Emblica officinalis as a therapeutic supplement in Alzheimer’s disease

(1)

Emblica officinalis as a therapeutic supplement in Alzheimer’s disease

The effects of pre-onset administered amla extract on APP/PSI transgene mouse model

N. L. Chilcutt

Thesis for the Master’s degree in Molecular Biosciences 60 study points

UNIVERSITY OF OSLO

June 2019

(2)

II

Emblica officinalis as a therapeutic supplement in Alzheimer’s disease mouse model N. L. Chilcutt

http://www.duo.uio.no/

Published: Reprosentralen, Universitetet i Oslo

(3)

III

Acknowledgements

I would like to thank my advisors, Jens Pahnke and Bernd Thiede, for guiding me through this process. Dr. Pahnke treated me like a post-doc and allowed me to pursue paths that piqued my curiosity. While this often reminded me of my limitations and that I am indeed not a post-doc; it did reinforce the importance of being independent, asking for help I could not rely on myself, and knowing the difference between the two. Dr. Thiede was very helpful in editing my thesis drafts and giving constructive criticism as well as providing comic relief.

Thank you to the staff at KPM Radiumhospitalet for tirelessly working to maintain healthy and clean environments for experimental animals. I am also grateful to the professors at IBV and all the administrative staff (especially Anna and Tørill) who helped guide this clueless international student through these two years!

Specifically, I would like to thank Iván for breeding the animals requested for these experiments, introducing me to the animal facility at Radiumhospitalet, and helping me get a part-time job so I could stay in Norway and complete this project. Thanks to Surya for instructing me on water maze protocols and Mirjam/Luisa/Markus/Tini for training me in animal handling. Special thanks to Thomas who always entertained my questions and ideas in addition to guiding me through IHC stainings and analysis.

Helle Wangensteen was very gracious and accommodating during the week I spent in her lab for plant extraction procedures. Her passion for research and real-world application is inspiring as well as her ability to engage others in such discussions. From her lab, Nastaran was very kind in helping me with the equipment even after I broke a round bottom flask!

Sorry about that!

Outside our lab, I very much appreciated the help of Anne Schad Bergsaker at the statistics helpdesk in Forskningsparken for patiently explaining all my statistical analysis questions and encouraging me to read the IBM SPSS book. Kristian Lensjø was very kind to take time to meet with me after class and suggest further reading on memory and behavioural analysis.

Lastly, I thank you – the readers and reviewers – for dedicating time and energy to read my thesis. I am eager to hear any critiques and suggestions for improvement.

(4)

IV

Abstract

Alzheimer’s disease (AD) is the most common form of dementia, currently affecting 50 million people worldwide. While plaques of misfolded protein amyloid-beta (Aβ) are a hallmark of the disease, drugs targeted to reduce or eliminate plaques have not been successful in restoring cognitive function. Questions now arise as to if, in fact, the plaque deposition or inflammatory response to those plaques cause cognitive decline. Yet treatments developed to alter immune responses or immunize against disease associated Aβ had adverse side-effects. Because comorbidity of AD and type II diabetes (T2D) are common, some have abandoned the “amyloid hypothesis” – which suggests that the misfolded proteins are the root cause of the disease – in favour of AD as a metabolic disease instead. Though there are drugs that effectively manage T2D by lowering blood glucose or lipid levels, studies have shown that amla fruit is just as effective. Other studies support claims that this plant confers anti- inflammatory, neuroprotective, and other benefits as practiced in traditional Indian medicine.

The aim of this project was to test effect of Emblica officinalis or amla phytoextract on the behaviour and pathology of Alzheimer’s disease mouse models. In these experiments, the well-characterized C57Bl/6J strain of Mus musculus was genetically modified to express the humanized amyloid precursor protein (hAPP) which develops cerebral amyloid plaques to serve as a pathological AD model. The Morris Water Maze was performed according to established protocol and animal activity was monitored thereafter. Brain tissue was analyzed by Western Blot to detect insulin-degrading enzyme (IDE) and APP proteases α-secretase (ADAM10) responsible for normal protein processing and β-secretase (BACE-1) involved in pathogenic protein production. Disease pathology was assessed through immunohistology to quantify neuronal, astrocyte, microglial, and amyloid plaque coverage using NeuN, GFAP, Iba1, and 4G8 antibodies respectively. In behavioural tests, animals administered plant treatment showed improved spatial reference memory during the water maze. Post-mortem results showed increased microglia coverage percentage and count per 10 mm² in the cortex of water treated mice compared to their plant treated counterparts. Previously reported anti- inflammatory properties of amla fruit may be responsible for the reduced immunological response of mice administered the treatment. Neuroinflammation is now thought to contribute to mild cognitive impairment (MCI) more than amyloid plaques, therefore treatments that minimize cerebral inflammation may benefit patients.

(8)

2

Abbreviations

AD Alzheimer’s disease

ADAM10 a disintegrin and metalloprotease 10, α-secretase ANOVA analysis of variance

APP amyloid protein precursor

APP/PSI amyloid precursor protein/presenilin I

BACE-1 β-site amyloid precursor protein cleaving enzyme 1, β-secretase CAA cerebral amyloid angiopathy

CNS central nervous system DAB 3,3'-Diaminobenzidine GFAP glial fibrillary acid protein hAPP human amyloid protein precursor

Iba1 Ionized calcium binding adaptor molecule IDE insulin degrading enzyme

IHC immunohistochemistry MWM Morris water maze

MCI mild cognitive impairment NeuN neuronal nuclei

T2D Type 2 diabetes PFA paraformaldehyde PSI presenilin I PSII presenilin II

(9)

3

1 Introduction

1.1 Alzheimer’s disease

Alzheimer’s disease (AD) is the most common form of neurodegeneration affecting an increasingly large portion of the aging population (Hodson, 2018). The line between normal cognition and dementia is very subjective and can be difficult to define. While research aims to identify biomarkers and imaging techniques (Shaw et al., 2007) that would diagnose patients decades before dementia onset as shown in Figure 1, clinicians rely on self- reported abnormalities from patients (Petersen, 2009).

Figure 1 | AD physiological and behavioural markers as a function of disease progression. The severity of each disease hallmark increases with disease progression from normal cognitive function to mild impairment to dementia. Amyloid-β plaques are the first to indicate the onset of AD, often depositing in the brain 15-20 years before cognition suffers. Tau proteins form tangles that aggregate inside neuronal cell bodies and coincide more closely to cognitive impairment. Slight problems with memory or language – while not dramatically altering daily routines or independence – can signal mild cognitive impairment. Self-reported measures of mild cognitive impairment are used in AD prevention studies. Figure taken from (Drew, 2018) illustration by Mohamed Ashour from data gathered previously (Jack et al., 2013)

Mild cognitive impairments (MCI) – such as increased forgetfulness and reduced memory recall – can mark the transition between normal and abnormal cognition (Petersen, 2003). In the most recent World Alzheimer Report featured personal anecdotes to illustrate memory loss due to the progression of dementia: “I wouldn’t recognize people I had known for 20 years, then I started getting lost along familiar routes” (Patterson, 2018, pg. 6).

