polypeptides in the pyramidal neurons on the hippocampus of a triple transgenic murine model for the Alzheimer’s Disease.

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Instituto Tecnológico y de Estudios Superiores de Monterrey

Campus Monterrey

School of Engineering and Sciences

Effect of the inflammation on the aggregation of the Tau and amyloid-β

polypeptides in the pyramidal neurons on the hippocampus of a triple transgenic murine model for the Alzheimer’s Disease.

A thesis presented by

Pedro Ricardo Moreno Velez

Submitted to the

School of Engineering and Sciences

in partial fulfillment of the requirements for the degree of Master of Science

In Biotechnology

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Dedication

To my parents Ligia and Pedro and my brother Vico. Thanks for all your unconditional confidence, support, patience, and encouragement. You were my main motivation for pushing through this work.

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Acknowledgements

I would like to express my deepest gratitude to all those who have been side by side with me along this journey. To Dr. Miguel Ontiveros for his confidence and believing in me, for always convey his passion for neuroscience and encouraging me to be better. To Dr. Jose Luna and his group for making me feel welcome since the beginning, for helping me and caring for my work. I really enjoyed working with you guys. To Tecnológico de Monterrey for the support on tuition and CONACyT for the support for living.

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Effect of the inflammation on the aggregation of the Tau and amyloid-β polypeptides in the pyramidal neurons on the hippocampus of a triple transgenic murine model for the

Alzheimer’s Disease.

by

Pedro Ricardo Moreno Velez.

Abstract

Alzheimer’s Disease (AD) is the most important form of dementia. It represents 70%

of all dementia cases worldwide. On a molecular level, AD is characterized by the presence of two fibrillar lesions: the neurofibrillary tangles (NFT) due to the oligomerization of pathological forms caused by post-translational modifications (PTMs) of protein Tau and; the aggregation of peptide amyloid-β (Aβ), product of a bad-processing of amyloid precursor protein (APP) forming neuritic plaques (NP).

The triple transgenic mice (3xTg-AD) presents some pathological species of Tau and Aβ aggregations. 3xTg-AD mice received an ethanol treatment to evaluate the generation of proteolyzed forms of Tau through the induction of an exacerbated neurotoxic state Caspase 3 activity was observed accompanied by an increment of NPs in the hippocampus, as well as, the presence of neurofibrillary aggregations of Tau and Aβ.

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List of Figures

Figure 1. Pooled incidence of dementia. ... 12

Figure 2. Staining of brain tissue sections from an AD case. ... 14

Figure 3. Brain atrophy in Advanced AD. ... 15

Figure 4. Processing of APP and generation of Aβ by β-secretase and γ-secretase proteolysis. ... 17

Figure 5. Protein Tau domains and antibody epitopes. ... 19

Figure 6. Model of possible NFT formation and tau aggregation in AD. ... 19

Figure 7. Hippocampus of the 3xTg-AD transgenic mouse. ... 20

Figure 8. Effect of ethanol treatment on the activation of caspase-3 in the CA3-CA4 regions. ... 29

Figure 9. Effect of ethanol treatment on the activation of caspase-3 in the CA3-CA4 regions at 8 h post injection. ... 29

Figure 10. Effect of ethanol treatment on inflammation. ... 30

Figure 11. Increase of the extracellular Amyloid β plaques in the CA1 hippocampal region of the 3xTg-AD 17-month-old mouse injected with ethanol. ... 31

Figure 12. Increase of extracellular Amyloid β plaques and Tau P301L polypeptide in the pyramidal cells of CA1 in the 3xTg-AD mouse injected with ethanol. ... 32

Figure 13. Decrease of protein kinase GSK3β in the Subiculum neurons of the 3xTg-AD mouse injected with ethanol. ... 34

Figure 14. Increase in the expression of protein kinase CDK5 in the Subiculum neurons from the 3xTg-AD with ethanol treatment. ... 35

Figure 15. Increase in the expression of TNF-α and its correlation with astrocytic activity in the Subiculum of the 3xTg-AD mice injected with ethanol. ... 36

Figure 16. Effect of ethanol on fibrillar aggregations. ... 37

Figure 17. Visual representation of the watershed segmentation-based image processing software steps. ... 38

Figure 18. Hippocamp of a mouse which has received the ethanol neurotoxicity treatment and sacrificed at 11 months. ... 39

Figure 19. Hippocamp of a mouse which has received the ethanol neurotoxicity treatment and sacrificed at 20 months. ... 39

Figure 20. Close up of CA1 regions of 3xTg-AD mice. ... 40

Figure 21. Model of the possible mechanism by which Tau is sequentially hyperphosphorylated. ... 44

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List of Tables

Table 1. List of primary antibodies used in immunoassays. ... 27 Table 2. List of secondary antibodies. ... 27

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1. Introduction

1.1. Epidemiology of dementia

The rise of life expectancy in the last decades is accompanied by an intensification of the dementia incidence. According with the World health organization, there are around 50 million people with dementia. Every year there are around 10 million new cases. Alzheimer´s Disease (AD) represents nearly 70% of all the cases of dementia.

