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. © Erik Nylander 2019 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 mem- brane 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 mito- chondrial morphology: a live cell imaging study. Submitted man- uscript. 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 hor- mone 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 aminopep- tidase (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 ad- diction potential of anabolic androgenic steroids and the effects of growth hor- mone. 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