(10)

4

Patients affected by the later stages of AD or dementia are unable to form new memories and navigate familiar settings. Dr. Jennifer Bute, the general practitioner quoted in the

Alzheimer’s Disease International publication (Patterson, 2018), was forced by AD

progression to resign her medical practice. The march from MCI to dementia (Fig. 1) is slow but relentless, resulting in the loss of independence and the ability to care for one’s self. What exactly causes this disease progression has been the subject of over a hundred years of

investigation. Though there are hypotheses as to the causative agent, the questions to why and how still plague researchers today (Hodson, 2018, Makin, 2018).

The first reports of AD were published by the German physician, Alois Alzheimer, in 1911. He reported tangles and amyloid plaques in the brain autopsy of a 55-year-old patient who suffered from dementia (Purves, 2008). Interestingly, Downs syndrome and AD share pathological characteristics. Since Downs Syndrome stems from chromosome 21

abnormalities, it was thought that the source of AD could have similar origins (Hartley et al., 2015). The causative agent of AD amyloid plaques, called amyloid protein because of the misfolded proteins observed in post-mortem studies, was purified in 1984 (Glenner and Wong) resulting in the report of a 4.2 kilo Dalton (kDa) protein of 40-42 amino acids. This amyloid precursor protein (APP) gene was later cloned from chromosome 21 in 1987 (Kang et al.). Most cases are seen as late-onset, meaning the patient sporadically acquired AD around or after 60 years of age, but rare cases have been documented as early-onset in young patients around 20 years old who inherit AD causing genetic mutations (Costandi, 2018, Drew, 2018, Ohm, 1997). Abnormalities within the APP gene appear to cause to the early- onset familial AD which accounts for a small percentage of occurrences (Tilley et al., 1998), but produces the same AD pathology as sporadic late-onset cases.

1.2 Amyloid Precursor Protein

Physiological APP function is unclear, but studies in cell lines and mice have suggested a variety of functions. Evidence for involvement of full length APP in neuronal growth and development roles (Young-Pearse et al., 2007) have been suggested in mammals, while other studies have shown that APP overexpression resulted in enlarged neurons (Oh et al., 2009), and yet other studies saw no significant phenotypic alterations in an APP knock- out models (Heber et al., 2000). APP is expressed by platelets or neurons (Baranello et al., 2015, Devraj et al., 2016) and proteolytic processing can take place across endothelial

(11)

5 neuronal cell membranes respectively. Non-amyloidegenic APP processing begins with α- secretase cleavage at the +17 position inside the Aβ sequence. Processing of APP by α- secretases, proteins in the family of a disintegrin and metalloprotease (ADAM), reduces amyloid deposition and cognitive disfunction. At the membrane, APP may be processed in a way that produces normal peptides or toxic peptides. Cleavage by α-secretase followed by γ- secretase at the cell membrane produces Aβ peptides associated with healthy and normal cognition. Acetylation and epigenetic changes to the α-secretase gene associated with aging are thought to hinder healthy APP processing (Donmez et al., 2010). These discoveries formed the basis of the amyloid hypothesis that APP cleavage by β- and γ-secretase, seen in Figure 2, results in amyloid protein forms that aggregate and cause neurotoxicity.

Figure 2 | APP processing in the development of AD pathology. The transmembrane amyloid precursor protein (APP) is cleaved by β- secretases, like BACE-1. Two aspartate active site residues enzymatically cleave APP to release the soluble extracellular protein portion. Subsequent cleavage of APP close to the membrane anchor causes amyloid beta (Aβ) fragments to be released in the cytosol. These Aβ fragments are prone to aggregate and form the amyloid plaques characteristic of AD. Figure adapted from (Makin, 2018)

The alternative path (Fig. 2) begins with β-site of APP cleaving enzyme (BACE-1) activity. BACE-1 is expressed in endosomes, endothelial cells, and neuronal membranes throughout mammals (Devraj et al., 2016). Presenilin proteins I (PSI) and II (PSII) traverse the membrane, forming the γ-secretase catalytic complex containing aspartyl active sites for APP cleavage (O'Brien and Wong, 2011) within the membrane. APP cleavage by BACE-1 is followed by γ-secretase which produces Aβ peptides prone to plaque formation, innate immunity activation, and synaptic disruption (Baranello et al., 2015, O'Brien and Wong, 2011, Thinakaran and Koo, 2008). The driving force behind the preference towards the α- or β- proteolytic pathway of APP is not clear (Baranello et al., 2015, Lesne et al., 2006, O'Brien and Wong, 2011). Uncovering the molecular mechanisms of APP processing led to the hypothesis that amyloid plaques are the causative agent in AD progression.

(12)

6

1.3 The Amyloid Hypothesis

The amyloid hypothesis, which states that abnormally high Aβ concentration underlies neurodegeneration, is supported by the following observations: 1) amyloid deposition

precedes tau abnormalities 2) familial and sporadic AD share neuropathological features 3) Aβ oligomers are neurotoxic and 4) APP mutations that decrease Aβ production hinder AD development (Baranello et al., 2015). Mice with human APP develop amyloid plaques within approximately 45 days, display spatial memory impairment, and synaptic disfunction.

Immunotherapy applied to AD models shows constant Aβ levels but prevents tau tangle development and APP mice crossed with tau knock-out models result in normal memory function despite amyloid deposits (Heber et al., 2000). This indicates that tau tangle development is downstream response to rather than co-occurring pathology with amyloid plaque accumulation. Amyloid peptides were observed to cause neuron death in cell cultures within 24 hours of injection which is attributed specifically to Aβ42 monomers(Butterfield, 2002) and insoluble amyloid plaques. Clearance of Aβ is recognized a potential causative process in the large Aβ42 : Aβ40 ratio and change in Aβ42 concentration in the CSF compared to the brain seen in AD patients.

Research to develop AD treatment has been inspired by the amyloid hypothesis; drugs targeting the production of amyloidogenic peptides and their production have targeted various steps in the pathway. Though it seems like elimination of Aβ42 – a neurotoxic form of the amyloid peptide – production is an obvious treatment option, immunotherapy against this specific protein caused encephalitis in human patients (O'Brien and Wong, 2011) among other side-effects. The three most affected subjects showed almost no amyloid plaque deposition upon post-mortem examination, yet exhibited significant AD clinical progression related to behaviour and memory as well as neuroinflammation (O'Brien and Wong, 2011). Because 80% of AD cases exhibit amyloid deposits on the blood vessel walls in the CNS also known as cerebral amyloid angiopathy (CAA), BACE-1 function could be implicated as a culprit (Devraj et al., 2016). Increased BACE-1 activity may be encouraged by oxidative stress and has been observed in early AD tissue (Guglielmotto et al., 2009). Therapies aimed at BACE-1 may have wide-reaching implications as experiments with BACE-1 knock-out mice showed improved cognitive function through behavioural tests, but abnormal neuronal myelination (Devraj et al., 2016, Nikolaev et al., 2009). Treatments targeting β-secretases or BACE-1 inhibitors have been developed in hopes of hindering amyloidogenic peptides (Baranello et

(13)

7 al., 2015). Immunotherapy aimed at γ-secretase inhibition reduces amyloid plaque load, but dramatically alters T and B cell maturation and immune function. Amyloid plaques have been seen as a risk factor for dementia, but autopsies and neuroimaging have shown that

cognitively normal elderly patients can exhibit AD and cerebrovascular pathology without AD symptoms (Makin, 2018). Thus, researchers and physicians began to expand their search beyond the amyloid itself.