As a form of dementia, AD is characterized by the progressive decline of brain functions such as memory, orientation and learning capacity among others.

Accordingly to the World Health organization, there are 50 million people with dementia around the world and every year 10 million new cases appear (WHO, 2019). AD represents 60-70% of all the cases of dementia (Duthey, 2013). Incidence of dementia increases significantly and as age progresses from the age of 65 and onwards for both sex populations (Van Der Flier & Scheltens, 2005).

Figure 1. Pooled incidence of dementia. Adapted from (Van Der Flier & Scheltens, 2005).

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1.2. Alzheimer’s Disease

Alzheimer´s Disease is a neurodegenerative disorder characterized by cognitive impairment, memory loss and the deterioration of other abilities such as speech, writing, visuospatial orientation and locomotion, among others (Ropper, Samuels, Klein, & Prasad, 2019). There is no definitive and opportune diagnosis for this disease. The only confirmatory diagnosis for AD is by postmortem histopathological diagnosis. This is based on the finding of the two molecular hallmarks of AD: the presence of extracellular neuritic plaques (NP) and intracellular neurofibrillary tangles (NFT) (Figure 2.). It has been suggested that the progressive accumulation of these fibrillar lesions in the AD brains is the cause of the degeneration of the cortical, subcortical and hippocampal neurons leading to synaptic and neuronal loss (Campos Peña & Meraz-ríos, 2014).

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Figure 2. Staining of brain tissue sections from an AD case. Staining with silver impregnation of A) a NFT and C) a NP. Staining with Thiazine Red of a B) NFT and D) a NP. Taken from (Luna-Muñoz et al., 2008).

1.3. Histopathogy of AD

The brain of the patients in an advanced stage of this disease present a weight reduction by 20 % or more. The convolutions become narrowed and the sulci widens.

There is also an increase in the ventricular area in the third and lateral ventricles (Figure 4.). Ones of the areas that present extreme atrophy is the hippocampus (Ropper et al., 2019).

On a cellular level, there is massive loss of neurons. On early stages of this disease, this is most notable on the entorhinal cortex. There is a noticeable loss of cholinergic

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neurons in the hippocampus, the parahippocampal gyri and the subiculum (Ropper et al., 2019).

Figure 3. Brain atrophy in Advanced AD. Taken from (Bagad, Chowdhury, & Khan, 2013)

1.4. Etiology of AD

Even though the precise etiology of Alzheimer’s Disease is unidentified, it is known that age is the most important risk factor (Campos Peña & Meraz-ríos, 2014). There are two known origins of AD. The Familial Alzheimer´s Disease represents around 10% of all cases and is characterized by the presence of mutations in three genes that are the amyloid precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2) leading to premature progress of AD (Campion et al., 1999). The sporadic form of Alzheimer’s Disease represents 90% of all cases which development depends mainly on non-genetic risk factors. Although there are several other factors that could influence of AD such as atherosclerosis, diabetes, head trauma, metabolic disorders, and hypertension (Chen, Lin, & Chen, 2009). The most important risk factor is age. The incidence of AD increases by 5% once the age of 65 is reached and by 20% over the age of 80.

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1.5. Amyloid precursor protein and amyloid-β aggregation

The amyloid precursor protein (APP) seems to control interactions with signaling systems that modulate the development of dendritic and axonal processes as well as synaptic maintenance (van der Kant & Goldstein, 2015). APP processing occurs by two different pathways from which the amyloidogenic pathway leads to the generation of amyloid-β (Aβ) peptide (Nalivaeva & Turner, 2013).

The amyloid cascade hypothesis implies that the fibrillar aggregation of Aβ peptide induces AD neuropathologies, even the formation of neurofibrillary tangles (NFTs) resulting in vascular damage, neuronal death and dementia (Tan & Gleeson, 2019).

Amyloid precursor protein is cleaved by secretases into alpha and beta oligomers.

Amyloid-β peptide of 40 and 42 amino acids tend to form aggregate and form amyloid plaques in the extracellular milieu (Shah, Morris, & Wray, 2020).

In the amyloidogenic pathway APP undergoes a sequential cleavage by the β and γ-secretases (Serrano-Pozo, Frosch, Masliah, & Hyman, 2011). The γ-secretase complex is formed by the γ-secretase enzyme and its catalytic core presenilin 1 (PSEN1) or presenilin 2 (PSEN2) and some accessory subunits (Nalivaeva &

Turner, 2013),(Tan & Gleeson, 2019). It is through this sequential cleavage that the Aβ is generated. The Aβ42 possesses a higher insolubility and fibrillization rate and is more abundant within the plaques (Serrano-Pozo et al., 2011).

