Studies on the cysteine protease legumain in the central nervous system during neuroinflammation

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legumain in the central nervous system during neuroinflammation

Cathrine Lilleheil Christiansen

Master Thesis for the title of Master of Pharmacy

Section for Pharmacology and Pharmaceutical Biosciences

45 credits

Department of Pharmacy

The Faculty of Mathematics and Natural Sciences UNIVERSITY OF OSLO

June / 2021

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Studies on the cysteine protease legumain in the central nervous system during neuroinflammation

Cathrine Lilleheil Christiansen

Master Thesis for the title of Master of Pharmacy

Section for Pharmacology and Pharmaceutical Biosciences

45 credits

Department of Pharmacy

The Faculty of Mathematics and Natural Sciences UNIVERSITY OF OSLO

June / 2021

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IV

2021

Studies on the cysteine protease legumain in the CNS and under neuroinflammatory conditions

Cathrine Lilleheil Christiansen http://www.duo.uio.no/

Print: University Print Centre, University of Oslo

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V Lactate, a metabolite in cellular respiration, has shown to play an important role in brain angiogenesis and neurogenesis through activation of the lactate receptor HCA1. To examine the HCA1-dependent effects in the brain after stroke, we used a stroke model and measured stroke lesion volume. In total, 76 mice either HCA1 deficient (knockout; KO) or controls (wild-type; WT), were subjected to one of four treatments: high-intensity interval training (HIIT; WT n = 8, HCA1 KO n = 11), medium intensity interval training (MIIT; WT n = 7, HCA1 KO n = 8), lactate injections (WT n = 9, HCA1 KO n = 9), or saline injections (WT n = 12, HCA1 KO n = 12) 5 days a week for 7 weeks. Results from the stroke volume

measurements showed no statistically significant difference between the groups (one-way ANOVA; p = 0.984, α = 0.05).

Preliminary data from the group has shown upregulation of legumain, a cysteine protease reported to be involved in several pathological conditions involving inflammation, in cells close to stroke lesion in a stroke model. Since this upregulation was consistent with

upregulation in response to neuroinflammation, we investigated another disease model where neuroinflammation is a key characteristic: the 5xFAD mouse model of Alzheimer's disease (AD). Immunohistochemistry (IHC) was performed on brain tissue from 5xFAD mice (n = 6) and control mice (WT n = 4) using a marker for legumain, astrocytes (glial fibrillary acidic protein, GFAP) and cell nuclei (4′,6-diamidino-2-phenylindole, DAPI). Results from IHC showed colocalization between the astrocyte- and legumain markers in nearly all astrocytes, with legumain staining strongest in the soma of the astrocytes. These findings suggest an upregulation of legumain in the hippocampus in AD-affected brain tissues, with the most increased staining in stratum oriens of the hippocampus. The same tendency was not seen in WT mice (Fisher Exact test, p = 0.048). GFAP-negative staining of legumain was observed in the CA3 region of stratum pyramidale of the hippocampus, cortex, and choroid plexus.

To investigate secretion of legumain from different neuronal cells, fibroblast skin cells and embryos were differentiated into microglial and neuronal cells. The cultured media from the cells were harvested and analysed for legumain concentrations by enzyme-linked

immunosorbent assay (ELISA). Conditioned media contain all extracellularly secreted proteins. Microglial cells differentiated from fibroblast skin cells from four different individuals were cultivated for 7, 8 and 12 days. Neuronal cells were differentiated from

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VI human pluripotent stem cells from embryos for 4-6 weeks; followed by cultivation for 7-8 days. The cultures contained mainly neurons and some immature astrocytes. Results showed secretion of legumain from both microglial cells (mean ± SD; 1.467 ± 1.013 ng/ml) and neurons and astrocytes (0.484 ± 0.224 ng/ml).

Both exercise and ischemic stroke are conditions related to inflammation. Previous results from the ProTarg research group indicate an increase in legumain in thrombus material in patients with acute ischemic stroke. Exercise is one of the best preventative strategies to avoid stroke. We wanted to investigate how the plasma level of legumain is affected by these two conditions. Plasma samples from stroke patients hospitalized with acute ischemic stroke (n = 42) were harvested within 24 hours of admission. Healthy, elderly people (n = 57) were used as controls, and blood samples from these participants were sampled both with participants resting, immediately after, and 30 minutes after HIIT. Samples were analysed by ELISA.

Results showed a high degree of variation with no statistical difference in plasma levels of legumain between resting levels of healthy participants (mean ± SD; 3.644 ± 1.480 ng/ml) and stroke patients within 24 hours of hospitalization (3.413 ± 1.471 ng/ml) (p = 0.473;

student’s t-test). The HIIT exercise regime had no measured effect on plasma legumain levels (p = 0.2683; repeated measures ANOVA).

The results in this thesis increase our knowledge about legumain in the CNS and under AD, ischemic stroke and exercise.

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VII

Acknowledgement

The work presented in this thesis was performed as a part of a collaboration between research groups ProTarg (Proteolytic Enzymes as Drug Targets) and the Neurobiology and Toxicology group at the Section for Pharmacology and Pharmaceutical Biosciences, Department of Pharmacy, University of Oslo.

First, I want to show my appreciation towards both research groups for letting me be a part of two impressive research environments. It has been educational to work alongside you and I particularly want to thank my supervisors associate professor Cecilie Morland, doctoral candidate Alena Hadzic, professor Rigmor Solberg, and postdoctoral fellow Ngoc Nguyen Lunde. Your contributions and guidance have been appreciated.

I also want to thank everyone that has contributed to the samples and tissues used in this thesis. Without their contribution, this thesis would not be half as interesting.

This thesis would not have been possible without the constant support from friends and family, thank you for always believing in me and encouraging me when I needed an extra push. Lastly, I want to thank myself for all the hard work and many hours I have put into this work, and for not giving up when the work was at its hardest. I finally made it.

Cathrine Lilleheil Christiansen Oslo, June 2021

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Abbreviations

Aβ Amyloid-beta

AD Alzheimer’s disease

AEP Asparaginyl endopeptidase

APP Amyloid precursor protein

BASE1 Beta-site APP cleaving enzyme 1

BBB Blood-brain barrier

Bp Base pair

BSA Bovine serum albumin

CA Capture antibody

CA3 Cornu ammonis 3

CAA Cerebral amyloid angiopathy

CNS Central nervous system

CSF Cerebrospinal fluid

CV Cresyl violet

DA Detection antibody

DAMP Damage associated molecular patterns

DAPI 4,6-diamidino-2-phenylindole

dMCAO Distal middle cerebral artery occlusion

ELISA Enzyme-linked immunosorbent assay

GFAP Glial fibrillary acid protein

HCA1 Hydroxycarboxylic acid receptor 1

HIIT High-intensity interval training

Iba1 Ionized calcium-binding adapter molecule 1

IgG Immunoglobulin G

IHC Immunohistochemistry

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i.p. Intraperitoneal

iPSC Induced pluripotent stem cells

KO Knock out

mAb Monoclonal antibody

MCAO Middle cerebral artery occlusion

MECT Maximal exercise capacity test

MIIT Medium intensity interval training

mRNA Messenger ribonucleic acid

NaPi Sodium phosphate

NCS New-born calf serum

NFT Neurofibrillary tangles

NPC Neural progenitor cells

pAb Polyclonal antibody

PBS Phosphate buffered saline

PBST PBS + 0.05 % Triton X-100

PFA Paraformaldehyde

PP2A Protein phosphatase-2A

RD Reagent Dilution

TIA Transient ischemic attack

tPA Tissue-type plasminogen activator

VRE Vacuolar Repressing Enzyme

WT Wild-type

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1 Introduction

1.1 Neuroinflammation

Neuroinflammation refers to inflammatory processes in the brain and spinal cord. In neuroinflammation, the immune system of the central nervous system (CNS), including microglia and astrocytes, is activated as a response to a disruption of the homeostasis of the brain (DiSabato et al., 2016). A myriad of trauma and diseases such as Alzheimer’s disease, multiple sclerosis and ischaemic strokes are directly or indirectly linked to neuroinflammation.