1.4 Brain Response to A β

The brain is potentially the most complex biological system. It is responsible for information processing, storage, and retrieval. Nervous system evolution began with bacterial ion gradients, ion channels, and K⁺ or Ca²⁺ voltage-gated channels designed to respond to environmental stimuli (Purves, 2008). Prokaryotic chemotaxis and chemosensitivity was the basis for neurotransmission within the first sensory neural elements. Central nervous systems (CNS) in animals with specialized organs led to further specialization and complexity

(Verkhratsky et al., 2010). The CNS consist of two cell types: neurons and glia. Neurons conduct electrical signals over long distances from the cortex to the periphery (Purves, 2008).

Glial cells were thought to simply serve as the glue or structural support in neural

development. As investigatory tools evolved, so did the knowledge of glial cell function. Now it is believed that glia play a role in waste removal from the brain parenchyma (Nedergaard, 2013), mediation of the inflammatory response (Heneka et al., 2015), and connection to cerebral blood flow (Perez-Nievas and Serrano-Pozo, 2018, Verkhratsky et al., 2010).

The CNS response to molecular injury such as amyloid plaque build-up could uncover new paths for preventing or treating AD. The characteristic neuropathology of AD patients includes 3 key features: 1) neurofibrillary tangles of the tau protein 2) amyloid plaques of the Aβ protein and 3) neuronal loss (Drew, 2018). The development of tau tangles and amyloid plaques contribute to neuronal loss and glial response through the mechanisms depicted in Figure 3.

(14)

8

Figure 3 | The amyloid hypothesis of AD development. Amyloid precursor proteins are processed in the neurons and released in to the

extracellular space as amyloid-β (Aβ).

Amyloid oligomers enter the bloodstream and activate the innate immune responses of microglial cells.

The release of inflammation response molecules by microglia may also contribute to neuron disfunction. Aβ oligomers form larger structures called amyloid plaques that eventually cause neuronal death. Apolipoprotein E (ApoE) supports amyloid plaque formation and regulates immune function. Plaque numbers and size may be reduced by Aβ-degrading enzymes like insulin degrading enzyme (IDE). Another protein Figure adapted from (Mucke, 2009)

Though APP is produced in large quantities, it is quickly processed by the ER and Golgi, sent to axons, and transported to the axonal terminal (Lee and Warchol, 2008).

Clearance mechanisms involve degradation by lysosomes or Aβ degrading enzymes. Axonal pruning or neuronal degenerative roles have been suggested from the interactions reported between the APP extracellular domain binding to death receptor 6 which activates caspase 3 and caspase 9 causing cell body apoptosis and axonal apoptosis respectively (Nikolaev et al., 2009). Because the amyloid peptides and subsequent oligomers are found in the extracellular space, an intricate interaction between glial and neuronal cells – as seen in Figure 3 – occurs during the development of AD. In clinical studies, APP interactions with ApoE in the cerebral spinal fluid of AD patients has been reported. ApoE, a cholesterol chaperone found in the blood stream, was shown to bind an APP fragment in protein experiments (Purves, 2008).

One ApoE allele, ε4, has been identified as a risk factor for AD because the allele is four times more prevalent in those who develop the disease and ε4 homozygous individuals are 8 times more likely to develop AD (Tang and Gershon, 2003). Though ApoE ε4 increases AD susceptibility, it is not sufficient to cause AD like PS1 and PS2 mutations (Tang and Gershon, 2003).

The brain’s first immune defence relies on microglial activity (Heneka et al., 2015).

Microglia initiate the innate immune response to neuron death and debris as well as maintain neural circuits by protecting and remodelling synapses. Phagocytosis of Aβ by microglia degrade peptides through endosomal pathways and extracellular proteases (Heneka et al.,

(15)

9 2015, Mucke, 2009) like insulin degrading enzyme (IDE) (Tundo et al., 2013). Microglial priming, the increased sensitivity to neuroinflammation resulting in increased cytokine and reactive oxygen species production and phagocytic activity, occurs in neurodegeneration and AD mouse models. Reactive astrocytes and activated microglia are found surrounding Aβ deposits in AD pathology and are the largest source of cytokines, which are proteins secreted by immune cells to act locally as immunoregulatory agents (Perez-Nievas and Serrano-Pozo, 2018, Sofroniew and Vinters, 2010, Verkhratsky et al., 2010). Those Aβ peptides can restrict cerebral blood flow by entering the blood stream affecting functional hyperaemia in neuronal activation responses. Reduced cerebral blood flow hinders oxygen delivery causing cerebral hypoxia, neuronal injury, and directly affects APP processing to favour amyloidogenic peptides (Heneka et al., 2015). Microglia respond to the abnormal proteins by releasing inflammatory mediator molecules or acting as macrophages and engulfing the peptides by phagocytosis (Heneka et al., 2015, Nedergaard, 2013).

Another class of glial cells, astrocytes, are also activated during AD progression (Heneka et al., 2015, Perez-Nievas and Serrano-Pozo, 2018, Sofroniew and Vinters, 2010, Verkhratsky et al., 2010). Astrocytes are specialized glial cells that support synaptic

transmission and circuit formation. Human astrocytes are approximately double the size with 100 times more synaptic connections per cell compared to of those in rodents (Sofroniew and Vinters, 2010). Both types of astrocytes, protoplasmic found in grey matter and fibrous found in white matter, contact blood vessels. Astrocytes coordinate blood flow with neuronal activity by releasing compounds that dilate or constrict blood vessels in response to synaptic transmissions (Sofroniew and Vinters, 2010). Neurons release glutamate that activates Na²⁺

glutamate dependent transporters that activate lactate synthesis, which is then released as a metabolic substrate for neurons (Purves, 2008). Astrocytes uptake extracellular glutamate from neurons and convert it to glutamine. The feedback loop between astroglia and neurons illustrates the interconnected activity between the two cells which maintains homeostasis and prevents excitotoxicity (Verkhratsky et al., 2010).

Astrogliosis, which develops from neuronal injury, has been observed in multiple sclerosis (MS) and AD patients (Sofroniew and Vinters, 2010, Heneka et al., 2015). Glial fibrillary acid protein (GFAP) was isolated from MS patients and found to be highly

concentrated around demyelinated plaques and the main stem branches of reactive astrocytes (Rosengren et al., 1995). Though it serves as an astrocyte biomarker, GFAP does not

(16)

10

accurately represent the extent of astrocyte branching networks like Golgi staining, GFP, or fluorescent dyes (Sofroniew and Vinters, 2010). While reactive astrogliosis is proportional to cognitive decline, studies have shown mixed results when trying to establish differences in GFAP expression between AD and non-AD brains (Perez-Nievas and Serrano-Pozo, 2018).

Astrocytes react to neuronal injury in order to protect and repair the tissues, though the reactivity may contribute to functional impairment and hindering synaptic transmission.

Release of nitric oxide, cytokines, interleukins, and other potentially neurotoxic compounds may contribute to neuroinflammation in response to Aβ deposits. Astrocytes regulate Aβ degrading proteases such as insulin degrading enzyme (IDE) and are involved in ApoE lipidation, which encourages Aβ clearance by microglia (Heneka et al., 2015). The concert of pathological developments during AD contributes to the decline in cognitive function

associated with the disease.

