Protective Capabilities of Allopregnanolone Against Induced Toxicity in SH-SY5Y Cells Relative to Alzheimer´S Disease

VT 2020

Protective capabilities of allopregnanolone against induced toxicity in SH-SY5Y cells relative to Alzheimer´s disease

Mohamed Mustafa

Degree Project in Pharmaceutical pharmacology, 30 hp, Spring semester 2020

Examiner: Mathias Hallberg

Department of Pharmaceutical Biosciences Division for Pharmacology Faculty of Pharmacy Uppsala University

Abstract

When the brain is exposed to a traumatic injury, the brain produces high amounts of neurosteroids like allopregnanolone and progesterone which show protective and neurogenic capacities. Alzheimer’s disease patients also have lower amounts of these neurosteroids in brain tissue. Neurosteroids act on GABAA receptors and cholesterol receptors which is interesting since both the cholesterol transporter ApoE and excitotoxicity seems to be issues plaguing the patients. To study if there is a relationship between Alzheimer’s disease and neurosteroids, there are ongoing phase one studies but neurobiological studies are equally important in order to understand the mechanism. In this work protective capabilities of allopregnanolone on induced toxicity was investigated in human neuroblastoma SH-SY5Y cells. Protection and induced toxicity were assessed by studying cell viability with MTT assay. Toxins used were the oxidative stress inducing agent t-BHP, excitotoxic glutamate and amyloid β25-35. Previous studies have found allopregnanolone to induce neurogenesis, decrease ROS levels, inhibit apoptosis and to have immunoregulatory capabilities. The present study did see an increase in cell viability when treated to 1x10-8 M allopregnanolone but this effect was not observed when the concentration was increased further to 1x10-7 M and 1x10-6 M. When the SH-SY5Y cells were treated with toxins after pretreatment of allopregnanolone, additional decrease was seen when compared to cells only treated with toxins. The present study discovered the influence of components like cell density and cell generation which is of value for researchers planning future neurobiological studies. These neurobiological studies give insight of the correct mechanisms in the brain, opening up opportunities for new efficient drugs to be developed.

Contents ABSTRACT ...... 2 INTRODUCTION ...... 4 ALZHEIMER’S DISEASE ...... 4 NEUROSTEROIDS ...... 5 ALLOPREGNANOLONE’S MECHANISM OF ACTION ...... 6 ALLOPREGNANOLONE IN CLINICAL TRIALS ...... 7 AIM ...... 7 MATERIALS AND METHODS ...... 8 RESULTS ...... 9 DISCUSSION ...... 13 CONCLUSION ...... 16 SVENSK POPULÄRVETENSKAPLIG TEXT ...... 17 REFERENCES ...... 18

Introduction In the event of a traumatic brain injury, an induced physiological shift is observed. In mice, the neurosteroid progesterone and its metabolite allopregnanolone which is also a neurosteroid, were significantly higher in brain tissue after injury (Lopez-Rodriguez et al., 2016). Interestingly, an increase of neurosteroids is attributed to neuroprotection as a higher degree of neuroprotection in pregnant female rats versus male rats with lower hormone levels could be detected in a previous study (Meffre et al., 2007). The natural increase of progesterone and allopregnanolone in pregnant rats is believed to have granted protective and rehabilitation properties for brain tissue. This coupled with the fact that a decrease in neurosteroids have been spotted in Alzheimer’s mice raises the question of the possible relationship of neurosteroids, neuroprotection and Alzheimer’s disease (Naylor et al., 2010). Alzheimer’s disease Alzheimer’s disease is characterized by dementia caused partly by a disrupted production and clearance ratio of amyloid proteins. The unsolvable plaques of amyloid β act as reservoirs for soluble oligomers which are believed to be the actual harmful perpetrator. Amyloid β with 40 and 42 amino acids are the main components of amyloid plaques (Ibanez et al., 2004). The exact mechanism of toxicity caused by amyloid β peptides is disputed but some pathways have been discovered. It has been seen that amyloid β oligomers disrupts the cell-membrane by producing breaches in it (Kayed and Lasagna-Reeves, 2013). Others have come to the conclusion that it is in fact through activating NDMA-receptors the harm is done by decreasing long-term-potentiation (LTP) (Li et al., 2011). A third possible pathway has been found as well where amyloid β oligomers affect depolarization through Ca2+-channels which leads to excitotoxicity and sensitization of the mitochondrial membrane and finally apoptosis (Morkuniene et al., 2015). Apolipoprotein E (ApoE), a cholesterol distribution protein, have shown to have an effect on the levels of amyloid β. There is a polymorphism in the gene encoding the protein, 13.7% having the ApoE 3 allele, 77.9%, the ApoE 4 allele and the rest of the population the ApoE 2 allele (Farrer et al., 1997). Carrying the ApoE 4 allele have shown to come with greater risk for Alzheimer’s disease (Corder et al., 1993). The exact mechanism is not fully understood but ApoE 4 carriers suffer from greater amyloid β secretion by neurons to the interstitial space (Schmechel et al., 1993) and simultaneously decreased amyloid β clearance by microglia versus ApoE 3 carriers (Lin et al., 2018). Alzheimer’s disease and other dementias are currently impacting 50 million people worldwide and the prevalence is estimated to grow. Alzheimer’s disease is a rapidly