Neuritic plaques (NPs) are extracellular fibrillar deposits of insoluble Aβ. NPs are associated with neuronal and synaptic loss as well as the activation and recruitment of astrocytes and microglia (Serrano-Pozo et al., 2011), (Lane, Hardy, & Schott, 2018). Amyloid plaques tend to accumulate in the isocortex (Campos Peña & Meraz- ríos, 2014) and the entorhinal cortex and hippocampal formation (Lane et al., 2018).

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Figure 4. Processing of APP and generation of Aβ by β-secretase and γ-secretase proteolysis.

Adapted from (Nalivaeva & Turner, 2013).

1.6. Tau protein and neurofibrillary tangles

The neurofibrillary tangles (NFTs) are the second fibrillar lesion that define the Alzheimer´s Disease and are originated by the post-translational modifications of the protein Tau. Tau is a microtubule associated protein (MAP) which essential role is to give dynamic stability to the microtubules allowing for axon and dendrite formation.

Microtubule network is responsible for transport systems in the axons thus Tau non- functioning leads to synaptic disfunction (Muralidar, Visaga, Sekaran, Thirumalai, &

Palaniappan, 2020), . Tau protein presents four regions that are important for its study: N-terminus region, proline-rich domain, microtubule binding domain (MTBD) and C-terminus region as shown in Figure 5.

Tau is regulated by post-translational modifications (PTM) including phosphorylation and dephosphorylation, nitration, acetylation, ubiquitination, and truncation, giving

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Tau its functional characteristics. The most common PTM that plays a vital role on Tau´s function is phosphorylation/dephosphorylation by protein kinases (PK) (Mietelska-porowska, Wasik, Goras, & Filipek, 2014). On AD there is an increased level of this PTM leading to pathological forms of tau that tend to auto aggregate and form abnormal filaments.

It has been demonstrated that Tau’s PTM occur in a traceable sequence that starts with the dissociation of Tau from the MT and cytosolic concentration exceeds the minimal necessary to lead aggregation. This dissociation is mediated by a phosphorylation in a specific site. Aggregation and truncation inducers are thought to induce a conformational change that confers Tau an aggregation competent conformation, impacting in the formation of β-sheet structures and enabling the interaction forming Tau dimers(Meraz-Ríos et al., 2010). It has been suggested that phosphorylation acts as an initiator while cleavage functions as a propagator (Binder, Smith, Perry, & Garcı, 2008). Once these dimers adopt a stable conformation, they begin a nucleation process that precedes the elongation forming oligomers of aberrant Tau. The self-assembly of Tau is driven by the hyperphosphorylation of the C-terminus inducing the formation of paired helical filaments (PHF). The oligomerization of Tau into PHF comprises the interaction of phosphorylated and truncated forms of Tau species in the cytoplasm of affected neurons forming a PHF- core that triggers a protective response within the cell that involves the phosphorylation of normal Tau and the activation of caspases (Flores-Rodríguez et al., 2015). As the oligomerization continues, subunits of filaments are generated and PHF that consist of two protofilaments take form (Meraz-Ríos et al., 2010). The accumulation of PHF as the misfolded protein aggregation continues leads to the formation of NFT that occupy the somatic space causing neuronal death (Figure 6.).

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Figure 5. Protein Tau domains and antibody epitopes. Taken from (Flores-Rodríguez et al., 2015).

Figure 6. Model of possible NFT formation and tau aggregation in AD. Taken from (Luna-Muñoz, Chávez-Macías, García-Sierra, & Mena, 2007)

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1.7. Mouse model for the study of Alzheimer’s Disease

To study the interaction between Aβ peptide and Tau, back in 2003, Frank LaFerla and Salvatore Oddo (Oddo et al., 2003) developed a triple transgenic mouse model.

They microinjected two independent cassettes for two human mutated proteins: the Swedish mutation (K670D/M671L) for the APPβ and the human mutated Tau (4R/0N)(P301L) into single cell embryos from homozygous knockin mice for PS1 (M146V) to generate mice with the same genetic background. As a result, it was observed the presence of extracellular Aβ aggregation (plaques) and the presence of human protein Tau (Figure 7). They detected intracellular immunoreactivity with the antibodies that recognize Aβ in the hippocampus at 6 months, extracellular deposits were observed until 12 months in the hippocampus being positive to the thioflavin-S staining for fibrillar aggregations. Pathologic tau is present in the hippocampus at first, in the CA1 region.

Figure 7. Hippocampus of the 3xTg-AD transgenic mouse. A) presence of human Tau detected by the antibody MC1. D) presence of Aβ reactive plaques detected by the Aβ42- specific antibody.