Research on this area can be a basis for developing new treatments and improving the life quality of people affected by these diseases and conditions.

Neuroinflammatory responses can be involved in beneficial processes, such as learning and increased plasticity, or harmful processes, such as tissue damage and cell death (DiSabato et al., 2016), and there is a close relationship and balance between these processes. More comprehensive responses due to highly increased activation of glial cells of the CNS (mainly microglia and astrocytes) and subsequent production of cytokines are linked to more negative outcomes. Stroke is an example of a condition that normally lead to an inflammatory response with highly increased activation of microglia and astrocytes (DiSabato et al., 2016). Multiple mediators are involved in these processes, such as cytokines, chemokines, and reactive oxygen species. Many of these mediators are produced by activated astrocytes and microglia (DiSabato et al., 2016).

In the case of CNS injury, such as ischemia, the initial inflammatory response is often beneficial but the secondary and more long-lasting inflammation is damaging (DiSabato et al., 2016). In addition to stroke and acute CNS injury, Alzheimer’s disease (AD) is a disease that is also characterised by chronic pathological neuroinflammation. Pathological neuroinflammation is often followed by increased inflammatory mediators, activation of immune cells, oedema, and possibly cell damage (DiSabato et al., 2016).

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1.2 Astrocytes

Astrocytes are a cell type that can be found throughout the entire CNS with a continuous organization in both grey and white matter. This specialized glial cell plays key roles in both healthy and damaged brain tissue (Sofroniew and Vinters, 2010, Kimelberg and Nedergaard, 2010). Astrocyte types differ between protoplasmic astrocytes and fibrous astrocytes, due to morphology and localisation in different types of brain tissue (Sofroniew and Vinters, 2010, Kimelberg and Nedergaard, 2010). Astrocytes are named after the Greek word for star, “astra”, because of their characteristic star-like shape with branches reaching out from the cell body (Kimelberg and Nedergaard, 2010). These branches are called processes, and the cell body is often referred to as the soma. Astrocytic processes have different functions, such as intercellular communication, homeostasis and providing nutrition from the circulation (Kimelberg and Nedergaard, 2010). Specialised processes called astrocytic endfeet cover the surface of CNS blood vessels (Sofroniew and Vinters, 2010, Mathiisen et al., 2010). Through the release of mediators such as prostaglandins, nitric oxide and arachidonic acid, astrocytes are able to regulate blood flow in regard to neuronal and synaptic activity (Sofroniew and Vinters, 2010, Kimelberg and Nedergaard, 2010, Koehler et al., 2009).

Astrocytes also play an important role in CNS homeostasis. Perisynaptic processes take up neurotransmitters such as glutamate and GABA from synapses and the astrocyte converts these signalling molecules back to their precursor form and prevents accumulation of glutamate and GABA in the synaptic cleft (Sofroniew and Vinters, 2010, Kimelberg and Nedergaard, 2010).

There are increasing evidence towards astrocytes being a substantial regulator of the blood- brain barrier (BBB), illustrated by the fact that astrocyte endfeet contributes to and enhances these barrier functions (Koehler et al., 2009, Kubotera et al., 2019). The BBB limits the transportation of possible neurotoxic and infectious agents from the bloodstream into the CNS (Zhao et al., 2015) and maintains homeostasis (Ikeshima-Kataoka and Yasui, 2016). Ikeshima- Kataoka & Yasui (2016) suggested that activation of astrocytes contributes to the restoration of the BBB after brain trauma (Ikeshima-Kataoka and Yasui, 2016)

An extensively used marker for astrocytes across species is the glial fibrillary acid protein (GFAP). GFAP is an intermediate filament protein that is essential for the function of activated astrocytes (Sofroniew and Vinters, 2010, Kimelberg and Nedergaard, 2010). GFAP is also

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3 found in “healthy”, nonreactive astrocytes, but at lower concentrations, and some healthy astrocytes do not express GFAP at all (Sofroniew and Vinters, 2010).

In mild astrocyte activation, there is an upregulation of GFAP. Mild activation can occur due to immune activation and at areas at a distance from larger trauma (Sofroniew and Vinters, 2010). If further activation occurs, astrocytes proliferate and increase in size, for example as a reaction to CNS trauma (Eng, 1985, Sofroniew and Vinters, 2010). When astrocytes are highly activated, GFAP is upregulated, cell bodies and processes increase in size, and proliferation is increased to a larger degree than previous levels. Additionally, there is a formation of glial scars which consist of a dense collagen matrix that inhibits migration of cells (Sofroniew and Vinters, 2010).

1.3 Microglia

Microglia is the main intrinsic immune cell of the brain and therefore contributes to and regulates the immune system of the CNS. These cells are found in both white and grey matter of the brain, but at lower densities than astrocytes, constituting only 10 % of all brain cells (DiSabato et al., 2016). Microglia processes are used for monitoring the microenvironment and regulations of homeostasis in healthy tissue (DiSabato et al., 2016, Zhang, 2019), and microglia activation is a key component of neuroinflammation. Microglia are activated as a response to damage and produces proinflammatory mediators such as cytokines (IL-1β, TNFα, IL-6), anti- inflammatory mediators and growth factors (DiSabato et al., 2016, Zhang, 2019). The activation also involves a change in morphology and proliferation. Activation is often corresponding to the severity of the inflammation stimulating condition (Zhang, 2019). Microglial activation is mainly neuroprotective, but excessive activation can also be harmful (DiSabato et al., 2016), and over time the activation can become chronic (Zhang, 2019).

1.4 Stroke

Stroke is the second largest cause of death worldwide, taking the lives of nearly 5.8 million people each year (World Health Organization, 2018). This makes stroke a substantial contributor to economic and health-related burdens (Benjamin et al., 2019). There is, on average, one stroke every 40 seconds in the United States of America (USA) (Benjamin et al.,

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4 2019). With stroke prevalence increasing with age (Becerra-Calixto and Cardona-Gomez, 2017) and a growing fraction of elderly in the world (United Nations, 2019) the burden of stroke will only continue to escalate. Studies have also shown that the incidence of stroke is increasing in adults under the age of 50. This combined with better treatments and a higher overall survival rate leads to an increase in the number of people living with the consequences of stroke. This way stroke is an important cause of work disability at a low age, imposing an economic burden to the health care system (Ekker et al., 2018).

The term “cerebral stroke” can be divided into two main categories: haemorrhagic and ischaemic strokes. Ischaemia is a result of obstruction of a brain vessel, leading to oxygen and glucose deficiency in parts of the brain (D'Aliberti et al., 2017). Ischemic stroke accounts for 87 % of all strokes (Benjamin et al., 2019). Haemorrhagic strokes account for the remaining strokes and are characterised by bleeding in the brain due to rupture of small arteries or arterioles (Gross et al., 2019). Stroke risk factors include hypertension, diabetes mellitus, heart rhythm disorders, high blood cholesterol and lipids, tobacco use, physical inactivity, unhealthy diet, genetics, kidney disease, and more (Benjamin et al., 2019, Ekker et al., 2018).