1.5 Cognitive Function

Along with the complex mechanisms of molecular and physiological function in the brain, psychological processes like cognition and sleep are sophisticated subjects.

Investigating cognition is complicated by the inability to directly observe these functions.

Therefore, research relies on inferring cognitive function from behaviour (Robbins, 2011).

Declarative memories are categorized as episodic or semantic in nature. Episodic memories include those of time, events, and location that describe past experiences. In humans, episodic memories can be communicated to answer the questions of what, when, and where something occurred. Because we cannot communicate with mice to ask them to recall details of

declarative memory, the spatial navigation functions are tested by the Morris water maze (Morris, 1981).

The hippocampus readily integrates new information into the neural networks, but cortex works with the hippocampus before independently storing information so that it is independent of hippocampal function. Shortly after acquiring new information, the

hippocampus dictates how the cortex responds in remodelling networks. Imaging studies have shown that retrieval of recent spatial memories involved both the hippocampus and cortex, while recall of remote spatial memories activated mainly cortical regions. In studies of brain injuries, lesions in the hippocampus affected recent memories while cortex damage disrupted more distant information (Frankland and Bontempi, 2005).

(17)

11 Because different areas are affected at differing time points in AD progression with respect to the AD mouse model as demonstrated by Radde et al (2006), specific tasks during behavioural analysis addressed these functional areas. Probe trials employ spatial reference memory; mice must use distal cues to navigate to the platform area which was reinforced through training trials. Other water maze protocols use probe trials directly after training to measure recent memory (D'Hooge and De Deyn, 2001). The depiction of the MWM (Fig. 4) below represents the experimental setup.

Figure 4 | Morris water maze experimental setup. Distal cues mounted on each of four walls surrounding the pool may serve as navigational tools. The edges of the pool are marked with N, S, E, and W as reference when introducing the mouse to the appropriate starting position. A clear plastic platform is submerged so the mouse cannot see it and must use the external distal cues to orient itself to the target. The submerged platform can be moved for differing trial purposes but remain at the SW position during training. Mouse movement and position are recorded by an overhead camera; software such as BiObserve can generate measures such as latency, proximity to platform, and percentage of time spent near the platform. Figure generated in BioRender software.

Animals are thought to exhibit episodic-like memories that may be comparable to human episodic memory. The purpose of the maze is to test the function of hippocampal place cells which help rodents navigate spatial orientation tasks (O'Keefe, 1976). Poor performance during training trials implies individual or group memory impairments. Thanks to tracking software, multiple dimensions of subject performance are measured such as latency, swim

(18)

12

length, swim distance, strategy employed, and etcetera. Out of these measurements latency is most commonly used to assess learning and acquisition of the task, path length is most used for spatial strategy and navigation capabilities, and maze solving strategy as a qualitative measure is used to confirm success or failure of task (Guidi and Foster, 2012).

The best-characterized mouse model with respect to aging and neurobiology is

C57Bl/6J in making it a suitable background for genetic manipulation. Figure 5 compares the aging process in mice and humans.

Figure 5 | Comparative aging between C57Bl/6J mice and humans. Data was collected as a survival curve of 150 female and 150 male C57Bl/6J mice.

Maturational rates were based on physiological and biochemical aging markers. Mice are said to be young adults at 3 months of age because sexual maturity begins at approximately 35 days of age.

Mice past 6 months of age exhibit age-related biomarkers typical for senescent changes. The mature adult age for mice is approximately 3-6 months which corresponds to 20-30 years of age in humans.

Within this timeframe, the mouse also ages 45 times faster than humans. This would indicate that a day for a mouse at this age is approximately a month and a half in human time. Figure adapted from (Flurkey, 2007) originally published in (Fox, 1981).

Relative life phase with respect to age in C57Bl/6J mice and humans (Fig. 5) was based on physiological changes and biomarkers (Fox, 1981). One physiological change that marks the transition from adolescence to mature adulthood is sexual maturity which is seen in mice after 2 months. Thus, adulthood in mice is estimated around 3-6 months whereas humans begin adulthood at approximately 20 years old. Molecular biomarkers that signaled cell senescence were also considered in characterizing this mouse model (Flurkey, 2007). Because the C57Bl/6J mouse model, colloquially known as “black 6,”

(19)

13 is well characterized including the sequencing of its genome (Flurkey, 2007) it is an ideal model for aging and disease research through genetic modifications that produce the disease pathology of interest.

1.6 Modeling AD Pathology

Known AD mutations from early-onset heritable cases are used in genetic

engineering of mice with human APP (hAPP) transgenes. Leucine to proline mutation at position 166 of presenilin 1 (PS1) is the most aggressive form of familial AD mutation, which has been reported to cause the highest Aβ42 to Aβ40 ratio among all familial AD mutations (Alzforum, 2019). Figure 6A depicts the genetic mutations within APP found in Swedish patients exhibiting familial AD while Figure 6B shows the mutations in PSI that cause disfunction of γ-secretase and leads to an increased production of

amyloidogenic peptides.

Figure 6 | Genetic manipulations made in APP/PSI transgene model of AD. A) Depicts the amyloid precursor protein (APP) form found in familial AD cases from Swedish patients. The aptly named Swedish mutation is used in disease models based on the amyloid hypothesis that AD disease characteristics are caused by plaque accumulation. APP genetic mutations recode the 670^th amino acid lysine (K-crossed out) to asparagine (N-red) and methionine (M-crossed out) at position 671 is replaced with leucine (L-red). Purple text annotates the N-terminus of APP as well as the cleavage sites for α-, β-, and γ-secretases. B) Presenilin 1 (PSI) L166P mutation is found in heritable AD cases. The 166^th amino acid leucine (L-crossed out) is replaced with proline (P-red) causing tau proteins to aggregate in neurofibrillary tangles. Adapted figures from (Alzforum, 2019) to represent mutations in APP/PSI transgene model organisms.

(20)

14

The Swedish mutation substitutes lysine for asparagine at the 670 position and methionine for leucine at the 671 position (KM670/671LN). Disease related γ-secretase subunit mutation of PSI replaces the 166 amino acid leucine with proline. Genetically modified mouse models for heritable AD (Radde et al., 2006) use both the Swedish APP mutation and PSI mutation shown in Figure 6. In order to direct the expression of the human mutated genes to the rodent brain cells, the Thy1 promoter used to because of its neuron specificity. Thy1 promoter was used to achieve high levels of neuron-specific transgene expression after birth and causes a 3 fold increase of hAPP expression compared to endogenous murine APP expression (Radde et al., 2006). Figure 7 demonstrates the disease progression over an 8-month period with the APP/PSI transgene mouse model.

Figure 7 | Establishment of AD pathology in mouse model. APP/PSI transgene animals showed that A) amyloid deposition, detected with NT12 antibody, started at 6-8 weeks of age and increased dramatically with age in the cortex. B) Hippocampal amyloid deposition started later than in the cortex, but also increased with age. Amyloid coverage percentage of cortex and hippocampal areas was only reported as significantly different between 1-8 months. C) Microglial activation or microgliosis, detected by Iba1 antibody, occurred in locations corresponding to Aβ plaques. Enlarged or hypertrophic microglia were observed to envelop amyloid plaques; the number of microglia were significantly larger than non-transgenic animals starting at 4 months of age. D) Astrocytes positive for GFAP displayed similar activation to that of microglia. Rather than staying confined to amyloid plaque deposits, astrocytes covered the cortex at 8 months of age. Scale bar is 100 µm. Pictures modified from (Radde et al., 2006) to summarize findings relevant to this study.