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1.8. Inflammation in AD

The inflammatory process in AD originates from a response to the chronic stress produced by Aβ deposits that start a cascade of events characterized by the induction of microglia and astrocytes, activation of complement by the classic and alternative pathways and the expression of cytokines and interleukins (Heneka &

Banion, 2007). Astrocytes act in Aβ clearance and degradation. They give neurons trophic support and form a barrier between fibrillar deposits an neurons (Wyss-Coray et al., 1997). Inflammatory mediators, specifically cytokines are secreted by microglia and astrocytes that surround Aβ deposits. There are several cytokines that have been associated with AD such as interleukins (ILs), TNF-α and TGF-β.

Their function is to regulate the duration and intensity of immune response and their production seems to be increased in inflammatory states (Tuppo & Arias, 2005).

TNF-α has pro and anti-apoptotic properties, it is responsible of most of the neurotoxic activity from monocytes and microglia (Combs, Karlo, Kao, & Landreth, 2001).

1.9. Alcohol and Neuroinflammation

There is recent evidence that links alcohol and neuroinflammation. Its use could impact on the immune system, leading to the activation of microglia and the release of pro-inflammatory cytokines. There are increased brain levels of IL-1 and TNF-α (Qin et al., 2008). Microglial activity is suggested to be a key event in the neuroinflammation in both AD and alcohol misuse through the activation of inflammatory cascades and the release of cytokines leading to neuronal death (Venkataraman, Kalk, Sewell, Ritchie, & Lingford-hughes, 2017).

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1.10. Motivation

It was previously shown that, in Alzheimer´s Disease, pathological Tau undergoes certain posttranslational modifications following a sequence that leads to the formation of fibrillar aggregations. In the triple transgenic mice model 3xTg-AD it was observed that, although this PTM followed a similar sequence, there was no presence of fibrillar forms of Tau. It is possible to induce a neurotoxic state through an ethanol treatment that leads to the activation of caspase 3 and the generation of proteolyzed forms of Tau. Proteolyzed Tau are thought to precede the formation of fibrillar aggregations in Alzheimer´s Disease. The need arises to evaluate the pathological events generated from the induced neurotoxicity in the 3xTg-AD.

1.11. Problem Statement and Context

In patients with AD there is the presence of the two fibrillar lesions that define the disease: The neuritic plaques formed by the fibrillar aggregation of Aβ and the paired helicoidal filaments and neurofibrillary tangles formed by the fibrillar aggregations of Tau. Both fibrillar forms are positive to the thiazine red staining. The triple transgenic mice model 3xTg-AD presents some pathological species of Tau P301L and Aβ polypeptides. It was observed positive staining of thiazine red on Aβ confirming its fibrillar aggregations, as well as an inflammatory process as occurs in humans. It was also observed that Tau processing follows a similar sequence, but Asp421 truncated Tau species were not present. There was no positive staining of thiazine red on Tau species indicating the absence of fibrillar Tau on the 3xTg-AD.

1.12. Research Question

Can ethanol treatment induce a neurotoxic state where:

• Caspase-3 is active?

• There are proteolysis evets that lead to fibrillar forms of Tau?

• The inflammatory state will be exacerbated?

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1.13. Hypothesis

The administration of ethanol on the 3xTg-AD leads to the generation of proteolyzed forms of Tau through a neuroinflammatory state generating fibrillar aggregates of Tau.

1.14. Objectives

1.14.1. General objective:

To implement a model of inflammation for the study of the polymerization of the Tau and amyloid β polypeptides in the 3xTg-AD mice through the administration of ethanol.

1.14.2. Specific objectives:

1. To study the effect of ethanol on the aggregation of Tau.

2. To study the effect of ethanol on the aggregation amyloid-β.

3. To evaluate the inflammatory effect induced by ethanol administration.

4. To standardize a protocol for the quantification of pathological events in the model of inflammation.

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2. Experimental Protocol

2.1. Experimental animals

The triple transgenic model 3xTg-AD which carry the transgenes with the homozygous mutants of APPSWE, PS1M146V and TauP301L (Oddo et al., 2003) were donated by PhD. Frank LaFerla (University of California, US) to PhD. Sofia Diaz-Cintra (Instituto de Neurobiologia UNAM, Campus Juriquilla, Queretaro). Mice were housed 4 per cage and kept under optimal vivarium conditions: 12 h light/ 12 h dark cycles, a 20° C temperature, relative humidity of 40-50%, ad libitum diet (Purina Rodent Chow 5001 and water). Colonies were kept under the procedure described by Orta-Salazar and collaborators, 2014. Two females and one male were placed in polycarbonate cages (12x12x25 cm) in the mating and gestation periods. Mice were weaned on the 30^th day and separated by sex: 3 males or 3 females by cage until they reached the ages of study. Ethanol treatments were applied as described in the following sections.

Induction of neurotoxic state on 3xTg-AD mice

As a first exploration to evaluate the molecular events in the 3xTg-AD, two groups of n=3 mice of 17 months were assigned as Controls and Treatments. Treatments received a subcutaneous injection of a 20% ethanol solution while Controls were injected with saline solution. Mice from both groups were euthanized 12 h after the injection was applied.