Strokes can result in complications like paralysis, speech and vision problems, memory loss, dementia, epilepsy, depression and anxiety, and gait instability (American Heart Association, Benjamin et al., 2019), depending on the area of the brain affected by the stroke (D'Aliberti et al., 2017, Ferdinand and Roffe, 2016). Ischemic strokes are also the cause of multiple comorbidities connected to hypoxia such as post-stroke pneumonia, aspiration, sleep apnoea and pulmonary embolisms (Ferdinand and Roffe, 2016). Further in this master thesis, “stroke”

refers to ischemic stroke.

1.4.1 Ischemic stroke

Ischemic stroke accounts for 87 % of strokes (Becerra-Calixto and Cardona-Gomez, 2017, Benjamin et al., 2019). Obstruction of blood flow to the brain can be a result of different events, such as clots formed in other places in the circulation being transported to the capillaries in the brain or formation of local blood clots. The obstruction can be either permanent or transient, the latter implying that spontaneous reperfusion occurs. Oxygen is vital for the survival of brain tissue through metabolic processes, and depletion of oxygen, and thereby energy, can quickly result in neuronal death and damage. The brain is especially sensitive to oxygen and glucose

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5 depletion because it has no alternative source of these vital components in its ATP production (Ferdinand and Roffe, 2016). A small store of glycogen is present in astrocytes, but this is quickly depleted during ischemia (Bak et al., 2018). Cell death is facilitated through a range of mechanisms, such as an increased influx of calcium due to lack of ATP and malfunctioning ion pumps such as the Na⁺/Ca²⁺ pump which are directly or indirectly ATP-driven and thereby oxygen- and glucose-dependent. Several calcium-dependent intracellular cascades can result in both apoptosis and necrosis of neural tissue (Ferdinand and Roffe, 2016). Acute strokes also facilitate an inflammatory response that activates the immune system to further trigger inflammation. Neurons affected by ischemia release damage-associated molecular patterns (DAMPs). DAMP is the collective name of molecules that contributes to this inflammatory response and consists of molecules such as ATP, heat shock proteins and uric acid. Effects of these substances can be both neuroprotective and detrimental (Chamorro et al., 2012).

Neuroinflammation plays an important role in the pathology of ischaemia. As previously mentioned, microglia, astrocytes, and neurons are responsible for the increased production and secretion of cytokines that drive the immune response (Becerra-Calixto and Cardona-Gomez, 2017). Microglia are activated in the case of ischemia but are dependent on residual blood flow to conduct this activation. Activation also differs in different brain regions (Zhang, 2019). Even though microglia can drive neuroinflammation forward, studies with depletion of microglia showed increased severity of ischemic strokes, by increasing activation of astrocytes (Jin et al., 2017).

In the case of an ischemic stroke, the objective of treatment is to increase cerebral blood flow through reperfusion to reduce further loss of brain parenchyma (D'Aliberti et al., 2017, Stoll and Nieswandt, 2019). Reperfusion therapy includes thrombolysis and thrombectomy (D'Aliberti et al., 2017, Ekker et al., 2018), the latter being a more invasive treatment.

Thrombolysis should be started within 3-6 hours of stroke symptom onset (Benjamin et al., 2019, Stoll and Nieswandt, 2019) but has shown to have limited efficacy (Stoll and Nieswandt, 2019). Pharmacologically, thrombolysis can be achieved with the thrombolytic agent alteplase, which is a recombinant human tissue-type plasminogen activator (tPA) (Stoll and Nieswandt, 2019). Some patients are not eligible for reperfusion therapy, and some have unsuccessful results from the treatment, resulting in permanent ischaemia. If reperfusion is achieved the ischaemia is referred to as transient ischaemia (Stoll and Nieswandt, 2019). Reperfusion can in

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6 some cases lead to increased damage, a phenomenon called ischaemia-reperfusion injury (Stoll and Nieswandt, 2019).

1.5 Exercise in stroke prevention

Physical inactivity is a contributor to cardiovascular disease, and treatment often involves lifestyle interventions such as a healthier diet and physical exercise. Stroke is a cardiovascular disease where exercise lowers the incidence for both first-time occurrence and recurrent stroke (Garcia-Cabo and Lopez-Cancio, 2020). A meta-study showed that medium and high-intensity exercise reduced stroke risk by 20 and 27 per cent, respectively (Lee et al., 2003). This effect could be linked to multiple beneficial effects of exercise such as reduced cholesterol, blood pressure and other risk factors of cardiovascular disease. There is dissensus as to whether low- intensity physical activity influences stroke risk. Some studies suggest that amount and intensity of the exercise is proportional to the stroke-reducing effect (Sacco et al., 1998). Studies point to exercise leading to improved functional outcome after stroke and how pre-stroke exercise through its effect on effector molecules and physiological functions can reduce lesion volume and neuronal death (Chaudhry et al., 2010, Zhang et al., 2014a). It is not known exactly what mechanism drives these neuroprotective effects of physical exercise, but there is consensus in there being a multitude of factors involved and more research is needed. Increased angiogenesis and neurogenesis are physiological functions that have been suggested to be affected by exercise (Tang et al., 2018, Cheng et al., 2020, Morland et al., 2017). Exercise post-stroke has shown to better patient outcomes in terms of increased mobility function (Macko et al., 2005, Ivey et al., 2006), pointing to a possible effect of exercise on neuroplasticity.

1.6 Lactate and HCA

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Lactate is a base mainly known for its multiple physiological functions related to energy metabolism, with more recent findings describing signalling functions (Brooks, 2018). The words “Lactate” and “lactic acid” are often mixed in everyday use, but lactate is the weak base corresponding to lactic acid. There are two isoforms of lactate, L-lactate and D-lactate, with L- lactate being the primary lactate isoform in humans (Andersen et al., 2013). Lactate is produced

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7 by most tissue in the body, but skeletal muscle is responsible for the greatest production (Andersen et al., 2013). Lactate is also found in the brain (van Hall et al., 2009). Plasma levels of lactate increase in response to high-intensity exercise (Siegel et al., 2008). An increase in lactate levels has also been seen in patients with sepsis (Shapiro et al., 2005), cardiogenic shock (Chiolero et al., 2000), cardiac arrest (Orban et al., 2017) and more (Andersen et al., 2013).

There are increasing evidence towards lactate being a part of the previously mentioned angiogenesis-increasing effect of exercise through unknown mechanisms, although some has been suggested (Porporato et al., 2012, Ruan and Kazlauskas, 2013, Morland et al., 2017).

Angiogenesis has also been suggested to be regulated by stimulation of the lactate receptor hydroxycarboxylic acid 1 (HCA1) receptor, where a study showed that both exercise and lactate injections led to increased angiogenesis in some areas of the brain in wild-type mice but not in HCA1 deficient mice (Morland et al., 2017). The location of the HCA1 in the brain has only recently been discovered by our research group (Lauritzen et al., 2014). Activation of the HCA1

receptor has shown to increase expression of the angiogenesis-stimulating factor vascular endothelial growth factor A (VEGFA) in the brain (Morland et al., 2017). VEGFA also stimulates neurogenesis (Geiseler and Morland, 2018) that can contribute to the regeneration of tissue in the infarct area. In fact, HCA1 has been shown to regulated neurogenesis in the sub- ventricular zone but not in the hippocampus (Lambertus et al., 2021).