growing problem that more and more countries are in impinged by as the average life expectancy is increasing. The illness is costing governments 818 billion USD each year which is 1% of the total global GDP (Alzheimer’s Disease International, 2015). Even though the illness causes such a dent on the global economy and healthcare system, there are currently only treatments relieving symptoms. Galantamine and memantine are two commonly prescribed treatments. Galantamine contributes to better cognitive functions and memory by acting as an acetylcholinesterase inhibitor (Greenblatt et al., 1999). As acetylcholinesterase lowers the amount of acetylcholine in the brain, an inhibitor such as galantamine raises the brain levels of acetylcholine. This makes sense as a treatment as Alzheimer’s patient commonly suffer from degradation of the cholinergic pathways of the brain. The other neurochemical treatment, memantine which inhibits N-metyl-D-aspartate (NMDA) receptors instead, has also shown to been symptom relieving (Epperly et al., 2017). None of these drugs significantly slow down the progression of the disease on a reliable scale. Beta-secretase (BASE) inhibitors, were once promising prospects due to the fact that they limit the production of amyloid β (Chatila et al., 2018). But recently many studies have detected too severe adverse effect like suicidal ideations and even worsening the progression of the disease (Egan et al., 2019). Today the vision of a BASE-inhibitor as an Alzheimer’s disease treatment is diminishing. Neurosteroids The classification of neurosteroids embark every steroid that is synthesized in the brain. There is a large variety of neurosteroids with a variety of mechanism of actions. Some neurosteroids like DHEA-S have shown to have an impact on dendrite length but have yet to have an established mechanism of action known (Krug et al., 2009). Progesterone is a pleiotropic steroid and can act through many pathways like progesterone receptor (PR) (Kastner et al., 1990) and membrane progesterone receptors (mPRs) (Zhu et al., 2003). PR is a steroid nuclear receptor which acts through genome manipulation as it is a transcriptional factor. Allopregnanolone on the other hand do not act as a traditional steroid. It is a metabolite of progesterone but can act through ionotropic gamma-aminobutyric acid (GABA) receptors on the cell membrane (Gee et al., 1988). PR-/- and PR+/- knock-out mice had noticeably lower protection versus PR+/+ mice against stroke damage, which hints at progesterone offering protection through the PR receptor which allopregnanolone does not affect (Liu et al., 2012). There is contrary belief that the main protective capacity of progesterone is due to the conversion to allopregnanolone as allopregnanolone have shown to be more effective than

progesterone (Sayeed et al., 2006). This is supported by allopregnanolone’s many protective roles against neuroinflammation (Noorbakhsh et al., 2011) , apoptosis (Sayeed et al., 2009) and also induces neurogenesis (Wang et al., 2005). Allopregnanolone’s mechanism of action The many different mechanisms of action asserted by allopregnanolone is expected since it is a pleiotropic substance with many effects on the brain, some mentioned above. There are three main established mechanisms of action that allopregnanolone has been assigned; acting through GABAA receptors, cholesterol receptors (Chen et al., 2011) and mPR (Kelder et al., 2010). Acting through glycine receptors have also been suggested (Chesnoy-Marchais, 2009), but this is not yet an widely endorsed view. GABA receptors are a major tool for the body to exert inhibitory imperative.

There are many ways of subdividing GABAA receptors as there are different GABAA receptors prominent in different brain regions, cells or even location on the cell.

