated with cognition, such as inhibited neurogenesis (Eisch et al., 2000), cell-death (Mao et al., 2002; Perez-Alvarez et al., 2010), and volumetric changes in areas of the brain associated with cognition (Younger et al., 2011). To conclude, these studies indicates that the effects of long-term opioid treatment may affect cognitive capabilities, which are somewhat overshadowed by the increasing number of opioid-related deaths associated with the ‘opioid crisis’.

23 Aims

The overall aim of this thesis was to investigate the protective and restorative effect of GH in opioid-treated cell cultures. The specific aims were:

• To investigate and compare the effects of clinically used opioids on cell viability using various cell cultures.

• To investigate the protective properties of GH in cell cultures when co-treated with methadone.

• To investigate the restorative effects of GH and its mediator IGF-1 in methadone-damaged cells.

• To investigate the effects of GH on dendritic spines in hippocampal cell cultures.

24 Methods

Animals All experiments involving animals (paper I, III, IV, and V) were approved by the local animal ethics committee (C14/15 and 5.8.18-18550/2018) according to Swedish rules and guidelines regarding animal experiments (Animal Wel- fare Act SFS1998:56) and the European Communities directive (86/609/EEC).

Cell cultures Primary cortical and hippocampal cell cultures Primary cortical (paper I, III and IV) and hippocampal (paper V) cells were harvested from fetuses of pregnant Wistar (Charles River, Italy) or Sprague Dawley (Charles River, Germany) rats at embryonic day 17. A mixed population of neurons and glial cells were used to mimic a physiological culture condition. Hippocampi and cortices were carefully dissected from the fetuses and the tissues were enzymatically and mechanically dissociated to cell sus- pensions. Cells of each type were seeded into tissue culture plates and grown in media favoring neuronal growth supplemented with fetal bovine serum and B27 in a humidified CO2 incubator. Already at 1 day(s) in vitro (DIV), cells started to develop neurites extending from the cell body and at 7DIV, cells had developed a mature network of neurites. In the primary hippocampal cell cultures, dendritic spines were clearly visible at 14DIV. A representative image from the primary cell cultures are shown in Figure 4A. Primary cortical cell cultures were considered mature at 7DIV while hippocampal cultures were used at 14DIV when dendritic spines were considered to be mature.

NG108-15 cells The NG108-15 cell line (ATCC, Manassas, USA and kind gift from Malin Jarvius) is a hybrid mix of mouse neuroblastoma and rat glioma (Hamprecht et al., 1985) that share many physiological properties with neurons and glial cells. The NG108-15 cells are rather large when compared to primary cell cultures and display a uniform profile that enables detection of small changes in morphology (paper II). These cells differentiate to a neuronal-like morphology

25 with neurites extending from the cell body when subjected to dibutyryl cAMP (dbcAMP, 1 mM for three days) treatment. It is therefore possible to examine the effects of compounds on non-neuronal vs neuronal-like cells using the NG108-15 cells (paper I). The NG108-15 cells were cultured in T25 or T75 culture flasks and subcultured to either new flasks or tissue culture plates at approximately 80% confluency. A representative image from NG108-15 cells is shown in Figure 4B.

Figure 4. Representative images of primary hippocampal cell cultures and differentiated NG108-15 cells. Images were captured using a high-content screening device, ImageXpress. (A) Mixed neuronal/glial primary hippocampal cell cultures stained with the neurite marker microtubule-associated protein-2 (as shown in green), astrocytic marker glial fibrillary acidic protein (shown in red), and the nuclei stain DAPI (shown in blue); (B) NG108-15 cells differentiated with dibutyryl-cAMP and stained with the neurite marker βIII-tubulin (shown in green) and the nuclei stain DAPI (show in blue).

SH-SY5Y cells The SH-SY5Y cell line (kind gift from Dr. Anne-Lie Svensson) is derived from human neuroblastoma and is used to examine the effects of compounds in cells with human origin. These cells display a distinct morphology profile, which are larger than primary cell cultures. Similar to the NG108-15 cells, the SH-SY5Y cells were cultured in T25 or T75 culture flasks and subcultured to either new flasks or tissue culture plates at approximately 80% confluency. The SH-SY5Y cells were used to examine the effects of opioids on cell viability in paper I.

26 Cell viability assays Mitochondrial function The mitochondrial function was assed using the colometric 3-(4,5-Dimethyl- 2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay (Sigma-Al- drich, St. Louis, USA), which was used in paper I, III, and IV. In cells with functional mitochondria, MTT is metabolized to purple formazan crystals by mitochondrial dehydrogenases yielding a purple-colored solution when dissolved. In non-viable cells, no formazan products are produced yielding a blank-colored solution when dissolved. For the MTT assay, cells were cultured and seeded into 96-well tissue culture plates and treated with desired compounds prior to adding MTT to each well. The plate was placed in a humidified incubator for 30 min in order to let the metabolism occur. Later, cells were lysed using dimethyl sulfoxide and the amount of formazan products were quantified by measuring the absorbance at 570 nm using a plate-reader (FLUOstar Omega, Germany).

Membrane integrity The membrane integrity or membrane damage was assessed using the lactate dehydrogenase (LDH) assay, which was used in paper I, III, and IV. Naturally, LDH is located within the cell body and leaks out of the cell upon damage to the cell membrane. The impact of substance-induced damage to the cell membrane can therefore be determined by measuring the amount of LDH present in the cell media. By utilizing an enzymatic LDH reaction kit (Sigma-Al- drich), the amount of LDH present in the cell media can be quantified. The supplied compounds in the LDH kit are reduced to a red formazan product that correlates well with the amount of LDH present in the media. For the LDH assay, cells were cultured and seeded into 96-well tissue culture plates and treated with desired compounds prior to adding the LDH reaction kit to each well. The plate was placed in the dark, protected from light, for 30 min prior to measuring the absorbance at 492 nm using a plate-reader (FLUOstar Omega).