1.7 Alzheimer’s disease

The first reported case of AD was reported in 1907 (Alzheimer, 1907), and over a hundred years later we still have a lot of questions about AD pathology. No curative treatment is yet available.

AD is a progressive disease characterised by dementia, and it affects over 5.8 million Americans, with numbers increasing every year due to the increasing elderly populations (The Alzheimer’s Association, 2020).

Symptoms of AD range from memory loss, learning and language problems, to reduced ability to function in everyday life. AD can also lead to depression and apathy, disorientation and difficulty talking and swallowing (The Alzheimer’s Association, 2020). Patients develop these symptoms many years after disease onset (The Alzheimer’s Association, 2020). The greatest risk factor for AD is age, with prevalence increasing drastically above 75 years of age (The Alzheimer’s Association, 2020). Another risk factor is familial predisposition. Genetic

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8 mutations linked to familial AD are mutations on presenilin 1, presenilin 2, and amyloid precursor protein (APP) (Lane et al., 2018). Another cause of familial AD is mutations in the apolipoprotein (APO) gene. APO E4 heterozygote and homozygote gene mutations increase the risk of late-onset AD by three and eight to twelve times, respectively, and the latter increases the risk of early-onset AD as well (The Alzheimer’s Association, 2020). APOs translates to proteins involved in the transportation of cholesterol in the blood and are involved in amyloid β (Aβ) clearance. APOE E2 and E4 also provide an increased risk of cerebral intraparenchymal haemorrhages due to increased deposition of Aβ in blood vessel walls. These depositions can also lead to cerebral amyloid angiopathy (CAA). CAA-related haemorrhages are associated with an increased risk of stroke-related dementia (Gross et al., 2019).

Alzheimer’s disease (AD) pathology is mainly characterised by aggregation of amyloid β (Aβ) plaques and oligomers and neurofibrillary tangles, and subsequent loss of neuron communication and possible neuron death (Zhao et al., 2015, The Alzheimer’s Association, 2020, Lane et al., 2018). Aβ pathology is in early stages found in the neocortex, with AD progression leading to affection in the hippocampus, amygdala, diencephalon and basal ganglia as well (Tiwari et al., 2019). Aβ with 40 and 42 amino acids are abnormally folded and the resulting aggregates are neurotoxic plaques. Aβ is a product of APP-processing by proteases such as α-, β- and γ-secretase. Another secretase that is believed to be involved in the handling of Aβ in the brain is δ-secretase. This enzyme is a cysteine protease, also called legumain or asparaginyl endopeptidase, and has recently been found to play a role in AD pathology (in detail below). In AD there is often an imbalance between the production and elimination of Aβ (Lane et al., 2018). Aβ oligomers are soluble Aβ, and these oligomers exhibit neurotoxic effects (Lane et al., 2018).

Neurofibrillary tangles are products of hyperphosphorylated microtubule-associated tau protein (Lane et al., 2018, Tiwari et al., 2019). These tangles are produced inside neurons and inhibit the transportation of nutrients and neurodegeneration, which can result in cell damage and loss of neurons (The Alzheimer’s Association, 2020, Lane et al., 2018). Tau pathology usually starts in the entorhinal cortex and spreads to the hippocampus (Lane et al., 2018, Tiwari et al., 2019).

Over time the loss of function leads to dementia symptoms. In addition to the role in the degradation of Aβ, research also points to legumain playing a role in neurofibrillary pathology (Zhang et al., 2014b, Behrendt et al., 2019) (more information below).

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1.8 The cysteine protease legumain

Proteases are enzymes involved in protein catabolism and the generation of amino acids through hydrolysis of peptide bonds. In addition to generating amino acids, these enzymes are important for the modification of proteins through specific cleavage, thereby producing new protein products. The effects are numerous, in processes ranging from DNA replication to apoptosis and necrosis. Proteases are generally unspecific cleavers, but there are examples of specific proteases such as the angiotensin-converting enzyme (protein specificity) and legumain (peptide bond specific) (Lopez-Otin and Bond, 2008).

Legumain is a cysteine protease in the CD clan and C13 family (Chen et al., 1997) that specifically cleaves on the C-terminal end of asparagine and is the only mammalian protease with this attribute (Dall and Brandstetter, 2016, Chen et al., 1997). At acidic pH, cleaving of aspartic acid residues has also been described, reflecting caspase activity (Halfon et al., 1998).

Legumain was identified in humans in 1997 (Chen et al., 1997) but was first found in plant beans, Blastocystis, and trematodes (Dall and Brandstetter, 2016) This cysteine protease is also named asparaginyl endopeptidase (AEP) due to its cleavage of asparagine residues, δ-secretase due to its role in APP fragmentation and Aβ production in AD, and vacuolar repressing enzyme (VPE) for its activity in plants (Dall and Brandstetter, 2016, Zhang et al., 2020).

Legumain is ubiquitously expressed, but most abundantly in the kidney, testis, placenta, spleen, liver, and thymus (Dall and Brandstetter, 2016, Zhang et al., 2020). Legumain is also found in the CNS and expression in microglia (Behrendt et al., 2019), neurons (Zhang et al., 2015, Ishizaki et al., 2010) and astrocytes (Ishizaki et al., 2010) has been suggested. Mammalian legumain is mainly localized in endosomes and lysosomes (endo-lysosomal system) (Dall and Brandstetter, 2016, Halfon et al., 1998). In these compartments the pH is acidic, ranging from pH 4.5-6. Legumain is also shown to function outside of lysosomes, both in the cytoplasm and the nucleus (Haugen et al., 2013). Increased cytoplasmic legumain is associated with diseases such as AD (Basurto-Islas et al., 2013), and increased extracellular legumain is associated with cancer (Murthy et al., 2005).

Legumain is synthesized as prolegumain before translocation through the endoplasmic reticulum and Golgi (Dall and Brandstetter, 2016). Prolegumain (56 kDa) undergoes autocatalytic processing to mature active legumain (36 kDa) triggered by a fall in acidity (Dall

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10 and Brandstetter, 2016), thus legumain is dependent on an acidic environment both to gain and remain its active conformation. Legumain contains a high number of acidic amino acids which contributes to its protein folding (Dall and Brandstetter, 2016). In a neutral environment, these amino acids are electrostatically repulsive, changing the conformity of legumain. At pH levels above 6, legumain is destabilised (Chen et al., 1997), and pH levels above 7 lead to irreversible damage and inactivation of the enzyme (Zhang et al., 2015, Zhang et al., 2016). Legumain exhibits maximum enzymatic activity at pH 5.8 (Johansen et al., 1999).

1.8.1 Legumain in the brain

Since legumain is activated by acidic environments, this protease could be linked to multiple pathological states in the brain causing a drop in pH levels such as oxidative stress, apoptotic cell death (which occurs during and after a stroke) and AD (Fang et al., 2010). Increasing evidence point to legumain having a role in neurodegenerative diseases such as AD, Parkinson’s disease, and certain types of dementia (Zhang et al., 2020). Legumain cleavage of APP and promotion of the production of Aβ are two factors contributing to AD. Legumain has been shown to mediate neuronal cell death through the cleavage of the phosphoprotein SET (more information below). SET is a phosphoprotein that regulates neuronal death by inhibiting DNase activity (Liu et al., 2008). SET-fragments triggers aggregation of tau in multiple diseases such as AD, brain ischemia, and traumatic brain injury (Zhang et al., 2020, Zhang et al., 2016).