Allopregnanolone can act both as a ligand for ionotropic GABAA receptors (similarly to natural GABA) but also as an allosteric ligand (potentiator) for GABAA receptors, increasing the effect of GABA (similarly as benzodiazepines) (Gee et al., 1988)(Hosie et al., 2006). The receptors allopregnanolone acts on can be either synaptic or extra-synaptic GABAA receptors. Synaptic receptors induce fast polarizations of the receiving cell which results in phasic inhibition in which studies have shown allopregnanolone to prolong the inhibition duration (Haage and Johansson, 1999). The receptor can also be localized extra-synaptic where it is usually highly sensitive to low levels of ambient GABA and can be potentiated further with allopregnanolone (Stell et al., 2003). This kind of receptor activation leads to tonic activation of the cell which have shown to have an influence on cell-cycle, inducing proliferation and neurogenesis (Liu et al., 2005). As tonic inhibition lasts for longer duration (slower desensitization) than synaptic inhibition (Bianchi et al., 2001), its inhibitory capability may have an important role for excitatory-inhibitory homeostasis (Vogels and Abbott, 2009). It is common of the extra-synaptic receptors to include the neurosteroid sensitive δ-subunit (Wohlfarth et al., 2002). It is known that neurosteroids affect different subunit compositions differently (Belelli et al., 2002). The α6 subunit increases the sensitivity of allopregnanolone dramatically while none of the β subunits seem to not have an influence. The γ subunit was not required for allopregnanolone activity and is also believed to decrease the neurosteroid sensitivity.

Liver X receptors (LXR) and progesterone X receptors (PXR) are integral parts of the cholesterol homeostasis. The essential ApoE which facilitates the relocation of cholesterol throughout the body is manipulated with the help of LXR ligands which act as a transcriptional factor, inducing synthesis (Liang et al., 2004). The similar PXR receptor which also plays an critical role in cholesterol homeostasis (Guo et al., 2003), have shown to protect neurons from inflammation (Langmade et al., 2006). Allopregnanolone have shown to act as an ligand for both LXR and PXR and affecting cholesterol levels in the brain (Chen et al., 2011). The mPR is one of the paths that progesterone is usually known for exerting its many effects. It is a membrane receptor which induces a rapid inhibitory change on cellular cyclic adenosine monophosphate (cAMP) levels (Sleiter et al., 2009). This in turn leads to an increase in proliferation as the decrease in cAMP signals anti-apoptotic effects (Dressing et al., 2010). mPRα is a progesterone receptor but it maintains 7.6% of the binding affinity with allopregnanolone (Kelder et al., 2010). This could explain some of the effects allopregnanolone maintains even with the lack of GABAA receptors. Allopregnanolone in clinical trials When doing research on neurosteroids, Dr. Roberta Brinton is a name that is impossible to not come across. Brinton has been paving the way since the start of the twenty-first century by arguing for the possibility of neurogenesis through neurobiological studies (Wang et al., 2005), preclinical animal studies (Chen et al., 2011)(Singh et al., 2012)(Wang et al., 2020) and now clinical phase 1 studies. Brinton completed a randomized clinical trial in 2018 treating 24 participants with allopregnanolone to uncover which intravenous doses are relevant for Alzheimer’s disease patients (NCT02221622). Estimated to end in late 2020, an intramuscular clinical study is also performed by Brinton (NCT03748303). But even with these clinical trials, neurobiological studies which study the physiological mechanism of neurosteroids are still equally important to understand the correct mechanism in order to develop an efficient drug.

Aim The aim of the present study was to examine protective capabilities of allopregnanolone on induced toxicity in human neuroblastoma SH-SY5Y cells.