To implement immunoreactivity quantification protocols, two groups of n=3 mice of 3, 11 and 20 months in each group were assigned as Controls and Treatments.

Treatments received a subcutaneous injection of a 20% ethanol solution while Controls were injected with saline solution. An injection of ethanol or saline solution was administered on days 1, 8 and 15. Mice from both groups were euthanized on day 21. Animal handling was supervised under veterinary license in accordance with the Guide for the care and use of laboratory animals published by the National

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Institute of Health (NIH, US) and approved by Comite de Bioetica from Instituto de Neurobiología (UNAM, Mexico).

2.2. Brain extraction and tissue preparation

Mice were anesthetized with ketamine/xylazine (85 mg kg^-1/9.5 mg kg^-1).

Transcardial perfusion was performed using paraformaldehyde 4% (PFA) in PBS 1X with 1000 UI heparin and 0.1% procaine. After the procedure, brains were removed and fixated for 24 h in PFA 4% at room temperature. Brain slices (50 µm) were obtained, in the sagittal plane, with a criostate (Leica SM 2000R). Brain sections were kept at 4°C in PBS 1X with sodium azide 0.01%.

2.3. Immunofluorescence and confocal microscopy

Brain tissue sections were washed three times PBS 1x for 5 min at room temperature. Unspecific sites were blocked with BSA 0.2% (Sigma) for 20 min at room temperature. Tissue sections were washed with PBS-T for 10 min and incubated overnight at 4°C with a mix of 2 or 3 primary antibodies with different isotope and from different species to avoid crosslinking with the secondary antibody.

Next day, sections were washed 3 times with PBS-T for 10 min. Sections were incubated for 1.3 h at room temperature with a mix of the correspondent anti-isotope and anti-species secondary antibodies (1:100). Secondary antibodies are coupled with fluorescein (FITC), tetramethyl rhodamine (TRITC) or Cy5 (Jackson Immuno Research Laboratories, Inc.). Samples were washed 3 times with PBS-T for 10 min.

They were incubated with 1 μM TO-PRO3 (Life Technologies) to stain DNA. Tissue samples were mounted on Vectashield (Vector Labs). Samples were analyzed with a confocal microscope SP8 (Leica) under 40 X and 100X objectives. In some cases, a solution of Thiazine Red (TR) 0.001% was applied to stain the fibrillar aggregations of Aβ or Tau (Edwards, Walker, & Klug, 1988)(Edwards et al., 1988; Mena, Edwards, Harrington, Mukaetova-Ladinska, & Wischik, 1996),(Mena et al., 1996). A series of optical sections (500 nm) were obtained through the Z axis using the corresponding filter combination to obtain the fluorescence signals for FITC, TRITC, Cy5 and TR.

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Prescence, distribution and co-localization of the antibody immunoreactivity with Tau, Aβ, and proinflammatory molecules in the tissue sections were captured individually or in combination with the primary colors green (FITC), red (TRITC or TR) and blue (Cy5 and TO-PRO3). TR is a fluorescent red dye (554-576 nm) that presents affinity with β-sheet conformed protein structures such as the fibrillar Tau and Aβ (Uchihara et al., 1995) . Some tissue samples were counterstained with TO- PRO3 (620-650 nm) that has affinity with DNA to satin the nucleus.

2.4. Immunohistochemistry

Brain sections were washed 3 times with PBS 1X for ten minutes. Endogenous peroxidase activity was blocked by the addition of hydrogen peroxide 0.3% in PBS 1X for 20 min. Tissue sections were washed 3 times with Triton X-100 0.02% in PBS 1X for 10 min. Tissues were incubated overnight at 4°C with a dilution of 1:200 of the primary antibody in PBS-T. Next day, sections were washed 3 times with PBS 1X. Tissue sections were incubated with the secondary antibody for 1.5 h at room temperature. Reaction was developed with 400 μL of a mixture of 6 mg 3-3’- diaminobencidine in PBS 1X and 20 μL of hydrogen peroxide 3% for 8 min at room temperature. Peroxidase reaction was stopped with PBS-T. Tissues were counterstained with 1 mL of hematoxylin solution for 8 min at room temperature and rinsed with water for 5 min. Fixated tissues were dehydrated by incubation in ethanol 70%, 80% and 90% (v/v) for 2 min in each solution. Then, samples were incubated in a mixture 1:1 of xylol-ethanol for 5 min followed by xylol for 5 min. Samples were mounted in DPX resin and observed with a bright field microscope Olympus FSX100.

Hippocampal areas of interest were focused under 10X and 20X objectives and images were captured to be processed by the quantification software.

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Table 1. List of primary antibodies used in immunoassays.