(21)

15 Figure 7A shows the progression of amyloid deposition over time in APP/PSI

transgene mice. Significant deposition is seen at month 8 in the cortex and was described as devastating to the brain region. Hippocampal deposition starts later as shown in Figure 7B where 8 months of age does not show the same degree of plaque coverage as in the cortex. In response to the high degree of coverage within the cortex, as the transgene mouse passes from mature adulthood into middle age, glial cells such as astrocytes and microglia are activated to respond to the proteinaceous infection. Microgliosis, or the activation of microglia, in Figure 7C is pronounced in immunohistochemical analysis at 8 months of age. The brain immune cells are enlarged and appear to be in the process of phagocytosis in response to the amyloid proteins. Astrogliosis, or the activation of astrocytes, coincides with the increased expression of GFAP shown in Figure 7D by the antibody directed staining that target the reactive protein. No changes in neuronal count were observed in this study, thus it was concluded that neuronal loss does not occur in this AD model up to 8 months of age. The pathological features accompanied behavioural deficits in tasks like the water maze (Serneels et al., 2009).

1.7 Phytoextracts as Treatment

Animal models like these have been promoted as useful tools for investigating therapeutic options. Other fields of curative research have shown us that, though mouse models may mimic pathology found in humans, the two organisms are fundamentally different (Flurkey, 2007, King, 2018) especially with respect to metabolism. Drug

development efforts, designed and tested in mice, have been thwarted by severe side effects or inefficient bioactivity in humans. Difficulties such as blood-brain barrier crossing complicate pharmaceutical intervention (Husain et al., 2019). While familial AD is useful in research as a model to investigate disease mechanisms, it is exceedingly rare compared to late-onset

sporadic AD which suggests that most cases arise from environmental factors. Relatives of those affected by sporadic AD are more susceptible to the disease which implies that the disease is epigenetic and not genetic; lifestyle and habits are a factor which tilts the argument towards nurture rather than nature (Kivipelto et al., 2018, Sohn, 2018).

The widespread and prolonged nature of AD makes the prospect of affordable and attainable treatment an important endeavour. Traditional medicinal plants have been used to alleviate wide-ranging ailments from diabetes to malaria (Bhattacharya et al., 1999, Variya et

(22)

16

al., 2016). One such traditional plant remedy is amla or Emblica officinalis, which has been used recently in several different human trials as an alternative to pharmaceutical remedies.

Promising studies have been conducted to suggest that plants contain active moieties that contribute to clearing of amyloid plaques typical of AD (Hofrichter et al., 2016, Husain et al., 2019, Justin Thenmozhi et al., 2016, Yuan et al., 2016). Potential mechanisms of success may be attributed to microbial metabolites that have direct or indirect effects on cognition and pathology via the gut-brain axis (Mayer et al., 2015, Pistollato et al., 2016).

Amla fruit, the common name for Emblica officinalis, administered orally at 3 g daily in human trials worked as well as glibenclamide (Daonil®) medication in diabetic patients in lowering blood glucose levels and outperformed the drug in improving lipid profiles (Akhtar et al., 2011). Daonil® administered twice daily (5 g) was compared to amla powder (1, 2, or 3 g) administered orally once a day over a 21 day period. Analyzed blood glucose levels and lipid profiles showed that 3 g amla powder daily resulted in similar health affects as Daonil®.

Type II hyperlipidemic patients were used to compare the effects of amla (500 mg) to statin (20 mg) over a 42 day period (Gopa et al., 2012). Analyses conducted in these two studies showed that amla significantly reduced total cholesterol, triglycerides, and blood pressure in humans suggesting potential utility as a therapeutic option (Akhtar et al., 2011).

Studies conducted in animal models have shown that amla fruit exhibits

neuroprotective, anti-oxidant, and anti-inflammatory properties (Golechha et al., 2012, Koshy et al., 2012, Ojha et al., 2012, Singh et al., 2014, Malve, 2015). These studies tested memory performance by inducing amnesia through scopolamine and assessing the counteractive effects of amla. Other studies have shown that amla extract reduces biomarkers of oxidative stress in heart muscle cells (Ojha et al., 2012), prevention of insulin resistance in high fructose diets (Koshy et al., 2012), and protected against oxidative stress by arsenic administration (Singh et al., 2014) in liver cells. They used varying methods of treatment from

intraperitoneal injection to oral administration by gavage. The range of extract concentration included 100-600 mg/kg body weight of mice when using ethanol extraction. Thus, amla seems to be a promising avenue of AD prevention and symptom alleviation.

(23)

17

1.8 Overview of Study

Previous work has demonstrated that amla extract administered to rodents protects against memory impairment (Golechha et al., 2012, Malve, 2015). The experiments from the current study are depicted in Figure 8 which shows the treatment or test performed respective to the age of mice involved.

Figure 8 | Schedule of control and phytoextract experiments with respect to mouse age. Mice were transferred from the breeding facility at

approximately 35 days old and treatment began at 40 days during the pre-onset of amyloid deposition. Establishment of this model (Radde et al., 2006) showed that amyloid deposition started 6-8 weeks after birth represented here as 45 days.

Morris water maze testing started after mature adulthood was reached,

approximately 3 months, and continued for 9 days. Mice slept in their home cages after completing water maze protocol and were placed in individual activity monitoring cages where movement was counted by InfraMot sensors. Figure created in BioRender software.

The design of this study included treatment via gavage pre-onset of AD pathology.

Behavioural tests were designed to take place during the timeframe of mild cognitive

impairment in order to assess the earliest time possible for detecting cognitive abnormalities.

The preventative model aims to detect slight deficits such as those self-reported by patients, which impair normal function but not dramatically enough to affect daily life. Monitoring daily activity of AD models is used to assess deviations from the normal circadian rhythm – which is opposite in mice because they are nocturnal – because reported sleep disturbances are also observed in AD patients (Gravitz, 2018). Brain tissues show the molecular and cellular progression of AD and techniques like western blotting and immunohistochemistry allow researchers to analyze treatment effects. Tissue collection at 110 days of age in this experiment aimed to see the early effects of AD and potential therapeutic action of treatment.

Correlating behavioural performance with pathological progression, theories of memory and cognitive function play an important role in data interpretation. Injuries and cognitive loss in humans have provided case-study insights into the connection between brain physiology and function. Henry Molaison was one such case-study (Squire, 2009). To treat epilepsy, his hippocampal regions were surgically removed. While the hippocampal removal

(24)

18

relieved epileptic seizures, he lost the ability to form new memories. This loss of working memory was not combined with the loss of older, remote memories that were made and solidified before his surgery. Networks connecting the cortex and hippocampus work in concert to acquire new information from the surroundings and consolidate that information into long-term storage. Because his hippocampus was surgically removed, this network was disrupted and memory retrieval of recent events post-surgery was not possible. The

acquisition and consolidation of information into long-term storage is also dependent upon the prefrontal cortex. The cortex is responsible for integration of sensory stimuli into cognition.