Legumain is found to be expressed both in astrocytes and microglia (Ishizaki et al., 2010).

1.8.2 Legumain and stroke

The increased acidity occurring during strokes leads to activation of legumain (Zhang et al., 2016). Upregulation of legumain is seen in and around the ischemic core after transient middle cerebral artery occlusion (MCAO) (Ishizaki et al., 2010, Liu et al., 2008), which is a frequently used model for ischemic stroke. Ishizaki et al. have found that legumain was processed to its active form in the periinfarct area and was mainly found in astrocytes (Ishizaki et al., 2010).

Ishizaki et al. also showed that there was no difference in infarct volume between wild-type and legumain deficient mice, indicating that legumain does not play a part in acute stroke but rather in the regulation of neuroinflammation (Ishizaki et al., 2010). Liu et al. 2008, on the other hand, found that DNA damage was increased in wild-type mice compared to legumain deficient

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11 mice, suggesting that legumain plays a role in the early phases of the stroke (Liu et al., 2008).

Hence, the roles of legumain in stroke remains unresolved.

Previous studies in our research group showed that patients with carotid atherosclerosis had increased levels of legumain in plasma after stroke and transitory ischemic attacks (Lunde et al., 2017b). Later, the group also found legumain in thrombus materials from patients after acute ischemic stroke, and that increased levels of legumain were associated with improved outcome at 70 months follow-up (Lunde et al., 2020).

Another way legumain could influence stroke conditions is through the cleaving of SET. As levels of mature legumain increased with lowered pH related to stroke, legumain could contribute to increased DNA damage and resulting apoptosis during and after stroke. Further supporting the findings that legumain is responsible for the cleaving of SET, legumain deficient mice treated with a neurotoxic agent is shown to have significantly decreased neuronal damage caused by cleaving of the protective protein SET, compared to wild-type mice (Liu et al., 2008).

1.8.3 Legumain in AD

Legumain has three different pathological mechanisms related to AD. The first is that APP is a legumain substrate, and increased processing and cleavage of APP produces neurotoxic fragments. Legumain cleaves APP at N373 and N585 residues in AD, making fragments that are absent in legumain deficient mice (Zhang et al., 2015). Zhang (2015) has shown that the APP1-373fragment has specific neurotoxic effects and leads to neuronal cell death by inducing apoptosis.

Two of the fragments produces by legumain cleaving of APP, APP374-695 and APP586-695, are further processed by beta-site APP cleaving enzyme 1 (BACE1). The second pathological mechanism is the ability of these fragments to produce neurotoxic Aβ and the fact that legumain influences the processing of APP by other enzymes (Zhang et al., 2015). Legumain produces an APP1-585 fragment, and this processing of APP has been shown to increase BACE1 processing of the APP586-695 fragment (Zhang et al., 2015). This way legumain acts as a trigger for BACE1-mediated Aβ production. The levels of Aβ40 and Aβ42 is also decreased in legumain deficient neuron cultures compared to normal cultures. Zhang et. Al (2015) found that the neuroprotective α-secretase produced fragment sAPPα was higher in conditioned media from legumain deficient neurons.

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12 The third mechanism of legumain in AD is tau pathology. In human tissue legumain processes tau by cleavage after the N368 residue to form neurofibrillary tangles (NFT). The result is a tau1-368fragment that is prone to aggregation and is neurotoxic. The same cleaving was not seen in legumain deficient mice (Zhang et al., 2014b). Legumain also cleaves tau at N167 after uptake in microglia (Behrendt et al., 2019). In addition, legumain cleaves the earlier mentioned phosphoprotein SET after N175 (Liu et al., 2008) into two fragments that are both inhibitors of protein phosphatase-2A (PP2A) (Wang et al., 2010a, Arnaud et al., 2011). These fragments are overexpressed in AD animal models (Tanimukai et al., 2005). PP2A is a phosphatase that regulates the production of NFTs by dephosphorylation and keeps it from hyperphosphorylation and subsequent formation of NFTs (Wang and Liu, 2008). This means that increased legumain activity could indirectly contribute to the formation of these neurotoxic tangles.

It has been discovered that legumain is found in the cytoplasm of neurons to a higher degree in AD brains than in healthy brain, where legumain normally is mostly found in lysosomes (Behrendt et al., 2019, Basurto-Islas et al., 2013). Cystatin levels are lower in AD brains than in healthy brain, for example, cystatin C is reduced in AD (Kaur and Levy, 2012). Since cystatin C, E/M and F are endogenous legumain inhibitors, a reduction of cystatins could explain the increased legumain activity in AD.

Studies on legumain deficient mice show a reduced accumulation of AD plaques and neurofibrillary tangles, and these findings suggest that legumain plays a crucial role in the onset of the disease. Legumain could also be upregulated as a result of cell damage and increased tissue acidity. As previously mentioned, one of the biggest risk factors of AD is age. Legumain activity has been shown to increase in an age-dependent matter, due to increasing levels of mature legumain. The production of legumain-derived APP-fragments increases concurrently (Zhang et al., 2015). There are increased legumain activity in AD-affected brain, shown both in brain tissue from AD patients and a mice AD model (5xFAD mice) when compared to healthy individuals (Zhang et al., 2015). This increased activity could be linked to disease progression and onset.

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13

2 AIMS OF THE STUDY

There are emerging evidence toward the cysteine protease legumain playing an important role in neuroinflammation and different neurodegenerative disease (Zhang et al., 2015, Ishizaki et al., 2010, Zhang et al., 2017). After experimental stroke in mice, it has been found that legumain was upregulated around the infarct area after ischemic stroke (Ishizaki et al., 2010).

Furthermore, legumain has shown to be associated with increased cell death after experimental stroke (Zhang et al., 2016). Our research group has recently shown that legumain is present in intracerebral thrombi obtained during acute cardiovascular events and associated with blood platelets and macrophages(Lunde et al., 2020). In addition, we have shown that the serum level of legumain is increased in patients with acute cardiovascular events (Lunde et al., 2017b) but the levels in stroke patients are not known.

To strengthen our knowledge about legumain in the CNS, this thesis aims to investigate:

1) Which neural cells express legumain?

2) Do human brain cells in cultures secrete legumain and, if so, which cell types?

Based on the effects of legumain in macrophages and the suggested role of legumain in brain pathology, we aim to study the effects of neuroinflammation on brain legumain levels. We will use two well-established models for conditions in the brain where neuroinflammation is a key component to investigate:

3) Does legumain increase selectively in astrocytes surrounding the lesion area after stroke?

4) Does legumain increase in response to AD and, if so, in which specific brain regions involved in AD pathology?

5) Is legumain plasma levels altered in patients after stroke?

Physical exercise is reported to prevent both stroke and AD. One hypothesis is that exercise acts by inducing partial preconditioning, meaning that it represents small and transient stress triggering brain protection against neuroinflammation. We, therefore, want to study:

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14 6) Whether seven weeks of high-intensity exercise prior to a stroke is protective and, if so,

whether legumain levels in cells in/around the stroke core is altered?

7) Whether exercise affects plasma levels of legumain.

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15

3 Material and methods

3.1 Chemicals, solutions and equipment

Listed in Appendix I, II, and III.