Antibody Epitope Isotype

Alz50 aa:5-15, 312-322 Mo IgM

Tau-C3 Truncationin Asp421 of Tau Mo IgG

499 aa: 25-36 of N-terminal portion of Tau Mo IgG PS-396 Phosphorylation in Ser396 of Tau Rb IgG PS-404 Phosphorylation in Ser404 of Tau Rb IgG

6E10 aa: 3 to 8 of Aβ. Mo IgG

GFAP Glial Fibrilar Acidic Protein Rb IgG

TNF-α Tumor Necrosis Factor α Mo IgG

p-CdK5

(pY15) Phosphorylation in Tyr15 Rb IgG

p-GSK3β GSK3β activated by phosphorylation RbIgG

BAM10 Beta-amyloid protein MoIgG

Rz3 Phosphorylation in Thr231 of Tau MoIgG

Table 2. List of secondary antibodies.

Antibody Chain Emision (nm)

Anti-Mouse

(MoIgG)-CY5 γ-specific (blue) 620/650

Anti-Mouse

(MoIgG)-FITC μ (green) 494/520

Anti-Rabbit

(RbIgG)-FITC H+L (green) 494/520

2.5. Quantification of pathological events

Quantification of affected neurons is performed by image processing captured from the immunohistochemistry essays. Method was developed by adapting the image watershed segmentation methods (Cloppet, Boucher, Cloppet, & Boucher, 2011), (Lefèvre, 2010), (Abdolhoseini, Kluge, Walker, & Johnson, 2019), (Byrne, Ross, Faull, & Dragunow, 2009) for the quantification of immunostained neurons. Program was developed by PhD. Andres Gutiérrez from the Department of Engineering and Sciences from Tecnológico de Monterrey, Campus Toluca. Once the image from clear field microscopy is uploaded, the software takes apart the components to be measured, in this case the immunostained neurons from the rest of the image and it

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gives back a number corresponding to the percentage of stained neurons. This makes possible to quantitatively compare how many neurons are being affected by pathological forms of Tau in a specific area of the brain from mice of different ages.

3. Results

3.1. Effect of ethanol administration on caspase 3 activation

To study the activation of caspase 3 in the hippocampal region of the 3xTg-AD, an immunoassay was performed Figure 8. It is evident the presence of activated caspase 3 in the transition of CA3-CA4 hippocampal regions at 12 h post injection.

Immunoreactivity with the antibody TauC3, that recognizes the proteolysis of Tau in Asp 421 was not observed. Comparing a wildtype control with the 3xTg-AD mice model with the ethanol treatment, caspase 3 is activated as a response to the presence of ethanol. In this immunoassay there was not immunoreactivity in the Subiculum and CA1 where the presence of pathological forms of Tau and amyloid- β are characteristic.

As shown in Figure 9. the activation of caspase 3 is accompanies with astrocyte activation, evident for the immunoreactivity of GFAP. Even though caspase 3 is active, there is no presence of Asp421 cleaved tau detected by TauC3 and there is no positive staining by Thiazine Red indicating the absence of fibrillar forms of protein Tau.

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Figure 8. Effect of ethanol treatment on the activation of caspase-3 in the CA3-CA4 regions. Triple immunostaining with the antibody that recognizes the active caspase-3 (green channel), TauC3 to measure the presence of cleaved Tau (red channel) and Alz50 to recognize Tau (blue channel).

Coronal plane sections of brains from the17-month mice injected with ethanol.

Figure 9. Effect of ethanol treatment on the activation of caspase-3 in the CA3-CA4 regions at 8 h post injection. A) Double immunostaining with the antibody that recognizes the active caspase-3 (green channel), TauC3 to measure the presence of cleaved Tau (red channel) and Alz50 to recognize Tau (blue channel). B) Double immunostaining and counterstaining with the antibody that recognizes the active caspase-3 (green channel), Alz50 to recognize Tau (blue channel) and Thiazine Red (red channel). Coronal plane sections of brains from the17-month mice injected with ethanol.

Casp-3 TauC3 Alz50 Merge

B A

GFAP TR Alz50 Merge

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3.2. Effect of ethanol on inflammation

The 3xTg-AD mice model is known to present an inflammatory reaction characterized by the activation of astrocytic cells, observable by the presence of active GFAP in immunofluorescence assays. The ethanol treatment on the 17-mont 3xTg-AD mice model exerts an exacerbated neurotoxic state where there is an increase of activated astrocytes. Figure 10. shows the presence of fibrillar plaques of Aβ positive to the Thiazine Red staining and the increase of astrocyte counting in the 3xTg-AD mice that received the ethanol treatment compared to the control group that was injected with saline solution.

Figure 10. Effect of ethanol treatment on inflammation. Immunostaining and counterstaining with anti- GFAP antibody to measure astrocytic activity (blue channel) and Thiazine Red (red channel). Coronal plane sections of brains from the 17-month 3xTg-AD mice injected with saline solution a) and ethanol treatment b). c) Astrocyte counting in the shown sections from 3x-Tg-AD mice with and without ethanol treatment.