Apoptosis Apoptosis is a programmed cell death carried out by enzymes within the cells. This is a normal regulation of cell death that occurs in many organisms however abnormal initiation of apoptosis, such as drug-induced apoptosis, can cause severe dysfunction of many organs. Apoptosis is regulated and executed by caspases, where caspase 3 and 7 are the executioner enzymes. A high activity of these enzymes indicates ongoing apoptosis (Brentnall et al., 2013; Thornberry et al., 1997).

27 Apoptosis was assessed using a caspase 3/7 assay in paper III where the activity of these enzymes was investigated using a luminance kit (Caspase GLO, Promega, Wisconsin, USA). The kit contains a caspase 3/7 substrate, which is cleaved by active caspase 3/7 if present. This metabolism produces a compound that further reacts with the supplied luciferase that in turn produces a luminance signal. For the caspase assay, cells were cultured and seeded into 96-well tissue culture plates and treated with desired compounds prior to adding the caspase luminance kit to each well. The contents were later carefully removed and placed in a white 96-well culture plate and the luminance signal was quantified using a plate-reader equipped with a filter lens (FLUOstar Omega).

Immunocytochemistry Immunocytochemistry was performed in paper I (targeting opioid receptors) and in paper V (targeting neurons, astrocytes, and dendritic spines). Cells were fixed with 4% paraformaldehyde, permeabilized with triton-X100, and blocked with normal donkey serum prior to adding antibody of interest. For the detection of the MOP receptors in primary cell cultures, NG108-15, and in the SH-SY5Y cells, an anti-MOP receptor antibody (Abcam, Cambridge, USA) was used. For the detection of neurons, two specific antibodies were used; microtubule-associated protein-2 (MAP2, Merck Millipore, Burlington, USA) and βIII-tubulin (Sigma-Aldrich). For the detection of astrocytes, a specific antibody targeting the astrocytic glial fibrillary acidic protein (GFAP, Thermo Fisher Scientific, Waltham, USA) was used. For the detection of spines, an antibody targeting the specific dendritic protein drebrin (Enzo Life Sciences, Farmingdale, USA) was used. Antibodies were added to the cells and incubated for one hour prior to adding an appropriate fluorescent-conju- gated secondary antibody. Finally, the nuclei marker 4′,6-diamidino-2-phenylindole (DAPI, Merck Millipore) was added in order to visualize the cell nuclei.

Gene expression Quantitative poly chain reaction (qPCR) was used to examine the mRNA expression of NMDA receptor subunits GluN1, GluN2a, and GluN2b in paper III. Briefly, primary cortical neurons were cultured in 6-well tissue culture plates for 7DIV and treated with opioids or GH for 24 hours prior to mRNA extraction and transformation to cDNA. Selective forward and reverse primers against genes coding for GluN1, GluN2a, and GluN2b were used to amplify and calculate the mRNA expression.

28 High-throughput screening assays ImageXpress High-throughput screening (HTS) was applied in paper II and V using Im- ageXpress (Molecular Devices, San Jose, USA), a high-content screening device. The system is equipped with a CO2/air pump and temperature control that enables live-cell imaging in an environment suitable for various cell cultures.

Mitochondrial morphology Mitochondria are essential for cell survival as it is generally considered the ‘powerhouse of the cell’. The mitochondria were visualized by staining cells with the mitochondrial marker MitoTracker™ Red (Thermo Fisher Scien- tific), which accumulates in live cells depending on membrane potential. For the mitochondrial morphology study in paper II, treated cells were subjected to live-cell imaging for four hours with images of the mitochondria acquired every 30 min using a 40x objective. The acquired images of mitochondria were later analyzed using automated image analysis where the morphology was compared. Four variables related to the morphology were investigated: total mitochondrial area, mitochondrial skeleton network, number of mitochondrial objects, and mean area of mitochondrial objects.

Dendritic spines Spine density was evaluated in paper V using immunocytochemistry in 96- well tissue culture plates containing treated cells. Images of dendritic spines were acquired using ImageXpress equipped with a 40x objective and later analyzed using automated image analysis. Three main variables were investigated: number of dendritic spines, spines per neurite length, and neurite length.

Image analysis Image analysis was performed using ImageJ software (version 1.52h) with macros developed by the author. The analysis macro was written and adjusted for automated image analysis using images acquired from the ImageXpress. The general principle for the detection of desired parameters was using thresh- olding and detection of various region of interests for analysis. Automated image analysis was carried out in paper II and V. An example of the image analysis in paper II is shown in Figure 5.

Figure 5. Example of image analysis of mitochondrial morphology. (A) Raw images; (B) Binary segmented images as a result of the analyzing macro.

Statistical analyses Statistics were calculated using GraphPad Prism software in all papers in- cluded in the thesis. In most of the cases, a randomized block analysis of variance (ANOVA) was used to compare the different treatment groups followed by an appropriate post-hoc comparison. Using primary cell cultures, cells harvested from each individual rat was considered one culture (n=1). For all cal- culations, a minimum of three independent cultures was used. Statistics were carried out on raw data with treatment and experiment (culture ID) as factors to account for differences between cultures. Non-linear regression was applied in paper I to compare the different lethal-dose 50% (LD50) values for each treatment using the extra sum-of-squares F test. Differences were considered statistically significant when p < 0.05.

30 Results and discussion

The brain is a fascinating organ susceptible to environmental- and substance- induced stimuli, and capable of adapting or changing its function thereafter. In this thesis, I have evaluated the effects of opioids and GH in cell cultures using various cell viability assays to determine 1) whether or not opioids are capable of inducing changes in mixed neuronal cell cultures leading to reduced cell viability and 2) whether or not GH acts protective or restorative in in damaged and healthy cells.

Opioids reduces cell viability In paper I, the effects of the following opioids; fentanyl, hydromorphone, ketobemidone, methadone, morphine, and oxycodone were evaluated with respect to their impact on cell viability in four different cell cultures. The mitochondrial function (as assessed in the MTT assay) and the membrane integrity (as assessed in the LDH assay) were used as cell viability markers in primary cortical cell cultures, human neuroblastoma (SH-SY5Y) cells, as well as in differentiated and undifferentiated hybrid neuroblastoma/glioma (NG108-15) cells. The different cell cultures were treated with 1-1000 µM opioids for 24 hours prior to data analysis. The results revealed a similar toxicity profile from the opioid treatments in all assays. For the mitochondrial function assay, the data was analyzed using non-linear regression and the LD50 was compared between the different opioid treatments. The calculated LD50 values from the four different cell cultures and all opioid treatments are shown in Table 1.