Materials and methods Materials. 3-4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), tert-butyl hydroperoxide(t-BHP), glutamate and amyloid β25-35 were purchased from Sigma-Aldrich, Sweden AB. Cell culture. The human neuroblastoma SH-SY5Y cell line was obtained from American Type Culture Collection, Manassas, USA and were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 100 units of penicillin/streptomycin. These were obtained from Invitrogen, Life Technology. Trypsin used for detachment of cells were obtained from Invitrogen, Life Technology. MTT assay. The cell viability was studied by measuring the amount of MTT broken down by mitochondrial dehydrogenases to formazon (Präbst et al., 2017). Cells were cultured in 96- well Sarstedt plates. A countess II automated cell counter was used to obtain the preferred density of cells in each well. The day after the transfer to the 96-well plates, the cells were treated with different agents. The following day after the treatment, the treatment’s effect on cell viability was studied by draining the supernatant of the wells and adding 100 µl of 1 mg/ml MTT in DMEM solution. After the cells were exposed to MTT for 30 minutes, the supernatant was drained again and refilled with 100 µl DMSO. The formazan created, dyed the solution in the wells purple. The change in color by the formazon was measured at the absorbance of 570 nm using a FLUOstar OMEGA microplate reader. Statistical analysis. Statistical significance of differences between treated cells and non- treated control cells were confirmed using the Student’s t-test. P-values for determining the significance of the differences were: *P<0.05, **P<0.01 and ***P<0.001.

Results Dose-response relationship. To find a viable concentration of t-BHP that induces 60% cell viability, a dose-response study was made. In Figure 1, it is shown how the concentration of t-BHP and cell viability has a dose-response relationship as the cell viability drops with higher concentrations of t-BHP. The toxicity of t-BHP was studied by inflicting SH-SY5Y cells with concentrations of t-BHP ranging from 1x10-6 to 1x10-3 M. It can be seen in Figure 1 that 2x10-5M t-BHP induced a decrease in cell viability to the desired amount, 60% (P<0.001).

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Figure 1. Toxicity effects of 24 hours treatment t-BHP on SH-SY5Y cells (generation P21) in a rising concentration gradient. The cell viability of the cells treated with t-BHP were reported in percentages in relationship to the non-treated control cells “- “. Data from the dose-response curve are means ± SEM of 8-24 wells in one to three 96-well plate. The cell viability of the cells was measured with MTT assay. The probability of the toxicity induced difference of cell viability versus the control cells beings significant was determined with Student’s t-test (**P<0.01 and *** P<0.001).

Cell density study. To investigate the appropriate cell density to use in further treatments, two different cell densities were treated with the same concentration of t-BHP. A concentration of t-BHP (5x10-5M) that was chosen induced at least 60% cell viability in the previous studies. The selected concentration of t-BHP was used to perform two new plates, one with 30 000 cells per well and one with 20 000 cells per well. To have the exact same conditions and amount of plates studied, the cell density data were analyzed independently from data obtained from previous and latter cell culture generations. As seen in Figure 2, t-BHP at 5x10-5 M induced only 14% decrease in cell viability versus the non-treated control cell when added to the cell culture of 30 000 cells per well. The same concentration of t-BHP induced a 44% (P<0.001) decrease when added to the 20 000 cells per well cell culture. The lesser cell density of 20 000 cells per well resulted in the cell culture to be more vulnerable to the toxicity of t-BHP than the higher cell density of 30 000 cells per well.

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0 - t-BHP - t-BHP Cells per well 30 000 cells 20 000 cells Figure 2. Toxicity effects of 24 hours treatment of 5x10-5M t-BHP on SH-SY5Y cell cultures (generation P22) with the cell density of 30 000 and 20 000 cells per well. The cell viability of the cells treated with t-BHP were reported in percentages in relationship to the non-treated control cells “- “. Data from each density study are means ± SEM of 8 wells in one 96-well plate. The cell viability of the cells was measured with MTT assay. The probability of the toxicity induced difference of cell viability versus the control cells beings significant was determined with Student’s t-test (*** P<0.001).

Treatment of allopregnanolone. For the purpose of establishing the effect of allopregnanolone alone on the cell viability, SH-SY5Y cells were treated with allopregnanolone. The concentration used to treat the cell cultures were increased in a manner of increasing concentrations of a factor of 10. It can be seen in Figure 3 that allopregnanolone in a concentration of 1x10-8 M significantly increased the cell viability with 14% (P<0,001) compared with non-treated cells. But the protective effects did not correlate to the concentration allopregnanolone. Further increase of allopregnanolone concentrations did not affect the cell viability compared to non-treated cells.

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Figure 3. Effects of 24 hours treatment of a neurosteroid, allopregnanolone on SH-SY5Y cell cultures. A rising concentration gradient of allopregnanolone from 1x10-8 to 1x10-6M were applied cell cultures with a cell density of 20 000 cells per well. The cell viability of the cells treated with allopregnanolone were reported in percentages in relationship to the non-treated control cells “- “. Data are means ± SEM of 72 wells from nine 96-well plates. The cell viability of the cells was measured with MTT assay. The probability of the protection induced difference of cell viability versus the control cells beings significant was determined with Student’s t-test (*** P<0.001).