3.3. Effect of ethanol on Aβ aggregation

In Figure 11. it is observed the effect of ethanol treatment on the aggregation of Aβ polypeptide in the CA1 hippocampal region of the 3xTg-AD mice with ethanol treatment, compared with the control group that was injected with saline solution. It

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stands out that there is an increment on the formation of Aβ plaques in the mice that received the ethanol treatment. This increase of Aβ aggregates is evident in Figure 12. making a close up on the stratum piramidale- stratum oriens transition and the stratum radiatum where green signal, that corresponds to the Aβ fragments, increases, and shows an aggregation pattern.

Figure 11. Increase of the extracellular Amyloid β plaques in the CA1 hippocampal region of the 3xTg-AD 17-month-old mouse injected with ethanol. Double immunostaining with the antibodies BAM10 that recognizes the Aβ40-42 fragments (green channel) and the antibody 499 that recognizes a fragment of the N-terminal region of protein human protein Tau with no chemical modification (red channel). Coronal plane sections of brains from 17-month mice treated with saline solution a) and ethanol b).

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Figure 12. Increase of extracellular Amyloid β plaques and Tau P301L polypeptide in the pyramidal cells of CA1 in the 3xTg-AD mouse injected with ethanol. Double immunostaining with the antibodies BAM10 that recognize Aβ40-42 fragments (green channel) and the antibody 499 that recognizes a fragment of the N-terminal region of protein human protein Tau with no chemical modification (red channel). Coronal plane sections of brains from 17-month mice treated with saline solution and ethanol. a) CA1; b) Sp-So: Stratum piramidale-Stratum oriens; c) Sr: Stratum radiatum.

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3.4. Effect of ethanol on protein kinase activity

As shown in Figure 13. there is a decrease in the expression of GSK3β in the hippocampus of the 3xTg-AD 17-mont mice when compared to the control group that was injected with saline solution. It correlates with an increase of GFAP in the ethanol treated group as an indicative of the activation of astrocytes surrounding the fibrillar Aβ formations. In Figure 14. there is evidence of an increment in the expression of the protein kinase CDK5 in the hippocampus of the 3xTg-AD mice compared with the saline-solution-treated mice. This increase in CDK5 expression correlates with an increment of recruited astrocytes and the formation of Aβ fibrillar aggregates.

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Figure 13. Decrease of protein kinase GSK3β in the Subiculum neurons of the 3xTg-AD mouse injected with ethanol. Double immunostaining and counterstaining with the antibodies that recognize the protein kinase GSK3β (green channel), GFAP to measure astrocytic activity (blue channel) and Thiazine Red (red channel). Coronal plane sections of brains from 17-moth mice injected with ethanol and saline solution.

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Figure 14. Increase in the expression of protein kinase CDK5 in the Subiculum neurons from the 3xTg-AD with ethanol treatment. Double immunostaining and counterstaining with the antibody that recognizes the protein kinase GSK3β (green channel), anti-GFAP to measure astrocytic activity (blue channel) and thiazine red (red channel). Coronal plane sections of brains from the17-month mice injected with ethanol and saline solution.

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3.5. Exacerbation of the neuroinflammatory state

It stands out in Figure 15. the increase in the expression of tumoral necrosis factor TNF-α in the hippocampus of 3xTg-AD 17-month mice with the ethanol treatment. It correlates with an increase in the fibrillar Aβ aggregates positive to Thiazine Red staining as well as an increase in astrocytic activity evident by the expression of GFAP.

Figure 15. Increase in the expression of TNF-α and its correlation with astrocytic activity in the Subiculum of the 3xTg-AD mice injected with ethanol. a) Double immunostaining and counterstaining with the monoclonal antibody that recognizes tumoral necrosis factor TNF-α (blue channel), an antibody that recognizes GFAP to measure astrocytic activity (green channel) and Thiazine Red (red channel). Coronal plane sections of brains from the17-month mice injected with ethanol and saline solution. b) Amplification of astrocytes and their interaction with fibrillar plaques of Aβ.

3.6. Effect of ethanol on fibrillar aggregations

This immunofluorescence assay was made to evaluate the presence of fibrillar aggregations of protein tau in the hippocampus of the 17-month 3xTg-AD mice model that was injected with ethanol. Results in Figure 16. show the presence of TauP301L with the PTM of phosphorylation in the Ser396 and Ser404 and antibody 499 that recognizes a fragment of the N-terminal region of protein human protein Tau with no chemical modification, giving an insight of the processing that Tau is

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undergoing. It stands out that in the 17-month 3x-Tg-AD mice there was a positive staining of Thiazine Red indicating the presence of fibrillar Tau.

Figure 16. Effect of ethanol on fibrillar aggregations. Double immunostaining and counterstaining with the antibodies PS-396 that recognizes a phosphorylation on Ser396 on Tau (green channel), the antibody 499 that recognizes a fragment of the N-terminal region of protein human protein Tau with no chemical modification (blue channel) and Thiazine Red (red channel). Coronal plane sections of brains from 17-month mice treated with ethanol.