Because MWM navigation is dependent upon hippocampal function (Moser and Moser, 1998), post mortem analyses were carried out in the hippocampal regions in Figure 9.

Figure 9 | Analysis of AD pathology from mouse brain tissue. Brains from experimental subjects were collected and sectioned coronally at the position indicated by the yellow bar and detailed representation taken from The Mouse Brain (Paxinos, 2007) reference book. Pink and orange areas indicate the cortex and hippocampus regions of interest, respectively. Created in BioRender software.

The APP/PSI transgene model has been shown to develop robust plaque pathology in the cortex earlier than in the hippocampus; both regions are integral in memory recall and cognitive function. In humans, the development of AD pathology starts in the forebrain and continues its destructive march towards the brain stem (Braak and Braak, 1991). Hippocampal regions are crucial to spatial navigation as evidenced by the molecular confirmation of

cognitive maps (Fyhn et al., 2007, Hafting et al., 2005). The aim of this project was to test the behavioural and physiological effect of phytoextract intervention in AD mouse models using APP/PS1 mouse model treated with amla fruit extract to assess the efficacy of phytoextracts on amyloid deposition and AD progression.

(25)

19

2 Methods

Two independent experiments were performed: first, a control experiment to establish the difference between wild type and transgene mice and second, the phytoextract experiment to assess differences between water and plant treated transgene mice. Water treatment for the control experiment did not require phytoextraction contrary to the Figure 10 depiction of experimentation workflow. Nevertheless, the experiments were conducted in the same manner by treating animals during AD pre-onset at approximately 40 days old. Behavioural tests were administered at the age of 95 days approximately. Tissue collection at a consistent mouse age of 110 days was essential for comparable pathological and biochemical results. Finally, protein and immunohistochemistry analyses were conducted to assess the disease progression of each group.

Figure 10 | Experimental Workflow. The graphic outlines the time points and procedures performed for testing amla extract in APP/PSI transgene mice. Control experiment using C57Bl/6J and APP/PSI transgene groups followed the same outline, but did not include the phytoextraction step because both groups were treated with water. Behavioural experiments (MWM & InfraMot) began at an average of 95 days of age. Brains were collected at 110 days of age so that disease progression was consistent with respect to age for each subject.

Brain hemispheres were stored separately with respect to the downstream analysis of either western blotting or IHC.

(26)

20

2.1 Alcoholic Extraction of E. officinalis

Extraction with ethanol results in the most comprehensive collection of moieties.

Ethanol is known as the universal solvent because its hydroxyl group binds to hydrophilic molecules like tannins while the ethyl group binds hydrophobic compounds. Thus, ethanol extraction was performed on amla powder to collect a broad range of compounds.

Biochemical analysis was performed to give a general overview of the moieties present and confirm previous characterization of amla fruit components. Analysis of functional groups within Emblica officinalis have included nuclear magnetic resonance (NMR), high

performance liquid chromatography (HPLC). NMR and HPLC-DAD have been used in flavonoid identification because of the separation efficiency of HPLC with the sensitivity of NMR (Zhang et al., 2011). Outputs including the UV chromatogram and heat map illustrate the movement of extracted moieties. The UV chromatogram shows how the extract was eluted from the column while the heat map represents three dimensions of the elution by showing retention time vs wavelength absorption vs intensity or concentration of the sample moiety. Flavonoids register as two peaks in the heat map around 300-380 nm and 240-280 nm as absorption peaks.

2.1.1 Phytoextraction

Extraction by Thermo Scientific Dionex™ ASE™ 350 Accelerated Solvent Extractor was performed on 300g amla fruit powder seen in Figure 11 (Ayushya Panchakarma Home Health & Research Centre Ltd) while 41g was set aside for biochemical analyses. The system was rinsed with acetone before use to remove residual plant material. Extraction was

programmed using a 4:1 ethanol to water protocol where the temperature was raised to 60°C for five minutes, then five minutes static time, and solvent was purged for 100 seconds at 50%

rinse volume. Diatomaceous earth (DAE) powder was used to reduce pressure inside the extraction cell. Plant mass to DAE mass was mixed at a 3:1 ratio and cells were packed using filter paper at the bottom of the cell and DAE surrounding the plant-DAE mixture.

(27)

21

Figure 11 | Powdered amla fruit used in Phytoextraction.

Emblica officinalis Gaertn.

(additionally called Phyllanthus emblica, Indian gooseberry, or amla) was obtained as 500 g powdered fruit sample by Ayushya Panchakarma Health Home & Research Centre Ltd, packaged January 15, 2016. Dried fruit samples were included.

Evaporation of the ethanol-water solvent was performed using the Wilmad-LabGlass IKA RV10 Rotary Evaporator 10 B S40 from VWR. Pressure was reduced using the pressure vacuum Chiron AS and kept within the 120-180 Mbar range. A 200 mL glass round bottom flask was used to incrementally evaporate ethanol from the plant extract. An average temperature of 40 °C and rotation of 100 rpm was used. After evaporation, the sample was kept at 4 °C for 48 hrs and then transferred to -20 °C before freeze-drying with the Martin Christ Alpha 1-2 LDplus Entry Freeze Dryer Package system.

2.1.2 Treatment Preparation

After reviewing relevant literature, a mid-range dosage was chosen based on those previous studies and their positive results (Malve, 2015, Singh et al., 2014, Dhingra et al., 2012). Solutions were prepared from plant extract material and ddH2O to a concentration of 300mg/kg respective to body weight. Calculations were made with regard to the duration of treatment, number of mice, and average body weight.

(300𝑚𝑚𝑚𝑚 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒)/(𝑘𝑘𝑚𝑚 𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 𝑤𝑤𝑒𝑒𝑤𝑤𝑚𝑚ℎ𝑒𝑒) × 1𝑘𝑘𝑚𝑚/1000𝑚𝑚× 20𝑚𝑚/𝑚𝑚𝑏𝑏𝑚𝑚𝑚𝑚𝑒𝑒= (6𝑚𝑚𝑚𝑚 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒)/𝑚𝑚𝑏𝑏𝑚𝑚𝑚𝑚𝑒𝑒 6𝑚𝑚𝑚𝑚/𝑚𝑚𝑏𝑏𝑚𝑚𝑚𝑚𝑒𝑒× 10𝑚𝑚𝑤𝑤𝑒𝑒𝑒𝑒× 100𝑏𝑏𝑒𝑒𝑏𝑏𝑚𝑚= 6𝑚𝑚 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑛𝑛𝑒𝑒𝑒𝑒𝑏𝑏𝑒𝑒𝑏𝑏

0.1𝑚𝑚𝑚𝑚/𝑚𝑚𝑏𝑏𝑚𝑚𝑚𝑚𝑒𝑒/𝑏𝑏𝑒𝑒𝑏𝑏 × 10𝑚𝑚𝑤𝑤𝑒𝑒𝑒𝑒 × 100𝑏𝑏𝑒𝑒𝑏𝑏𝑚𝑚 = 100𝑚𝑚𝑚𝑚 𝑚𝑚𝑏𝑏𝑠𝑠𝑚𝑚𝑒𝑒𝑤𝑤𝑏𝑏𝑛𝑛 𝑛𝑛𝑒𝑒𝑒𝑒𝑏𝑏𝑒𝑒𝑏𝑏

Thus 3 g of plant extract in 50 mL ddH2O or 1.5 g plant extract in 25 mL ddH2O could be prepared and aliquoted for use. The dosage of 300 mg/kg of body weight corresponds to

(28)

22

60 mg/mL administered in 0.1 mL dosages to each mouse. Estimating that each mouse weighed approximately 20 g, the 0.1 mL dosage per mouse resulted in 6 mg per mouse per day.