31 Table 1. The lethal dose 50% (LD50) results from the mitochondrial function assay (MTT) sorted from lowest to highest. The LD50 value was calculated using non-linear regression. A minimum of four (n=4) independent cell cultures were used. Mean log LD Cell type Opioid 50 Mean LD ± Std. Error 50 Differentiated Methadone -4.59 ± 0.11 28 µM NG108-15 Fentanyl -4.02 ± 0.17 96 µM Ketobemidone -3.73 ± 0.14 186 µM Oxycodone -3.21 ± 0.16 622 µM Hydromorphone -3.09 ± 0.16 807 µM Morphine -2.22 ± 0.81 >2000 µM1 Undifferentiated Methadone -4.25 ± 0.13 56 µM NG108-15 Fentanyl -3.94 ± 0.10 115 µM Ketobemidone -3.52 ± 0.13 300 µM Hydromorphone -3.15 ± 0.10 701 µM Morphine -2.94 ± 0.08 1142 µM1 Oxycodone -2.94 ± 0.11 1158 µM1 SH-SY5Y Methadone -4.18 ± 0.10 66 µM Fentanyl -3.67 ± 0.12 213 µM Ketobemidone -3.52 ± 0.13 305 µM Oxycodone -3.46 ± 0.13 348 µM Hydromorphone -3.13 ± 0.09 742 µM Morphine -3.01 ± 0.11 987 µM Primary cortical Methadone -3.83 ± 0.20 148 µM cell cultures Fentanyl -3.54 ± 0.09 288 µM Oxycodone -2.94 ± 0.15 1143 µM1 Hydromorphone ~2.91 ± n/a ~1223 µM1,2 Ketobemidone -2.87 ± 0.13 1300 µM Morphine -2.27 ± 0.63 >2000 µM1

1 LD50 value exceeds maximum concentration used (1000 µM). This is a theoretical calculated value.

2 Ambiguous curve-fit meaning approximate value.

The results from the mitochondrial function assay clearly demonstrated that methadone and fentanyl were the most potent opioids with respect to inducing mitochondrial dysfunction. In all cell types, methadone had the lowest LD50 followed by fentanyl, indicating that a much lower concentration of these opioids is needed to induce mitochondrial deficits. Ketobemidone was ranked as the third most potent opioids when compared to the other treatments. This was observed in three of the four tested cell cultures. Next to ketobemidone, oxycodone treatment was ranked in the middle while hydromorphone and morphine induced little to no effects with respect to mitochondrial function. Similar results were observed in the membrane integrity studies, although the effects were not as prominent as seen in the MTT experiments. Thus, opioids may therefore be more effective in reducing mitochondrial function rather than causing membrane damage, which warrant further investigation. The

32 key findings from the LDH assay, however, clearly displayed that methadone and fentanyl induced the most potent effects on membrane integrity. This was observed using the two highest doses (100 and 1000 µM) while no differences were observed between any of the opioid treatments at the two lowest doses (1 and 10 µM). Oxycodone and ketobemidone induced a greater effect on membrane integrity when compared to hydromorphone and morphine, similar to that of the results in the MTT assay. The results obtained from both of the assays ranked the opioids, with regards to opioid potency on cell viability, as follows; methadone, fentanyl, ketobemidone, oxycodone, hydromorphone, and morphine. The main goal for the present study was to evaluate and compare the effects of various clinically used opioids on cell viability. With that stated, it is still of interest to speculate as to why these differences in opioid-induced toxicity occurs. The effects of methadone, in particular, may be associated with its antagonistic properties to the NMDA receptor (Ebert et al., 1995) and not via the MOP receptor. This may also explain the high toxicity induced by ketobemidone that also exert NMDA receptor antagonism (Ebert, et al., 1995). The suggested hypothesis is based on the fact that morphine and hydromorphone, the opioids with the highest affinity to the MOP receptor (Volpe et al., 2011), induced little to no effects on cell viability and that the MOP receptor was expressed in all three cell types. In fact, the affinity ranking of the MOP receptor binding profiles by Volpe et al. of all tested opioids (except ketobemidone) is the opposite of the cell viability ranking obtained in this study. Similar affinities are seen with the KOP and DOP receptors as well (Codd et al., 1995; Maguire et al., 1993; Raynor et al., 1994). These observations indicate that the toxicity is mediated through another pathway, possibly via the NMDA receptor. However, this does not explain the high toxicity induced by fentanyl as seen in these experiments. Fentanyl may perhaps mediate toxicity through a mechanism associated with the NMDA receptor, a mechanism suggested to occur in morphine tolerant mice (Mao, et al., 2002). The authors of this study observed a morphine-induced increase in pro-apoptotic proteins leading to neuronal cell death that was associated with a tolerance-induced increase in spinal glutamatergic activity. Granted, this study was carried out in vivo using chronic morphine treatment, but since the potency of fentanyl is approximately 100 times higher than that of morphine, perhaps a similar and faster mechanism occurs in vitro. Additional studies using naloxone and NMDA receptor antagonists are needed to further understand the molecular mechanism behind these effects.

Opioids disrupt mitochondrial morphology The next step was to examine the negative effects on the mitochondria, in more detail, using the most potent opioids fentanyl and methadone. It was also deemed interesting to further compare the effects of these opioids with that of

33 morphine. This was performed in paper II where the mitochondrial morphology was evaluated in NG108-15 cells using HTS and live-cell imaging. The cells were treated with 25-100 µM opioids for four hours together with the mitochondrial dye MitoTracker™ prior to acquiring images using ImageX- press. The results revealed that both fentanyl and methadone had significant effects on mitochondrial morphology while morphine treated cells remained sta- ble. Fentanyl and methadone decreased the mitochondrial skeleton network during the four-hour exposure (Figure 6A and 6B, respectively), an effect that was time and dose-dependent with no effects seen from the morphine treatment (Figure 7A). Representative images from the segmentation obtained from the automated image analysis are shown in Figure 7B.