3.2 The effect of lactate injections and exercise on lesion volume through HCA

1

General information

The following part of this master thesis partly proceeds five previous master projects in the Neurobiology and Toxicology group. Some of the experiments were hence performed before I started this master project. This includes genotyping, animal experiments, sectioning, and cresyl violet staining. Unless otherwise stated, these methods were performed by postdoctoral fellow Samuel Geisler or former master’s students Camilla Brox (2020), Kimberly Dungdung Phan (2020), Teresa Dang Nguyen (2020), Karl Martin Forbord (2019), or Ghazal Sajedi (2019) before I started my master project. These methods are described briefly below. My first experimental contribution to the project was to analyse brain lesion volumes (chapter 3.2.1).

All procedures and experiments were done in accordance with national ethical guidelines, EU- directive 2010/63, and PREPARE guidelines, and reported according to the ARRIVE guidelines. The use of animals was approved by the Norwegian Animal Research Authority (FOTS applications ID12521 and ID14204) and Animal Use and Care Committee of the Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo. Animal experiments were performed by the Federation of Laboratory Animal Science Association Category C (FELASA C) certified personnel.

The study included 144 C57Bl/6N mice of both wild-type (WT) and hydroxycarboxylic acid 1 (HCA1) deficient (knock out; KO) mice (Ahmed et al., 2010) to investigate any HCA1- dependent effects of exercise. The mice were acquired from the lab of Stefan Offermann at Max-Planck Institute for Heart and Lung Research, Department of Pharmacology, Bad Nauheim, Germany.

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16 The animals were bred, housed and cared for at the animal facility at the Department of Comparative Medicine, Faculty of Medicine, University of Oslo. Male and female mice were housed in separate cages, with females housed with up to eight animals per cage and males individually to minimise aggressive behaviour. The animals were kept at a 12-hour light/dark cycle, with interventions done during the light part of the cycle for practical reasons. Animals had unlimited access to water and food, but no running wheels because this could affect the results of the interventions.

Genotyping

Genotyping is a technology used to find small genetic differences between individuals.

Genotyping of HCA1 and LacZ (replaced the HCA1 gene in the deficient mice) was done prior to interventions and was the foundation for the semi-randomization of approximately the same number of HCA1 KO and WT in each group.

Ear biopsies were collected at 4-6 weeks of age, and the tissue from these was used for genotyping. Genomic DNA was extracted after lysis of the biopsies, HCA1 and LacZ were amplified by polymerase chain reaction using specific primers and analysed by gel electrophoresis. WT mice showed a band of 195 base pairs (bp) representing the HCA1 gene and HCA1 KO mice showed a band of 507 bp representing the LacZ gene in the HCA1 KO mice. Heterozygote mice with bands of both 195 and 507 bp were excluded from the study.

Interventions

Interventions were started at 5-7 weeks of age. Both HCA1 KO and WT mice were exposed to one of four interventions: lactate injections, saline injections (control), high-intensity interval training (HIIT), and medium intensity interval training (MIIT), resulting in eight semi- randomized groups. Each treatment group had an approximately equal ratio of HCA1 KO and WT mice and distribution of gender. Lactate or saline injections were administered five consecutive days followed by two days of rest for seven weeks. The administrations were intraperitoneal and were alternated between the left and right side of the abdomen to minimise injection discomfort and possible side effects to the skin. The lactate-treated group received 2 g/kg sterile sodium L-lactate (≥ 99.0 %, dissolved in 0.9 % saline; pH-adjusted to 7.4) in a

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17 volume of 10 μl/g body weight. The saline-treated group received 10 μl/g bodyweight of 0.9 % sterile saline. Doses were adjusted according to weekly weighing.

The animals semi-randomized to interval training ran on a treadmill for five consecutive days followed by two days of rest for seven weeks, either as HIIT or medium MIIT intensity interval training. The training consisted of a 10-minute warm-up followed by ten rounds of 4-minute intervals with 2-minute breaks. The HIIT group ran at 80% of maximal capacity and the MIIT group ran at 60 % of maximal capacity.

Middle cerebral artery occlusion

After the seven weeks intervention period the animals were subject to induced focal ischemia by permanent occlusion of the distal middle cerebral artery (dMCAO) as described (Llovera et al., 2014). Isoflurane was used to induce anaesthesia and to maintain anaesthesia during surgery. Buprenorphine (0.3 mg/kg) administered as an i.p. injection was used for analgesia.

After a surgical plane of anaesthesia had been ensured, the middle cerebral artery was occluded both proximal and distal to the M1 branch of the artery using electrocoagulation forceps. The site was checked for reperfusion after 30 seconds with a gentle touch to the artery and electrocoagulation was repeated if any recanalization was observed. The animals recovered from the anaesthesia in a nurturing box at 32 °C and were later transferred to their original cages. Additional i.p. injections of buprenorphine (0.1 mg/kg) were given daily for the following four days post-surgery.

Fixation

Three weeks after stroke operations, transcardial perfusion fixation with paraformaldehyde (PFA) was performed to maintain tissue integrity while extracting, preparing, and analysing the brain. PFA converts to formaldehyde in solution, and formaldehyde inactivates proteins by cross-linking. Fixation is done to stop microbial and enzymatic degradation and other effects of decay. Perfusion fixation is the gold standard of brain tissue fixation because it ensures instant distribution of the fixative through the circulation, utilizing the comprehensive vascularization to transport fixative to all tissue at a high rate (McFadden et al., 2019). This way it gives rapid preservation of the brain and a more even result in terms of fixation than for

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18 instance immersion fixation. In the latter, the entire brain, or pieces of the brain, is immersed in fixative.

Prior to fixation, the animals were put under deep anaesthesia with i.p. injections of 0.1 ml/10 g body weight ZRF anaesthetic mixture consisting of 3.3 mg/ml zolazepam, 3.3 mg/ml tiletamine, 0.5 mg/ml xylazine and 2.5 μg/ml fentanyl. The thorax was opened, and the fixative was infused (5 ml/min) into the left ventricle at a rate not exceeding mouse cardiac output (Kreissl et al., 2006). The right atrium was immediately punctured to allow blood and fixative to exit the circulation. This procedure causes concurrent termination and fixation of the animal.

After perfusion fixation for eight minutes, the brain was gently removed. The tissue was stored at 4°C overnight in 4 % PFA, before switching to 0.4 % PFA.

Cryoprotection, sectioning and mounting

The brain tissue needed to be frozen before sectioning to ensure the preservation of tissue integrity. The tissue was saturated for 24-48 hours in a cryoprotection solution (30% sucrose + 0.01% sodium azide solution) prior to freezing to inhibit membrane rupture and cell lysis that can occur during freezing. The sucrose is hypertonic and extracts water from the cells. Before sectioning, the brains were put on a “sucrose stage” on the microtome freezing unit, allowed to freeze at -20 °C and stabilized by dripping more sucrose around the brain. The brains were cut into 20 μm coronal sections and systematically transferred in sequence to four 24-well plates with 0.01 % sodium azide in NaPi buffer with the first section in A1, the second in A2 and so on, to ensure complete control of the order of the sections in the rostral-to-caudal direction for future analysis. The plates were stored refrigerated until mounting and analysis. Every sixth section was mounted on microscope glass slides.

Staining with cresyl violet

Nissl staining with cresyl violet (CV) is a method commonly used for neuronal staining and has a high degree of correlation when measuring cerebral infarct volume (Tureyen et al., 2004). CV binds to acidic components and rough endoplasmic reticulum (Cammack et al., 2008), which is found in high concentrations in the cell cytoplasm of neurons (Tureyen et al., 2004).