Plant extract suspension in water was stored in aluminum foil during transport to avoid potential photosensitive degradation, and the solid extract was slowly dissolved with magnetic stirrer to avoid potential oxidation by excessive exposure to air and bubbles. Solutions were stored at Komparativ Medisin (KPM) in Radiumhospitalet. The phytoextract samples were stored at -20°C in 1.5 mL aliquots to protecting plant extract activity and standardize treatment procedure.

2.1.3 Biochemical Analysis

Emblica officinalis extract (80% ethanol extraction, dissolved in 50% methanol, filtered through 0.45 µm pores) was separated by high powered liquid chromatography (HPLC, LaChrom Elite, Hitachi) using reverse phase C18 column (Phenomenex, 5µMm, 150x4.6mm) and analyzed with an L-2455 diode array detector (DAD). The extract was eluted through the column using a mobile phase of water with 0.1% triflouracetic acid and varying gradients of methanol (0-5 min 2%, 5-50 min 2-90%, 50-55 min 90%) with a flow rate of 1.0 mL/min. Absorbance values were recorded at 230, 280, 320, 350 nm and elution was carried out at 25 °C. Proton NMR spectrum was obtained in 128 scans using deuterated methanol on the Bruker AVII 400 spectrometer.

2.2 In Vivo Experimentation

The Morris Water Maze (MWM) maze is used as a standard rodent memory test specific for spatial orientation and hippocampal function (D'Hooge and De Deyn, 2001, Guidi and Foster, 2012, Malve, 2015). Morris measured path length or the distance swam from start to platform and latency or time elapsed during trial until completion as analytical measures (Morris, 1981). Training trials are used so the animal may learn to use visual cues in

navigating to the submerged platform in order to escape the maze. In probe trials the platform is taken away and the animal swims for 30s were utilized to assess learning acquisition and spatial preference for the target location (Guidi and Foster, 2012). Visual cue trials are employed to assess visual and motor capabilities of each subject to control for any pre- existing physical shortcomings.

(29)

23 Analytic strategies include repeated measures ANOVA (analysis of variance) for trial tests in measures of latency or time to reach the platform and path length to the platform. It has been suggested that multi-factored ANOVA may be used for probe trials and that one- tailed Student’s t-test may be used for spatial strategy and percent of time in platform

quadrant for testing trials and visual cue trials (Guidi and Foster, 2012). While these statistical models may all be employed in the given circumstances, this introduces the potential for inconsistencies in significance determination. Thus for the following experiments, one analytical method (repeated measures ANOVA) was employed for each measure to maintain continuity and allow comparison.

Two experiments were conducted: one referred to as the control experiment

comparing the wild-type C57Bl/6J to the APP/PSI transgene, and the phytoextract experiment which compared two APP/PSI transgene groups of water treated and amla treated mice. The purpose of the control trial was to establish a baseline of performance in animals that had been handled and gavaged. This trial was expected to show that C57BL/6J mice learned and remembered significantly different from APP/PSI transgenic positive mice. This was done to show that the experimenter’s handling of mice during gavage treatment and the execution of the water maze test was not a significant confounding variable. Genetic constructs were verified with polymerase chain reaction (PCR). At the appropriate age, treatment was

administered via oral gavage which directed the solution to the animal’s stomach. Analysis of memory was performed by water maze and activity was monitored thereafter. Finally, brains were collected for further analysis.

2.2.1 AD Mouse Model

The best-characterized mouse model with respect to aging and neurobiology is C57Bl/6J in making it a suitable background for genetic manipulation. Known AD mutations from early-onset cases found in Sweden and London were used in genetic engineering to produce the AD mouse model. Mice with human APP (hAPP) genetic constructs display similar AD neuropathology as seen in humans. Leucine to proline mutation at position 166 of presenilin 1 (PS1) is the most aggressive form of familial AD mutation, which has been reported to cause the highest Aβ42 to Aβ40 ratio among all familial AD mutations. The Swedish mutation substituting lysine for asparagine at the 670 position and methionine for leucine at the 671 position (KM670/671LN), is

(30)

24

combined with the PSI mutation associated with AD pathology construct under the Thy1 promoter used to regulate neuron-specific expression within the brain. Thy1 promoter was used to achieve high levels of neuron-specific transgene expression (Radde et al., 2006).

Animal genotypes were verified by polymerase chain reaction (PCR) of tissue biopsies designed to differentiate C57Bl/6J wild type (wt/wt) from APP/PSI transgene (wt/+) animals.

Ear biopsies were collected at approximately 4 weeks after birth. Tissues were lysed using buffer (50 mM KCl, 10mM Tris, 0.4% NP 40, 0.4% Tween 20) to pH 9 and proteinase K, incubated overnight at 55°C for protein digestion, and then one hour incubation at 95°C to inactivate proteinase. Supernatant was collected after centrifugation (14,000rpm for 10 minutes at 4°C) for PCR. The primers shown in Supplemental Data were used for APP/PSI transgene verification. PCR products were separated by gel (agarose and TAE)

electrophoresis with the intercalating agent, ethidium bromide, for visualization.

Mice were bred from a pure C57Bl/6J background and APP/PSI transgenic animals originated from previous studies (Radde et al., 2006). Food (RM3, Special Diet Services, UK) and water were provided ad libitum for each cage which housed 8-10 females in enriched environments including paper and wood chip bedding as well as plastic tube housing. The animal facility (KPM Radiumhospitalet) maintained consistent temperature (21-22°C) and 12h light/dark cycles. For the control experiment comparing C57Bl/6J mice to APP/PSI transgene, 8 females were assigned to each group and resided in respective cages. In the phytoextract experiment comparing APP/PSI transgene animals that were treated with water to APP/PSI transgene mice treated with amla extract, 10 animals were assigned to each group and caged according to treatment.

2.2.2 Oral Gavage Treatment

During the control trial, both wild-type and transgenic animals were given 100µL water daily for approximately 70 days. All animals in the control trial started gavage on the same day for simplicity’s sake. The average age was approximately 40 days old and water was administered until the tissue was collected at 110 days of age. During experimental trial, both transgenic groups were treated for exactly 70 days with a dosage of plant extract

according to approximate weight (300 mg/kg body weight, 60 mg/mL, or 6 mg per mouse) while the placebo group was given an equal volume (100 µL) of water. Treatment protocol

(31)

25 performed in the control experiment comparing C57BL/6J mice to APP/PSI transgenic mice deviated from the standard phytoextract treatment protocol by treating all the mice starting on the same day rather than at the same age because the measure in question was not histology, protein aggregation, or other physiological markers that would have been collected at a specific age, but rather the performance during a behavioural test which started at a specific date. Thus the duration between treatment and experiment was controlled for by starting each with respect to a date rather than mouse age. In the phytoextract treatment trial, each animal started treatment with respect to their age (40 days) because important downstream analyses were performed on tissue samples collected at a specific age (110 days).