Figure 6. The effects of fentanyl and methadone on mitochondrial morphology in NG108-15 cells. Mitochondrial morphology was evaluated using live-cell imaging with images acquired every 30 min prior to automated image analysis. (A) The effects of fentanyl; (B) The effects of methadone. Statistics were calculated using repeated measurement two-way ANOVA followed by Dunnet’s post hoc test. All data are presented as mean ± SEM from three (n=3) independent cultures. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

These results provide additional data as to how methadone and fentanyl may reduce cell viability as seen in paper I. The changes in mitochondrial morphology occurred at much lower concentrations in comparison with the data obtained from the cell viability assays, with significant effects observed at 25 µM for fentanyl and 75 µM for methadone. Furthermore, the negative effect on mitochondrial morphology was initiated rather fast. Using the highest dose, 100 µM, the first significant effect appeared at 90 min following methadone and fentanyl treatment. Together, this data indicate that fentanyl and methadone induce fast and potent effects affecting mitochondrial morphology that may lead to the reduced cell viability as observed previously. However, the present study did not determine whether or not the effects on mitochondrial

34 morphology is caused by a direct effect on the mitochondria or if it is due to a secondary effect caused by reduced cell viability. The findings that methadone and fentanyl induce cellular changes affecting mitochondrial morphology have not been studied before. However, there are published data that support the results seen in this study, such as studies demonstrating negative effects on mitochondrial respiration, metabolism, and activity (Djafarzadeh et al., 2016; Ficklscherer, et al., 2013; Perez-Alvarez, et al., 2010; Vilela et al., 2009; Zamparelli et al., 1999) following methadone or fentanyl treatment.

Figure 7. The effects of opioids on mitochondrial morphology in NG108-15 cells. Mitochondrial morphology was evaluated using live-cell imaging with images acquired every 30 min prior to automated image analysis. (A) The effects of morphine; (B) Segmentation images from the 100 µM different opioid treatment. Statistics were calculated using repeated measurement 2-way ANOVA and all data are presented as mean ± SEM from three (n=3) independent cultures.

It is well recognized that mitochondrial function is important to maintain. Sev- eral neurodegenerative diseases are in fact partly caused by impaired mitochondrial function (Nunnari and Suomalainen, 2012). There are specific mechanisms in the brain that removes damaged or injured mitochondria in order to maintain integrity and function of the cell, called mitophagy (Lemasters, 2005). If this is not maintained properly, mitochondrial debris accumulates and can potentially induce toxic effects (Batlevi and La Spada, 2011; Wong and Cuervo, 2010). Although this was not thoroughly studied in the present study, both fentanyl and methadone treatment did in fact increase the mean area of mitochondrial objects (Figure 8A and 8B, respectively) indicating that mitochondrial accumulation occurs. An observation that was not seen in morphine treated cells. However, it is difficult to interpret this data as it is unclear whether or not the mitophagy system is active in the NG108-15

35 cells. It is for example known that certain macrophages are not present in various cell cultures and therefore the results need to be evaluated accordingly. Nevertheless, the impaired mitochondrial morphology as well as the reduced cell viability may be the cause of neuronal cell death that in turn causes impaired cognitive function in long-term opioid users.

Figure 8. The effects of (A) fentanyl and (B) methadone on the mean area of mitochondrial objects. Statistics were calculated using repeated measurement two-way ANOVA followed by Dunnet’s post hoc test. All data are presented as mean ± SEM from three (n=3) independent cultures. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

It is important to consider that the concentration of the opioids used in paper I and II are high and do not mimic physiological concentrations in the brain following administration in vivo. However, the use of these doses enabled us to compare the effects of the different opioids and to detect cellular injury that was the primary aim for these studies. The results were obtained from 24-hour treatment (paper I) and 4-hour treatment (paper II) and these doses would have been significantly lower if chronic administration for a longer time period was possible. However, due to the fact that cells in culture can only be maintained for a certain time, it is hard to conduct experiments with chronic administration of opioids without compromising cellular health. It is possible that these effects occur in vivo following long-term treatment, although this statement warrant further studies.

The protective effects of GH As accumulating evidence suggests that GH may act neuroprotective in the CNS, the protective effects of recombinant human GH (rhGH) were examined in opioid-treated cells. The opioid methadone was selected to induce damage because of the hypothesis that the NMDA receptor could be involved due to methadone’s antagonistic effects on the receptor. This is particularly

36 interesting because the potential mechanism behind GH’s beneficial effects is suggested to be mediated via the NMDA receptor. In paper III, methadone was added both alone and in combination with 0.01-1000 nM rhGH to primary cortical cell cultures for 24 hours. Prior to examining the protective effects of rhGH, the hormone was administered alone to the cells in order to evaluate the general effects of rhGH. These experiments revealed no effects in the mitochondrial function assay but a significant LDH decrease in the membrane integrity assay was observed using the highest doses (100 and 1000 nM). This data indicate that an excess of GH may counteract spontaneous natural cell death affecting membrane integrity and that GH exerts general positive effects on cultured cells. Next, rhGH was given as a co-treatment together with methadone to investigate if GH could protect cells damaged by methadone. These experiments revealed that 1) methadone induced significant damage to mitochondrial and membrane function (confirming previous observed results in paper I and II) and 2) rhGH effectively protected the cells against methadone-induced damage (Figure 9). The protective effects of rhGH were present in both the mitochondrial function assay (Figure 9A) and the mitochondrial function assay (Figure 9B), indicating an important role of rhGH in maintaining mitochondrial function and membrane integrity. In the membrane integrity assay, rhGH was capable of reducing the LDH levels back to control levels. These data support the hypothesis that GH acts as a neuroprotectant in the CNS and confirms previously published studies investigating the protective role of GH (Alba-Betancourt, et al., 2013; Mitsunaka, et al., 2001; Scheepens, et al., 2001; Svensson, et al., 2008). However, the inability of GH to protect cells from methadone-induced caspase activation (data not shown) alongside the protective effects in reducing membrane damage, indicate that the hormone may be more efficient in protecting cells against necrosis rather than apoptosis. These results are supported by the fact that impaired membrane integrity commonly is observed in necrosis-related cell death (Proskuryakov et al., 2003). Although, this is difficult to interpret as both necrosis and apoptosis may occur at the same time (i.e. as it does naturally).