CV-staining of brain sections was done using following a strict protocol with multiple consecutive steps involving rehydration, staining, differentiating and dehydration. All steps in

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19 the procedure were conducted with EasyDip™ Slide Staining System. The sections were hydrated to ensure even distribution of CV in the tissue and staining was performed at 60 °C in a water bath. After staining the sections were washed and differentiated with 1 % glacial acetic acid in ethanol to subtracts CV from the cytosol. The last step was dehydration with 95 % ethanol and Neo-Clear to remove any remaining water. After allowing the sections to dry the sections were covered with a coverslip using either gelatine-glycerine or Eukitt® (Sigma- Aldrich, Merck) as mounting media.

3.2.1 Lesion volume measurements

All stained sections were digitalized using NORBRAIN slide scanner microscope Axio Scan.Z1 (Carl Zeiss Microscopy, Germany) at 20X magnification. Scanning was done by the Neural Systems (NESYS) research group, Institute of Basic Medical Sciences, University of Oslo.

In total, more than 3000 sections were analysed, and the work was divided between me and four other operators (master students Camilla Brox, Kimberly Phan, Teresa Dang Nguyen, and Erasmus student Hanne-Lise Doosje). Sections d from 1.645 rostral to bregma to 2.355 caudal to bregma (Allen Brain Atlas, 2008) were included in the analysis, with each operator responsible for an individual part of the brain in terms of distance to bregma. Furthermore, all operators were blinded to the genotype and treatment groups to minimise operator bias. To examine reproducibility and operator precision 79 of the sections were analysed twice. Fiji (ImageJ) image processing program was used to manually outline the cortex of each hemisphere in all scanned sections. Regions of interest were made to include only cerebral cortex tissue without scarring or signs of tissue death thereby accounting for both the lesion and any brain atrophy (fig. 3.1). Since the lesions were fragile, some sections completely lacked tissue in the stroke area. Some sections also appeared to lack parts of healthy tissue. Efforts were made to extrapolate as thoroughly as possible wherever sections were missing a piece, using the contralateral hemisphere or the nearest section as a template. Any folding of the sections was accounted for as far as possible.

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20

Figure 3.1 A: Cresyl violet-stained coronal section of one mouse brain, showing how the cortex was outlined using the region of interest (ROI) function in FIJI. Damaged or dead tissue in the stroke core was excluded from the ROI of the ipsilateral cortex (outlined in red). If the sections were torn, each piece of the tissue was outlined separately, and the area of each piece was summed to give the total cortical area (an example of this is shown in the medial contralateral cortex). The difference in cortical area between the ipsilateral (delineated by the red line) hemisphere and the contralateral (delineated by the yellow line) was the basis for the lesion volume calculations. B: The Allen Mouse Brain Atlas (2004) was used as a template when outlying the cortical area (light green; marked with the abbreviation CTX). The image is from 0.545 rostral to bregma and is a representative area for the section in A. Scale bar: 1 mm.

Both the ipsilateral (stroke) and the contralateral (healthy) side was measured. To calculate the lesion area in each section, the healthy area of the ipsilateral cerebral cortex was subtracted from the corresponding contralateral area. Since the lesion area was measured in every sixth section, the lesion area measured in each section was multiplied with the distance between the sections to calculate the lesion volume of each animal. The theoretical distance between every sixth section was 120 μm (20 μm/section x 6 sections), but to make calculations more accurate the theoretical distance of 4 mm from the anterior to the posterior section divided by the number of sections used was the intermediate distance for lesion volume calculations.

Exclusion criteria after digital analysis were 1) stroke extended past the CC (unsuccessful operation), 2) lesion volume below 3 mm³, and 3) extensive damage to sections that prevented proper outlining of the cortex. Groups were compared using one-way ANOVA statistical analysis with a significance level of 5%.

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21

3.3 Localisation of legumain in brain tissue

Stroke materials

Immunostaining was performed on brain tissues obtained in previous master projects (Forbord, 2019, Sajedi, 2019). The project shares a lot of similarities with the earlier mentioned, with a few exceptions: 1) the number of experiment animals, 2) treatments given included only intraperitoneal injections with either saline or lactate, 3) treatments were given post-stroke operation, as opposed to prior and 4) animals were terminated either one week or three weeks post-surgery. Brain sections were stored in 24-well plates in NaPi with 0.01% azide. Only sections from one (n = 6) and three (n = 6) week post-stroke saline-treated wild type animals were used for immunostaining of the cysteine protease legumain and astrocytes. Brain sections from the stroke mice were selected from approximately 0.745 anterior to bregma (Allen Brain Atlas, 2008).

Alzheimer’s disease (AD) materials

A frequently used mouse model of Alzheimer’s disease (AD) is the five-familial AD (5xFAD) transgenic mice model. 5xFAD mice express human transgenes that are linked to AD. The mutations are either linked to amyloid precursor protein (Swedish (K670N/M671L), Florida (I716V), and London (V7171)) or presenilin 1 (M146L and L286V) (Amram and Frenkel, 2017, Richard et al., 2015). The model does not express any tau pathology, but Aβ deposits are observed at eight weeks of age with increasing depositions with age, as well as increased inflammation compared to wild-type mice (Amram and Frenkel, 2017). The activity of legumain is increased in 5xFAD mice (Zhang et al., 2015).

Brains from the 5xFAD transgenic AD mice model (n = 8) and WT mice (n = 12) were used for fluorescent immunohistochemical staining of legumain and astrocytes. The mice were terminated at 39-49 weeks of age with perfusion fixation and brains sections were treated in the same matter as described above and selected from approximately 1.655 posterior to bregma (Allen Brain Atlas, 2008). The area was chosen since we wanted an area in the brain where the hippocampus and the neocortex were present since these areas show pathophysiological changes and neuroinflammation in subjects with AD (Fjell et al., 2014).

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3.3.1 Immunohistochemistry

Immunohistochemistry (IHC) is a method used for the localization of different proteins in cells or tissue through the binding of an antibody to an antigen. Immunostaining was performed to determine which brain cells expressed legumain. Therefore, co-localization with an astrocyte marker (glial fibrillary acidic protein, GFAP) and legumain in the brains of mice after stroke or with Alzheimer’s disease was performed.

Primary antibodies specific to GFAP and legumain were used to detect astrocytes and legumain, respectively. GFAP is the main intermediate filament protein of astrocytes, and the presence of GFAP is a well-used marker for the identification of astrocytes in IHC (Eng, 1985). GFAP is commonly used as a marker for non-reactive (resting) astrocytes as well as active astrocytes, but resting astrocytes express lower levels than reactive astrocytes (Sofroniew and Vinters, 2010). The legumain antibody binds prolegumain, intermediate form of legumain and mature legumain, and further reference to “legumain” refers to all three forms prolegumain, intermediate form of legumain and mature legumain. Staining for nuclei was performed with 4,6-diamidino-2-phenylindole (DAPI), which is a reagent that emits blue fluorescence when binding to adenine-thymine regions of deoxyribonucleic acids (DNA).

Method development and testing

As the procedures initially did not produce adequate results in terms of immunohistochemical staining, different methods were used (discussed further in section 5.2.1) The final method used was free-floating immunohistochemical staining. Additionally, the other method used, mounted staining, is explained briefly below.