Effects of allopregnanolone treatment before induced toxicity. Treatment of SH-SY5Y cells with 5x10-5 M t-BHP and 6x10-2 M glutamate induced a significant decrease in cell viability to 74% (P<0.05) and 69% (P<0.05) respectively (Figure 4a). No toxicity was inflicted by amyloid β25-35. The cell viability increased instead to 112%, although not significant. t-BHP and glutamate induced a more adequate decrease in cell viability than amyloid β25-35. To further investigate the protective effects of allopregnanolone, three different toxic insults were applied 10 minutes after the allopregnanolone treatment. The different toxic insults used were the mentioned above: the oxidative agent t-BHP, the excitotoxic agent glutamate and amyloid β25-35 peptide. This investigation provides more insight to what kind of toxicity allopregnanolone can protect against, if any. As demonstrated in Figure 4 b), c) and d), no substantial protection from allopregnanolone was observed in cells induced to any toxicity. For t-BHP and glutamate, allopregnanolone did more harm than good. Nothing can be said of the protective capabilities of allopregnanolone against amyloid β25-35 toxicity, since amyloid β25-35 alone did not induce any toxicity (Figure 4a and d).

Figure 4. a): Effects of 24 hours treatment of t-BHP (5x10-5 M), glutamate (6x10-2 M) and -5 amyloid β25-35 peptides (1x10 M) on 20 000 cells/well SH-SY5Y cell culture. The cell viability

of the cells treated with the toxic substances were reported in percentages in relationship to the non-treated control cells “- “. b), c) and d): Effects of 24 hours treatment of a neurosteroid, allopregnanolone on SH-SY5Y cell cultures together with a constant concentration of t-BHP (5x10-5), glutamate -2 -5 (6x10 ) or amyloid β25-35 peptides (1x10 M). A rising concentration gradient of allopregnanolone from 1x10-8 to 1x10-6M were applied cell cultures with a cell density of 20 000 cells per well 10 minutes prior to the application of the toxic substance. The cell viability of the cells treated with allopregnanolone and t-BHP together were reported in percentages in relationship to the control cells “Tox “. Control cells were only subjected to t- BHP with the absence of allopregnanolone treatment. Data from each toxic substance are means ± SEM of 8-16 wells in one to two 96-well plate. The cell viability of the cells was measured with MTT assay. The probability of the effect induced difference of cell viability versus the control cells beings significant was determined with Student’s t-test (** P<0.01).

Discussion Several previous studies have seen various kinds of neuroprotective capabilities of allopregnanolone. Allopregnanolone have shown to not only prevent cell death but also reduce inflammation and protect cell against oxidative stress. For example, allopregnanolone reduced swelling of brain mitochondria caused by excessive influx of Ca2+ which in turn lead to a decline in apoptosis through a decrease of the apoptosis promoter factors like cytochrome c and caspace-3(Sayeed et al., 2009). Through myelin preservation and lowering the amount of CD3ε (an integral factor for T-cell activation), allopregnanolone reduced neuroinflammation (Noorbakhsh et al., 2011). Ganaxolone, an analog of allopregnanolone, have shown immunoregulation capabilities showing the importance of GABA receptors in decreasing neuroinflammation (Paul et al., 2014). Both human fibroblast cells and SH-SY5Y cells have seen benefit of allopregnanolone which showed to lower intracellular ROS levels which gave the neurosteroid its antioxidant effects (Zampieri et al., 2009). These scientific findings suggest that allopregnanolone would lead to a raise in cell viability in a neurobiological study. The present study although suggests something else. Allopregnanolone did raise the cell viability when applied to the SH-SY5Y cells alone but this effect did not persist when toxic substances were co-applied. Both glutamate and t-BHP reduced the cell viability close to 70%. Preapplication of allopregnanolone lowered the cell viability further, close to 60%. Although allopregnanolone herein showed some additional toxic properties in contrary to previous studies mentioned, the present study’s portrayed difference had no statistical significance by the Student’s t-test. As for the amyloid β25-35 induced toxicity study, there was not a sufficient enough toxicity which the allopregnanolone could protect against. This renders the capability of concluding that allopregnanolone did not protect against