Figure 9. The protective effects of growth hormone in the (A) mitochondrial function assay and (B) membrane integrity assay following methadone treatment. Statis- tics were calculated using a randomized block ANOVA followed by Dunnet’s post hoc test. All data are presented as mean ± SEM from six (n=6) independent cultures. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

These results from the above-mentioned experiments motivated further studies investigating the potential mechanism as to how GH acts protective in the CNS. Primary cortical cell cultures were treated with methadone or methadone together with the opioid receptor antagonist naloxone, in order to examine the role of the opioid receptors in mediating cellular toxicity. These experiments revealed that methadone-induced toxicity still occurred to the same extent even though naloxone was present (Figure 10). Thus, the results indicate that methadone induces damage to cell cultures via another pathway, most likely via the NMDA receptor. This observation supports the hypothesis that methadone causes reduced cell viability via a NMDA receptor mediated mechanism, as mentioned previously.

Figure 10. The opioid antagonist naloxone did not inhibit the toxic effects caused by methadone. Thus, an impact of the opioid receptors in mediating methadone-induced cellular damage seems unlikely. All data are presented as mean ± SEM from the same three (n=3) independent cultures.

This hypothesis was further tested when the mRNA expression of the NMDA receptor subunits was examined. Methadone induced the expression of the NMDA receptor subunits GluN1, GluN2a, and GluN2b, an increase that GH treatment effectively inhibited (Figure 11). This raised several questions concerning the mechanism of GH as the hormone has earlier been shown to increase the expression of these subunits, which was linked to improved cognition (Le Greves, et al., 2002; Le Greves, et al., 2006; Ramis, et al., 2013; Studzinski, et al., 2015). Instead, the data gathered from the gene expression studies indicated that GH effectively restored the methadone-induced increase in mRNA expression. It is tempting to speculate that methadone may induce cellular damage by changing the conformation of the NMDA receptor. The role of GH in protecting these cells may therefore be associated with a modulating role of the NMDA receptor in order to counteract these effects. Perhaps GH modifies the NMDA receptor function differently depending on if the primary role of GH is linked to neuroprotection or improved cognition.

Figure 11. Growth hormone modulates the mRNA expression of the N-methyl-D-aspartate (NMDA) receptor subunits GluN1, GluN2a, and GluN2b following methadone treatment. (A) The mRNA expression of the NMDA receptor subunit GluN1; (B) The mRNA expression of the NMDA receptor subunit GluN2a; (C) The mRNA expression of the NMDA receptor GluN2b. Statistics were calculated using a randomized block ANOVA followed by Dunnet’s post hoc test. All data are presented as mean ± SEM from three (n=3) independent cultures. *p < 0.05, **p < 0.01.

39 The restorative effects of GH and IGF-1 The observed protective effects of GH led us to examining the restorative effects of the hormone in methadone-treated cells. This study was aimed to investigate if GH, or its mediator IGF-1, could potentially restore cellular function damaged by methadone. In paper IV, the restorative effect of GH and IGF-1 was addressed using primary cortical cell cultures exposed to methadone for 24 hours. After the initial methadone exposure, the opioid was removed from the cell media and GH or IGF-1 were added for 48 hours. The results revealed that both GH and IGF-1 successfully restored the mitochondrial and membrane function in cells pre-treated with methadone (Fig- ure 12). In the mitochondrial function assay, GH effectively restored the mitochondrial function at 1, 10, 100, and 1000 nM (Figure 12A). Similar results were observed using IGF-1 where the mitochondrial function effectively were restored using the same doses (Figure 12B).

Figure 12. The restorative effects of recombinant human growth hormone (rhGH) and insulin-like growth factor-1 (IGF-1) in cells pre-treated with methadone. (A) The effects of rhGH in restoring mitochondrial function; (B) The effects of IGF-1 in restoring mitochondrial function; (C) The effects of rhGH in restoring membrane integrity; (D) The effects of IGF-1 in restoring membrane integrity. Statistics were calculated using a randomized block ANOVA followed by Dunnet’s post hoc test. All data are presented as mean ± SEM from five (n=5) independent cultures. *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001.

40 Similar results were observed in the membrane integrity assay, where GH and IGF-1 treatment demonstrated a decline in LDH-release, indicating a membrane stabilizing effect (Figure 12C and 12D, respectively). This was the first study in this thesis using IGF-1, which is thought to mediate many of GH’s effects, particularly during growth and development (Le Roith et al., 2001). The results obtained from these experiments show that IGF-1 effectively restore both the mitochondrial function and membrane integrity in a similar fashion as that of GH. These observations indicate that there is a close relationship between the two hormones, but it is difficult to assess whether or not GH is solely responsible for the observed restorative effects or if it is due to an increase of GH-induced IGF-1 release from the cells. If this would be the case, a local regulation of the somatotropic system is likely present within the cells. This has been discussed in several studies and a few different theories have emerged, such as 1) the original hypothesis describing that GH’s effects are mediated via other growth factors, 2) the ‘dual-effector theory’ suggesting that GH and IGF-1 have specific direct effects, 3) that there is a local production/regulation of IGF-1 via endocrine/paracrine mechanisms, and 4) that the effect of brain IGF-1 differs from serum IGF-1 (extensively reviewed in Le Roith, et al., 2001; Torres-Aleman, 2010). However, these hypotheses are associated with wide array of different cells and are depending on whether or not the endpoint is of systemic or peripheral origin. These results warrant further examination of the GH/IGF-1 relationship using primary cell cultures. Nevertheless, this study does provide proof that IGF-1 alone act restorative in primary cortical cell cultures. The restorative effects of GH have previously been reported. In patients or animals suffering from various brain traumas caused by accidents or birth de- fects, GH act rehabilitating and improves cognition along with other related symptoms such as motor activity (Devesa, et al., 2016; Devesa, et al., 2013; Donze et al., 2018; Heredia, et al., 2013; Ong, et al., 2018). Many of these patients also display a pronounced GHD, indicating that treatment with GH is needed (Devesa, et al., 2013). As seen in the present experiments along with other studies within the field, these beneficial restorative effects from GH and IGF-1 are most likely associated with the effects these hormones carry out on neurogenesis, which opioids are known to inhibit (Eisch, et al., 2000). It is not surprising that GH increases cell differentiation and proliferation in embryonic rat cell cultures, an effect that is linked to locally produced IGF-1 (Ajo et al., 2003). Likewise, the brain weight, number of cortical cells, and the neuronal/glial cell ratio increased in offspring when GH was administered to pregnant rats (Zamenhof et al., 1966). However, there is a difference between embryonic neurogenesis and adult neurogenesis. The latter is of more importance with respect to the restorative effects as damaged or dead neurons regenerate slow, if at all, in the CNS. There are studies providing proof that GH may enhance the adult regeneration of cells. In adult hypophysectomized rats, GH treatment increased the number of newborn cells within the 6-day treatment