Free-floating procedure

The sections were placed in individual wells in two 24-well plates filled with NaPi with 0.01

% azide until analysis. The sections were rinsed in 1000 μl PBS for 2 x 10 minutes. The reagents were removed and added throughout the procedure using a pipette. This was done gently because of the risk of suctioning the brain sections into the pipette tip and damaging it.

Due to storage over time, heat-induced epitope retrieval (also called antigen retrieval) with citric acid was performed on the brain sections before washing and incubation with the primary

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23 antibody. Fixating tissue over time can lead to cross-linking of proteins, which can lead to masking of epitopes. After washing away the NaPi with azide the sections were incubated in citrate buffer (pH 8.6, 80 °C) at 80 °C for 30 minutes. This was done by transferring the sections to two other well plates with preheated citrate buffer and gently floating the wells in a water bath set to 80 °C. After 30 minutes the sections were rinsed with 1000 μl PBS for 2 x 5 minutes before incubation with the primary antibodies in blocking solution. The well plates were covered with a plastic film and set on gentle swirling on the Belly Dancer® overnight.

On the second day, the sections were rinsed with 1000 μl PBS for 6 x 10 minutes. The PBS was replaced between washes. The secondary antibodies were diluted in blocking solution and 300 μl was added to the wells and sections were incubated for two hours on the Belly Dancer®.

After another rinsing step with PBS for 3 x 10 minutes, the sections were incubated with 500 μl DAPI solution (1:5000 in PBS) for 15 minutes. The sections were then rinsed a last time for 3 x 10 minutes with PBS before mounting on microscope slides (Superfrost® Plus Microscope Slides) with ProLong™ Gold Antifade reagent mounting medium and 24 x 60 mm Corning®

Cover Glass.

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24 Table 3.1 Overview over the protocols used for mounted and free-floating

immunohistochemical staining for legumain and GFAP on mouse brain sections

Procedure Free-floating Mounted

DAY 1

Rinse with PBS 2 x 5 min 2 x 10 min

Block with 1% BSA + 3% NCS in PBST (blocking solution)

1 x 2 hours 1 x 2 hours Incubation with the primary antibodies in blocking

solution

Overnight Overnight Goat anti-legumain

Mouse anti-GFAP

1:300 1:1500

1:300 1:500 DAY 2:

Incubation with the secondary antibodies in blocking solution

1 x 2 hours 1 x 2 hours Donkey anti-Goat 1:1000 1:1000

Goat anti-Mouse 1:500 1:1000

Incubation with DAPI in PBS

DAPI

1 x 15 min 1:5000

The mounted procedure was performed essentially as described above for the free-floating sections, but preparation, incubation times and rinsing steps were slightly different. Antibody concentrations did also differ (see table 3.1).

Brain sections were mounted on microscope slides (Superfrost® Plus Microscope Slides, Thermo Scientific). A liquid blocker Super pap pen (Electron Microscopy Sciences, USA) was used to create a hydrophobic ledge around the tissue to keep the reagents in place on the slide while staining. The pap pen marked slides were dried overnight before starting the procedure.

All incubation steps of the procedure were done with the glass slides placed facing up in a

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25 Raaco box with wet paper along the base of the box, making it a humidity chamber. Antigen retrieval was done using EasyDip™ Slide Staining Jars and immersing the glass slides in the buffer in the water bath.

Analysis of IHC results

The sections were digitalised using Andor DragonFly confocal microscope with a 20X objective. Lasers were set for 405 nm, 488 nm and 561 nm corresponding to the wavelengths of the secondary antibodies used. Images were taken as Z-stacks ranging from 8-12 stacks per section, allowing for depth in the images.

Sections were visually compared for tendencies in healthy and diseased tissue and observed differences between genotypes were noted and used for simple statistical analysis. Due to covid- 19 and restricted access to microscope premises, the imaging was delayed, and quantification of astrocytes was not performed due to lack of time.

Statistical analysis was performed using GraphPad Prism 9. Tests were performed with a 5 % significance level. Fisher’s Exact Test was used to calculate statistics between categorical data due to small sample numbers when comparing traits of fluorescent staining in AD-affected and healthy brain.

3.4 Legumain in plasma and conditioned media from neural cells

Enzyme-linked immunosorbent assay (ELISA)

Enzyme-linked immunosorbent assays (ELISA) are generally used for the detection of specific antigens in a sample. There are different types of ELISA protocols, and for analysis of total legumain in various samples (table 3.2), a sandwich ELISA was used.

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26 Table 3.2: Overview of samples analysed for legumain concentration by ELISA.

SAMPLE TYPE Sample size

Conditioned culture media

Neuron- and astrocyte

culture 14 samples

Microglia 12 samples (3 samples x 4 individuals)

Plasma samples

Healthy, exercising

volunteers 171 samples (3 samples x 57 individuals) Stroke patients 42 samples (42 individuals)

The protocol contains multiple steps of antibody, sample, and substrate incubation (fig. 3.2).

The advantage of this protocol is that it has high specificity towards the legumain since the capture antibody is monoclonal.

Figure 3.2: Simplified illustration of the principle of sandwich ELISA. 1) A detection antibody is added to the wells of a microplate (illustration shows one well). 2) The unbound parts of the well are blocked using a blocking solution. 3) The sample is added, and the target antigen binds to the capture antibody. 4) A biotinylated detection antibody is added and binds to another epitope on the target antigen. 5) Streptavidin-horse radish peroxidase (HRP) is added and streptavidin binds to the biotin. 6) When a substrate solution (H2O2 and tetramethylbenzidine) is added, the HRP catalyses the reaction between the substrates, and colour or fluorescence is developed. The amount of HRP is directly linked to the amount of antigen in the sample and determines the rate of colour or fluorescence development. 7) Sulphuric acid (H2SO4) is added to stop the reaction.

Studies on the cysteine protease legumain in the central nervous system during neuroinflammation

legumain in the central nervous system during neuroinflammation

Cathrine Lilleheil Christiansen

Master Thesis for the title of Master of Pharmacy

Section for Pharmacology and Pharmaceutical Biosciences

45 credits

Department of Pharmacy

The Faculty of Mathematics and Natural Sciences UNIVERSITY OF OSLO

Studies on the cysteine protease legumain in the central nervous system during neuroinflammation

Cathrine Lilleheil Christiansen

Master Thesis for the title of Master of Pharmacy

Section for Pharmacology and Pharmaceutical Biosciences

45 credits

Department of Pharmacy

The Faculty of Mathematics and Natural Sciences UNIVERSITY OF OSLO

Acknowledgement

Abbreviations

Table of contents

1 Introduction

1.1 Neuroinflammation

1.2 Astrocytes

1.3 Microglia

1.4 Stroke

1.4.1 Ischemic stroke

1.5 Exercise in stroke prevention

1.6 Lactate and HCA

1.7 Alzheimer’s disease

1.8 The cysteine protease legumain

1.8.1 Legumain in the brain

1.8.2 Legumain and stroke

1.8.3 Legumain in AD

2 AIMS OF THE STUDY

3 Material and methods

3.1 Chemicals, solutions and equipment

3.2 The effect of lactate injections and exercise on lesion volume through HCA

3.2.1 Lesion volume measurements

3.3 Localisation of legumain in brain tissue

3.3.1 Immunohistochemistry

3.4 Legumain in plasma and conditioned media from neural cells

Enzyme-linked immunosorbent assay (ELISA)