amyloid β25-35 toxicity as there were none. Since none of toxicity induced studies had any statistical significance, there is of plenty room for improvement in the present study. The fact that 5x10-5 M t-BHP in the cell density study on generation P21 resulted in a less degree of cell damage than the initial dose-response relationship suggests the cell generation being significance. The SH-SY5Y cells might have underwent changes as tumorous cells are prone to not ensure consistency. A change in the latter cell generation could have happened in which the cells gained protecting against t-BHP. Another reason that might have tainted the results of the present study is lack of statistical power due to the low amount of plates studied. The cell density of the SH-SY5Y cells in the wells showed to have a major impact on the cell viability. The same concentration induced two vastly different drops in cell viability. With even the cell generation being the same and only the cell density being a difference, from 30 000 cells per well to 20 000 cells per well. The more vulnerable cells in the less dense cell culture of 20 000 cells per well had more of an impact from the same concentration of t-BHP. This difference of vulnerability might be explained by the fact of cell to cell communication being impinged on with lesser cell density which affects the cells means of self-protection. It can be seen that the effects of allopregnanolone tapers of as with raising of the concentration. This is expected, allopregnanolone and other neurosteroids have shown to have a U-shaped influence of neurogenesis on cells. The concentrations of 1x10-9 and 1x10-7 M have been seen to be optimal for inducing neurogenesis. The effects of allopregnanolone have shown to diminish at concentrations 1x10-6 M and more (Wang et al., 2005). This is also coherent with the highest normal serum concentration of allopregnanolone in humans. During the third trimester of pregnancy, human serum concentrations of allopregnanolone are elevated to close to 1x10-7 M (Luisi et al., 2000). Three different toxic substances were chosen in this present study to achieve a more complex picture of the strengths and weaknesses of the protective capabilities of allopregnanolone. t-BHP is an organic reactive oxidative species (ROS) inducing agent useful for studying oxidative stress on cell lines. It is comparable to using H2O2 but it is believed that t-BHP exerts its oxidative harm in a more complex manner by also inducing superoxide-like radicals which H2O2 does not (Slamenova et al., 2013). Analyzing protection against an oxidative agent is probably important in an Alzheimer’s disease neurobiology study since research points at the importance of antioxidants against Alzheimer’s disease progression (Halliwell, 2001). The soon to be mentioned toxic agent, glutamate, also eventually induces oxidative stress (Lafon-Cazal et al., 1993). The difference between glutamate and t-BHP is

that glutamate initially cause excitotoxicity which eventually cripples the cell’s normal redox function and t-BHP being a more direct oxidative agent. Amyloid β peptides have an even more complex way of inducing toxicity. Previously stated in the introduction, amyloid β’s toxicity has many different explanations. One is the potential damage it can do on sensitizing cell’s and then leading excitotoxicity similarly to glutamate. The present study’s research on amyloid β toxicity were performed on the 25-35 fragment of the peptide. This segment of the peptide have shown to be its most toxic part and there is research that the whole 1-40 or 1-42 peptides are not needed to exert toxicity and decrease in cell viability in concentration of -5 1x10 M (Ueda et al., 1997). Since the concentration of amyloid β25-35 in mentioned study and the present study are the same, this suggests that there were some other issue leading to the lack decrease of cell viability in the present study. Although the mentioned study exposed its cell culture of amyloid β25-35 for 24 hours more than the present study, the decrease in cell viability were too different to only be explained by the exposure duration. Other studies on SH-SY5Y cells achieved a decrease to 70% cell viability with 1x10-5M with the same exposure duration (24h) as the present study (Zheng et al., 2014). Previous studies have achieved vastly different results when inducing amyloid β25-35 compared to the present study’s issues with the matter. This together with low amount of plates studied, there might have been a human error in performing that study in the present study. The conclusions about cell viability, induced toxicity and protection were only based on the results from MTT in the present study. The decrease in cells would lead to a lower number of mitochondria and therefore lower MTT results. But in order to gain a more multidimensional view of the issue, a direct measurement on ROS levels in cells and caspase- 3 activity would be useful for future studies. ROS levels are a more direct way of studying the oxidative stress on a cell and caspase-3 activity can tell more about the inducement of apoptosis when allopregnanolone is applied. But as Alzheimer’s disease is believed to affect mitochondria as mentioned in the introduction and allopregnanolone have shown to protect mitochondria (Sayeed et al., 2009), MTT is an obvious choice to include in the list of measurements of cell viability but it should not have been the only one on said list. Using SH-SY5Y cells comes with some advantages but also some disadvantages. SH-SY5Y cells are easier to gain access to versus non-tumorous human cells which bring on an ethics issue to the study. SH-SY5Y cells have the benefit of being a human neuron-like cell line which is easier to handle and grow versus animal primary cells which do not express human proteins. As previously mentioned, the risk of the cells developing different properties each generation is a factor to consider as the SH-SY5Y cells are tumor