41 period and this effect lasted without any negative effects on survivability to the 28-day mark (Aberg N. D. et al., 2009). Examining the effects of GH in animal models using hypophysectomy may however yield results that are difficult to interpret, as an altered somatotropic system impact the feedback- mechanisms of many other related systems. However, these beneficial effects of neurogenesis appear in non-surgery treated animals as well. This has primarily been observed in exercise models where training induces secretion of GH that in turn prevents age-associated neurodegeneration in mice (Blackmore et al., 2009). This effect is associated with an increase of IGF-1 uptake from the bloodstream to specific brain areas (Trejo et al., 2001). Nev- ertheless, it is clear that both GH and IGF-1 exert important physiological effects on neurogenesis, which support the findings in these experiments where GH and IGF-1 effectively restored damaged cellular functions.

GH increases dendritic spines in cell cultures For the past 20 years, research has led to the suggestion that GH acts as a cognitive enhancer in both humans and animals (Nyberg, 2000; Nyberg and Hallberg, 2013). Cognitive function is difficult to assess, and even more chal- lenging (e.g. impossible) to do so in cell cultures. It is, however, interesting to study the formation of dendritic spines, as they are associated with higher functions in the brain, such as long-term-enhancement as well as learning and memory (Engert and Bonhoeffer, 1999; Kasai, et al., 2010). In paper V, the effects of GH were assessed by investigating the formation of dendritic spines using primary hippocampal cell cultures. Primary hippocampal cell cultures were treated with brain-derived neurotrophic factor (BDNF), a known inducer of spines (Chapleau et al., 2008; Gu et al., 2008), or 10-1000 nM GH for 24 hours and later stained with antibodies targeting drebrin, a protein abundantly expressed in dendritic spines, as well as βIII-tubulin, a neuronal marker. The treated cells were subjected to HTS using ImageXpress with thousands of images acquired prior to automated image analysis. The results revealed that the highest dose of GH (1000 nM) effectively increased the formation of dendritic spines as both the ‘number of spines’ (Fig- ure 13A) and ‘spines per neurite length’ (Figure 13B) increased when compared to control-treated cells.

Figure 13. The effects of growth hormone on dendritic spine formation in mature primary hippocampal cell cultures. Results from the analysis measuring (A) the number of spines per site and (B) the number of spines per neurite length. Statistics were calculated using a randomized block ANOVA followed by Dunnet’s post hoc test. All data are presented as mean ± SEM from five (n=5) independent cultures. *p < 0.05, ***p < 0.001.

No effect in the ‘mean area of spines’ was detected suggesting that the area of the spines was unaffected by the GH treatment. Furthermore, the parameter neurite length, used as a marker for general health, was unaffected following GH treatment (Figure 14). Representative images from the experiments are shown in Figure 15.

Figure 14. The effects of recombinant human growth hormone (rhGH) on neurite outgrowth in mature primary hippocampal cell cultures. Statistics were calculated using a randomized block ANOVA and all data are presented as mean ± SEM from five (n=5) independent cultures.

Figure 15. Representative images from the spine assay. Primary hippocampal cell cultures were treated with recombinant human growth hormone (rhGH) and brain- derived neurotrophic factor (BDNF) for 24 hours prior analysis of dendritic spines. Drebrin was used as a marker for dendritic spines and βIII-tubulin as a neuronal marker. (A) Control treated cells; (B) BDNF-treated cells; (C-E) rhGH treated cells.

44 Dendritic spines contain dense populations of NMDA and AMPA receptors that regulate synaptic strength and LTP (Matsuzaki, et al., 2004). These receptors, in particular the NMDA receptor, are involved in the mechanism behind learning and memory (Bourne and Harris, 2008; Kasai, et al., 2010) as blockade of the NMDA receptor inhibits LTP (Engert and Bonhoeffer, 1999; Frankiewicz, et al., 1996). In the present study, GH increased the number of dendritic spines, an effect that may be mediated via modulating the NMDA receptor as previously suggested. These results support the observed effects of GH on cognition (Enhamre-Brolin, et al., 2013; Gronbladh, et al., 2013; Le Greves, et al., 2002; Le Greves, et al., 2006; Ramis, et al., 2013; Rhodin, et al., 2014) and further support the hypothesis that GH act as a cognitive enhancer (Nyberg and Hallberg, 2013). The increase in dendritic spines may very well serve as the potential mechanism behind these effects. The doses used in the present study were in the supra-physiological range, indicating that an excess of GH is mediating these effects. Similar results were seen using GH in similar doses whereas the number of spines increased in the hippocampus following GH treatment in rats (Olivares-Hernandez et al., 2018). The potential mechanism that GH exerts in the hippocampal cell cultures may be directed to binding of the GH-receptor followed by increased activation of the JAK/STAT pathway. Activation of this pathway and subsequent downstream factors lead to increased production of proteins that may alternate the NMDA receptor conformation resulting in the emergence of new spines. It is also possible that the expression of certain subunits or the number of NMDA receptors (and AMPA receptors) may perhaps also increase in silent synapses that normally do not express these receptors. Nevertheless, the findings that rhGH increases dendritic spine density support the hypothesis that GH may be used as a novel treatment to diseases affecting cognition.