2.2.3 Behavioural Analysis

At approximately 95 days of age, animals started the Morris water maze to assess cognitive function. The white pool (120 cm in diameter) was filled with water (22 ±0.5 °C) so that the transparent platform (9 cm in diameter) was 1 cm submerged to allow for escape from the water while standing on the platform. The pool was placed in a corner of the room so that two white walls tangentially lined the S and W edges while white curtains completed the square enclosure. Each wall or curtain displayed one visually distinct distal cue that, according to Pythagorean Theorem, was available to the mouse’s line of sight while

swimming and thus could be used for navigation. Animal performance measured with camera and Viewer3 (BiObserve, Germany) software. Training trials were conducted by gently lowering the mouse into the pool where the mouse was allowed to swim freely for a maximum of 90 seconds. If the mouse failed to find the platform in quadrant 3 at the SW position (Figure 12A), it was guided there and remained for 10 s before being offered escape.

A towel-lined box was presented to the experimental subject for transport from the pool to the heating cage for inter-trial warming for 1 minute before resuming the next trial.

(32)

26

Figure 12 | Water maze protocol. A) The pool was arbitrarily marked with N, S, E, and W references. The pool was divided into quadrants for data collection. Platform position during training trials was kept at the SW demarcation, probe trials were conducted by removing the platform from the pool, and visual cue platform position varied. Days 1-8 include training trials with 3 probe trials at the start of day 4, 6 and 9. The final day of testing consists of visual cue trials conducted after the final probe trial. B) Starting positions for training trials are indicated by blue highlight, while probe trial starting positions are found in green. Platform position was

consistent throughout training trials and was removed during probe trials. Visual cue platform position alternated between NW and SE which corresponded to unique mouse starting positions E, SW, S, and NW. Mice were given new bedding and cages on days 1, 4, and 8. After filling the pool on day 1, the water was drained and replenished on day 5 and drained again on day 9following the completion of visual cue trials. Credit: Surya Rai.

Mice rested for one minute before starting the next trial; the starting position was semi-randomized for each day (Fig. 12B) but the platform remained in Q3 at the delineated position. The experiment included three probe trials (Day 4, 6, and 9) and one proximal visual cue trial. Water maze testing started when mice were approximately 95 days old. Training trials were prepared by heating the water to 22.5 °C and a transparent platform was located in the middle of the SW quadrant. The platform was submerged under 0.5 cm of water so that the animals could comfortably stand there after successfully locating the target. Tween 20 (Sigma-Aldrich) detergent was added to the water to a concentration of 1:10,000 with respect to the pool volume to minimize mouse floating. Previous water maze testing resulted in ear necrosis in some subjects; therefore precaution was taken by covering each animal’s ear with Vaseline (Ophtha, 800 mg) to prevent damage. Unfortunately, some animals still acquired ear damage and in those cases post trial treatment was conducted to minimize pain and suffering.

Four training trials were performed at random starting positions (N, E, NW, SE) each day for eight consecutive days. Successful trials were completed before 90 seconds which was set as the training trial time limit. If subjects did not find the platform in the allotted

timeframe, they were guided to the platform and remained there for 10 seconds before being

(33)

27 transferred to the heating cage. Animals were allowed to rest in the heating cage for 1 minute between trials. On days 4, 6, and 9 probe trials were administered by removing the platform and allowing the animal to swim for 30 seconds starting from the NE position.

Training and probe trials were conducted using distal cues; four black and red pictures as seen in the appendix were mounted on the walls surrounding the tub. The 9^th day was committed to the final probe trial and followed by visual cue trials where a 50 mL Falcon tube filled with blue dye was placed on the platform to control for vision and mobility. Distal visual cue pictures were removed after probe trials so that the only visual cue available was the proximal blue Falcon tube. The platform and blue marker were moved from NW to SE every trial and starting position varied from E, SW, S, and NE as shown in Figure 12 B.

Because latency is not relevant in assessing the success of probe trials, the percentage of swim time spent in the target quadrant was analyzed. Target quadrant percentage occupancy was also used in training trial analysis for consistency and easier comparison.

Training trials were analyzed to assess how the subjects learned the tasks according to the search strategy, latency, percentage of time spent in target quadrant, and proximity to target. Probe trials were analyzed by percentage of time spent in target quadrant, proximity to target, and target zone crossings (comparable to training trial latency) as measures for task acquisition and memory. Visual cue trial data was analyzed according to latency and search strategy so that animals lacking visual acumen or normal swimming ability could be excluded as abnormal.

Experimenter observations were noted according to a coding system (Supplemental Data). The raw data collected from the Viewer software was then adjusted accordingly if discrepancies were noted. Trials in which the animal was drowning (Z) and the experimenter manually stopped (G) the experiment were corrected by ensuring the task latency was 90s indicating failure of the task. If an animal climbed atop the platform (√), yet was not within the region of interest designated by the software (#), and the task was completed because the mouse stayed atop the platform for 10 s, then the experimenter manually stopped recording (G) and trials were corrected by subtracting 10s from recorded latency. Trials where the animal found the platform (√) and jumped off (B) before 10s had elapsed, then the final recorded time was accepted. When animals touched the platform (A) but did not climb atop (√) – if the software stopped recording and interpreted the touch as success – then the latency was corrected to show the actual time of platform discovery or failure of the task if the animal

(34)

28

did not return. Data was compiled in Microsoft Excel and analyzed through repeated measures two-way ANOVA in SPSS (IBM) software according to the relevant script (Supplemental Data).

2.2.4 Activity Monitoring

Physical activity was assessed by the infrared sensor-based InfraMot (TSE Systems) over 72 hrs, the first 24 of which were to acclimate animals to the new single-mouse cage environment. Animal movement was tracked by infrared sensors and analyzed (Phenomaster Software, TSE Systems) from 10 minute interval movement counts.

2.2.5 Collection of Brain Tissue

Whole brains were collected when each animal reached 110 ±1 days old. Animal age was tracked by the Microsoft Access program Animal Database generated by the Pahnke lab.

The margin of ±1 day is two-fold: animal ages are not precise (there’s no way to know exactly when animals are born) because they were estimated by a veterinarian according to physical characteristics depicted in the Jackson Lab guide. Animals were sacrificed by cervical dislocation and cardial perfusion was performed with PBS. Brains were extracted, olfactory bulbs and cerebellum areas were removed, and hemispheres were separated along the longitudinal fissure with a scalpel. One hemisphere was flash frozen in liquid nitrogen and stored at -80 °C for later Western Blot analysis while the other hemisphere was fixed in paraformaldehyde (PFA) for subsequent immunohistochemical staining.

2.3 Post-Mortem Analysis

After collection of brain tissue, one hemisphere was allocated for protein analysis via western blot while the other hemisphere was preserved for analysis of pathological markers by immunohistochemistry. Western blots employed primary antibodies to target BACE-1, IDE, and ADAM10 while secondary antibodies conjugated with horseradish peroxidase (HRP) were utilized to visualize proteins through chemiluminescence when developed in luminol and peroxide. In immunohistochemistry the primary antibodies Iba1, NeuN, GFAP, and 4G8 were used to identify microglia, neurons, microglia, and Aβ respectively.

Emblica officinalis as a therapeutic supplement in Alzheimer’s disease