cells. The present study used undifferentiated SH-SY5Y but if the study proceeds to study neurosteroids more in the future, a differentiation will be considered. SH-SY5Y can be differentiated to more neuron-like cells as well as cholinergic (Adem et al., 1987) which is relevant to Alzheimer’s disease studies due to the loss of the cholinergic cells being a prominent issue. Conclusion In summary, allopregnanolone did induce an increase in cell viability which can be either attributed to proliferation, neurogenesis or both. It cannot be concluded that it does or does not protect against induced damage from t-BHP, glutamate or amyloid β25-35 as no result of any difference in cell viability were found with enough statistical significance. Although the study did not suffice in examining the neuroprotective capabilities of allopregnanolone, it did discover the influence of the components in the study. Future studies on the subject can consider these discoveries during the methodological segment. Discoveries in interest may be the great influence of cell density and cell generation. In many previous studies, the cell density of the 96 well plates are not even mentioned. This is important to take in account as these neurobiological studies are critical and efficient studies are needed to develop an efficient drug in the future.

Svensk populärvetenskaplig text Vanligtvis när en person halkar och slår sig i huvudet, utsöndrar kroppen neurosteroider, bland annat allopregnanolon. Även bland gravida kvinnor så finner man en ökning av dessa neurosteroider i hjärnan. Men bland patienter som lider av Alzheimers sjukdom så har man hittat en lägre nivå av dessa skyddande neurosteroider. Detta är väldigt intressant då neurosteroider har visats erbjuda en rad olika fördelar för hjärnan, inte minst skydd mot skador och förhindrandet av celldöd. Även en möjlighet av nybildning av celler med hjälp av neurosteroider diskuteras i forskningsvärlden. Då det i nuläget inte finns något läkemedel som visats bromsa utvecklingen av sjukdomen så är dessa neurosteroider spännande. En stor del av skador som Alzheimers sjukdom orsakar tros vara på grund av plack som bildas. Dessa plack orsakar en rad olika problem i hjärnan så som hål i mitokondrier (cellens kärnkraftverk) och en för hög aktivering av hjärnans celler som leder till att celler till slut dör. Alzheimers sjukdom är en av de vanligaste orsakerna för demens. Forskning på människor angående möjliga doser av allopregnanolon har precis börjat i USA och förväntas vara klara i slutet av 2020. Däremot så är det fortfarande viktigt med cellstudier för att förstå helheten bättre och hitta ett smart och effektivt sätt att utnyttja dessa fördelar. Den här studien har undersökt om neurosteroiden allopregnanolon kan skydda mot toxiska ämnen. När dessa ämnen valdes fanns det i åtanke vad som är relevant för Alzheimers sjukdom. Ämnet som placken består av, amyloid-beta, var bland dessa ämnen som testades men även andra ämnen som orsakar liknande skador valdes ut. Flera olika ämnen testades då amyloid-beta är känt för att inte alltid orsaka skador i cellstudier så som man förväntat sig. Precis detta skedde också i den här studien då det blev svårt att få amyloid- beta att orsaka skada bland cellerna. För att se hur allopregnanolon eller toxiska substanserna påverkat cellerna mättes cellerna ämnesomsättning genom att mäta aktiviteten av mitokondrier. En ökad överlevnad hittades bland celler när de behandlades med endast allopregnanolone men inte tillsammans med toxiska substanserna. Dock så är det viktigt att veta att de väldigt få tester med toxiska substanserna kunde göras under den här studien. Men vad man även såg var hur viktiga olika komponenter var i testerna. Celldensitet, något som i tidigare forskning lätt överses med, visade sig ha en väldigt stor inverkan. Oavsett resultat är dessa studier viktiga för att underlätta framtida forskning som kan leda till ett potentiellt läkemedel.

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