45 Conclusions

The overall conclusion of this thesis is that GH can protect and restore cells from opioid-induced damage. Opioids, primarily methadone, were shown to reduce cell viability in various cell cultures, an effect that was effectively inhibited when co-treated with GH. Additionally, both GH and its mediator IGF- 1 were able to act restorative on cells damaged by opioids. More specifically, the thesis concludes that:

• Of the six tested clinically used opioids, methadone and fentanyl were ranked as the most potent opioids with respect to reduced cell viability. These observations, obtained from four different cell types, provide evidence that opioids reduce cell viability. When investigated further, the reduced cell viability caused by methadone and fentanyl may be associated with altered mitochondrial morphology.

• GH maintained cell viability when co-treated with methadone, an effect that was associated with normalization of the NMDA receptor subunits. This indicates that GH act protective via the NMDA receptor and that the hormone may potentially serve as a NMDA- receptor modulator.

• Both GH and IGF-1 effectively restored cell viability in cells pre- damaged by methadone, an effect that is indicative of the restorative properties of GH and IGF-1.

• GH treatment promotes the formation of dendritic spines, which are associated with pro-cognitive functions. This effect may serve as the underlying mechanism behind the beneficial effect GH has on cognitive function.

46 Populärvetenskaplig sammanfattning

Tillväxthormon är ett av de hormon som bidrar till en ökad tillväxt av bland annat fett, ben och muskulatur. Det frisätts från hypofysen i hjärnan i en na- turlig rytm som framförallt styrs av fysiologiska faktorer såsom sömn och trä- ning men även av andra hormonella förändringar. Eftersom tillväxthormon är viktig för tillväxt har människor och andra däggdjur en hög frisättning och höga nivåer av tillväxthormon under puberteten. Det är också därför som till- växthormon ges som ett läkemedel till små barn som inte upprätthåller en normal tillväxtkurva. Med åren har intresset för tillväxthormon ökat och flera forskare har börjat undersöka dess effekter i hjärnan, som idag fortfarande inte är klarlagda. Detta intresse började framförallt när rapporter om ett flertal barn, som hade brist på tillväxthormon, uppvisade symtom på försämrad kognitiv funktion som exempelvis nedsatt inlärning och minne. När dessa barn, men även vuxna, behandlades med tillväxthormon förbättrades den kognitiva funktionen. Ef- tersom dessa symtom förbättrades gjordes ett flertal studier på äldre männi- skor där det upptäcktes att de som har låga nivåer av tillväxthormon också visade en tendens till att ha försämrat minne. Detta kan försämra patienter som lider av olika demenssjukdomar, såsom exempelvis Alzheimers sjukdom, en sjukdom som drastiskt försämrar minneskapaciteten. Ett flertal olika studier genomfördes som visade att tillväxthormon eventuellt kan användas som en så kallad ”kognitiv förstärkare” för patienter som lider av försämrat minne. Utöver de goda effekter tillväxthormon har på inlärning och minne har även hormonet skyddande egenskaper i nervsystemet. Bland annat skyddas cellers funktion av tillväxthormon vid exempelvis syrebrist eller mot celldöd orsakat av andra substanser. Det pågår också forskning kring den läkande egenskap tillväxthormon innehar. Detta har testats hos människor som lider av hjärn- skador, exempelvis hos patienter som skadat sig allvarligt vid bilolyckor eller liknande trauma. Hos dessa patienter har det visat sig att tillväxthormon kan användas som en behandling under rehabiliteringen då både motoriska och minnesförbättrade egenskaper har påvisats hos dessa individer. Opioider är en grupp läkemedel som används vid måttlig till svår smärta när övriga smärtstillande preparat inte räcker till. Denna läkemedelsgrupp är oerhört effektiv och ges därför också som långtidsbehandling till patienter som lider av kronisk smärta, som exempelvis cancersmärta. Biverkningar från opi- oida läkemedel varierar från mildare symtom som förstoppning och trötthet till mer allvarliga symtom som exempelvis beroendeproblematik, andnöd och

47 död. Det senare har uppmärksammats i USA, där det just nu pågår en så kallad ”opioidkris” där många människor dött i opioidrelaterade dödsfall. Detta star- tade i början av 90-talet när det ansågs att opioider för långtidsanvändning generellt sett var ofarligt. Under samma tid lanserades läkemedlet OxyCon- tin® innehållandes den långtidsverkande opioiden oxykodon under en aggres- siv marknadsföringskampanj. Detta bidrog till att tusentals människor blev fast i ett beroende som senare ledde till flera dödsfall, en negativ trend som fortsatte med åren fram till idag. Senare har även syntetiska opioider, såsom heroin och fentanyl, varit involverade. Under 2016 skedde över 42 000 opioidrelaterade dödsfall i USA, vilket är en ökning med cirka 30% från 2015. I flera av dessa fall var fentanyl involverat, men även andra opioider, som exempelvis oxykodon, metadon och morfin. Utöver risken att hamna i ett beroende eller i värsta fall dödsfall har långtidsanvändning också en effekt på väl- mående. Senare studier visar att det eventuellt kan finnas en risk att patienter som står på lång opioidbehandling visar tecken på nedsatt funktion i inlärning och minne. Detta är idag inte helt klarlagt utan verkar uppstå i specifika individer. Det finns teorier om att denna nedsatta kognitiva förmåga orsakad av opioider är relaterat till ökad celldöd, nedsatt tillväxt och förändrade volymer i vissa regioner i hjärnan. Denna avhandling har haft som mål att undersöka om opioider kan orsaka skador på celler samt att undersöka om tillväxthormon kan agera skyddande eller läkande på cellulära funktioner. För att uppnå dessa mål användes nerv- cellskulturer av olika slag. Med försöken som bedrevs visade det sig att opioider, speciellt metadon och fentanyl, var särskilt skadande på celler. Cellernas funktion och stabilitet minskade drastiskt vid exponering av dessa opioider. Fortsatta studier visade att om tillväxthormon gavs samtidigt som metadon så minskade skadorna orsakade av opioiden. Vidare undersöktes om tillväxthor- mon kunde reparera de skador som orsakats av metadon. De skador som metadon inducerade under ett dygn behandlades sedan med tillväxthormon och det visade sig att cellernas funktion förbättrades avsevärt. Slutligen undersök- tes om tillväxthormon hade någon funktion i att öka antalet dendritiska utskott, som spelar stor roll vid inlärning och minne. Dessa försök visade att tillväxt- hormon faktiskt ökade dessa utskott och förstärker teorin om att detta hormon kan användas som behandling för nedsatt kognitiv funktion. Sammanfattningsvis visar denna avhandling att opioider såsom metadon och fentanyl har en negativ påverkan på cellers funktion och utseende. Detta kan förklara de negativa effekter som opioder har på kognitiva funktioner, exempelvis inlärning och minne. Uppkomsten av dessa effekter minskar om till- växthormon ges samtidigt som opioider, där dessutom cellfunktionen förbätt- rades vid fortsatt behandling av hormonet. Slutligen visar denna avhandling att tillväxthormon ökar antalet dendritiska utskott vilket har betydelse vid in- lärning och minne. Det sammanfattade resultaten i denna avhandling styrker teorin om att tillväxthormon kan agera skyddande, läkande och att hormonet

48 skulle kunna appliceras som en behandling för sjukdomar som försämrar kognitiva funktioner, såsom inlärning och minne.

49 Acknowledgement

The work described in this thesis was carried out at the Department of Phar- maceutical Biosciences and was supported by Kjell and Märta Beijer founda- tions, the Swedish Brain Foundation as well as the Swedish Research council. Special thanks to Apotekarsocieten and Smålands Nation for providing me with scholarships to travel to international conferences. This thesis could not have been produced without the wonderful help and support I have received during my time as a PhD student. To my main supervisor, Professor Mathias Hallberg; thank you for your endless support and for having me as your PhD student. We’ve had a great synergy during my time in our lab group and I’m thankful for you being there, answering all my questions and dealing with my ideas of new experiments. My co-supervisor, Dr. Alfhild Grönbladh, for being an excellent supervisor and researcher throughout my PhD studies. For our many discussions about growth hormone, experiments, but also all the other important off-topic chats. Sometimes, I cannot grasp how you almost always provide me with a satisfy- ing answer to all my concerns. The senior professor in our group, Fred Nyberg, for enlighten me with your experiences and all the good feedback I’ve received for all the papers and manuscript as well as the discussions regarding growth hormone and opioids. The rest of our lab group, Sofia, my fellow PhD colleague, thank you for being a wonderful roommate and for all our discussions in the office, whether it was about statistics our me trying to convince you to start playing video games. Although my attempts failed, I still have fond memories of the good times we’ve had in the lab and on our journeys abroad to international conferences. Past and present members of our group; Anna J, Frida S, and John, thank you for our interesting discussions, small talks, and helping building a wonderful atmosphere in our group. Erika, for good talks about our kids, PhD life, or interesting discussion regarding growth hormone. My co-author Lenka, for our great collaborations regarding opioids. For our former post-docs, Shanti, wow, you taught me all there is to know about cell culturing, lab- works, and many things more. I will never forget all the laughs we had in the lab or during our confocal microscopy sessions. Anna L, for all the good times in the lab and interesting discussion about everything from Harry Potter to SNI experiments. You guys are surely missed in the group! For all the past and present PhD students at FarmBio for making the PhD life fun and stimulating! To Nikita, Linnea, Shima, Sara, Fadi, Erik,

50 Theodosia, Elva, Emilia, Rekha, and Lisa for nice chats. Special thanks to Stina, for our great times during my time here and also during the two courses we went together (visualize your science and the long ‘introduction to PhD studies’). Patrik, for our great talks about basically everything and for always bringing a good mood to the lunch room. To all my past and present colleagues at Farmbio, thank you for providing me with a stimulating and fun work environment. Special thanks to Björn, Ola S, Tanya, Polina, Rein and Wes for our great discussions associated with Im- ageXpress. Magnus, for all the support and help with the technical things, Lena, for your help with basically everything involving labwork. Marina, Hannah, Mathias Ö, Elisabeth, Martin aka ‘the core of Farmbio’, for making my PhD life much easier and fluent. To all our master students I have unoffi- cially supervised. Thank you all for bringing joy to our little lab group.

To all my friends and loved ones outside of BMC

My family, Frida, for being the best wife ever existed and for your endless love and support! I love you! My kids, Gustav and Ebba, for bringing another perspective of life outside BMC. I have always loved coming home to you, shifting the thoughts from trying to understand how the brain works to talking about Star Wars or unicorns. Mum (Ingrid) and Dad (Olle), it turned out that I became just like you, with that scientific spark of interest. I cannot explain how much you mean to me. For being the best mum and dad, for taking care of the kids when me Frida needed it the most, and for your endless support. My little sister Karin, even though I’m older and muuuch wiser ☺, I still look up to you and sometimes wish I had the same dedication and positiveness you always bring. And, of course, that we still share these silly jokes we’ve had since childhood that no- body understands. Susan and Jan-Olof, for helping out with the kids and your helpfulness with all the practical things that both me and Frida really have appreciated! And for your kind hospitality during our lovely weeks up in Gvorrskäret every sum- mer. Gabriel and Leo, for reminding me about the life outside BMC with all our gaming experiences during the late evenings. Whether it is climbing the ranks in Apex, getting demolished by the young gods in Fortnite, or scoring those aerials in Rocket League. That reality escape has been important and hope our cozy gaming sessions never ends.

Erik Uppsala, November 2019

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Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 279 Editor: The Dean of the Faculty of Pharmacy

A doctoral dissertation from the Faculty of Pharmacy, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy”.)

ACTA UNIVERSITATIS UPSALIENSIS Distribution: publications.uu.se UPPSALA urn:nbn:se:uu:diva-393940 2019