3.7. Hippocampus mapping and quantification of pathological events

Later we proceeded with a mapping of the hippocampus to establish the methodology to quantify immunoreactivity by computational methods. We parted from the 3xTg-AD model with a chronic ethanol treatment described in the experimental protocol section. Images shown in Figure 18. and Figure 19. were taken from clear field microscopy. CA1 area close up images shown in Figure 20 were selected and input to the quantification software based on watershed

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segmentation (Figure 17). Software results are shown in Figure 20. c). Graph shows the percentage of immunostained area that correlates with the number of immunostained neurons. This technique makes it possible to compare the number of affected neurons through different stages of the development of the disease.

Figure 17. Visual representation of the watershed segmentation-based image processing software steps. The image is uploaded, brown channel is recognized, a gray filter is applied, and the image is segmented for its quantification.

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Figure 18. Hippocamp of a mouse which has received the ethanol neurotoxicity treatment and sacrificed at 11 months. Immunohistochemistry with HRP using an Rz3 (pThr231) monoclonal antibody.

Figure 19. Hippocamp of a mouse which has received the ethanol neurotoxicity treatment and sacrificed at 20 months. Immunohistochemistry with HRP using an Rz3 (pThr231) monoclonal antibody.

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Figure 20. Close up of CA1 regions of 3xTg-AD mice of a) 11 months and b) 20 months that received the ethanol treatment. Immunohistochemistry with HRP using an Rz3 (pThr231) monoclonal antibody. c) Quantification of immunoreactive neuros expressed as a percentage of the stained area.

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4. Discussion

There are several molecular events that characterize AD. At an extracellular level, it is observed the presence of Aβ polypeptide and its fibrillar aggregation in form of neuritic plaques (NP), as well as an inflammatory response characterized by the presence of activated astrocytes. Intracellularly, there are several posttranslational modifications such as hyperphosphorylation, glycosylation, nitration, ubiquitination, glycation, and proteolysis. It presents some molecular events that are observed in humans (Ontiveros-Torres et al., 2016). The triple transgenic mice model 3xTg-AD was developed to study the molecular events that may be implicated in AD (Oddo et al., 2003).

It was previously determined that Aβ aggregation in the 3xTg-AD model leads to the formation of fibrillar deposits at an extracellular level triggering the activation of astrocytes in an age dependent manner. As to pathological species of Tau, it was observed the presence an evolution of the ones detected by the antibodies 499, Alz50, PS-409, PS-400, PS-396, PS-199, AD2 in 3xTg-AD mice from different ages (3,9,11,18 and 28 months)(Ontiveros-Torres et al., 2016). Nevertheless, a proteolysis mediated by caspse-3 in Tau´s amino acid Asp421, one of the major early events that seems to be involved in the formation of NFT (Luna-Muñoz, Chávez-Macías, García-Sierra, & Mena, 2007) was not found in the 3xTg-AD by the absence of the proteolyzed form of tau recognized by antibody Tau-C3. More importantly there was no positive Thiazine Red (TR) staining on tau in the 3xTg-AD model indicating the absence of fibrillar Tau deposits.

In the search of adapting the molecular events that occur in humans on the 3xTg- AD model, a study was found where a neurotoxic state is induced in the C57BL/6 mice (Olney et al., 2002). Through ethanol injections they induced caspase 3 activation in the C57BL/6 mice and found the major concentration of active caspase 3 at 8 and 12 hours post injection. Caspase 3, as demonstrated by Gamblin (Gamblin et al., 2003), is capable of proteolytically cleave Tau at Asp 421. The ethanol treatment activates caspase 3 in the CA3-CA4 regions, but Asp 421 cleaved forms of Tau detected by antibody Tau-C3 were not found in the 3xTg-AD mice model

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treated with ethanol as shown in Figures 9 and 10. Surprisingly, fibrillar forms of Tau positive to TR that seem to be independent of Tau proteolysis in Asp 421 were found in the hippocampus of one 17-month 3xTg-AD mouse previously treated with ethanol.

The implementation of the ethanol treatment in the 3xTg-AD to generate a model of inflammation for the study of the polymerization of Tau and Aβ polypeptides lead to interesting results. As stated by Ontiveros-Torres, the accumulation of Aβ in the 3xTg-AD transgenic mouse and the formation of fibrillar deposits positive to TR triggers an increase in GFAP expression, characteristic of active astrocytes (Ontiveros-Torres et al., 2016). In the 3xTg-AD transgenic mouse treated with ethanol, there is an increase in Aβ accumulation (Figure 10) that is shown in detail in Figure 11. that correlates with an increase in GFAP expression and astrocyte number (Figure 9. c)). This is an indicative of an exacerbate immune response to the presence of ethanol in the hippocampus of the 3xTg-AD mice treated with ethanol when is compared with the control group that was injected with saline solution.

This exacerbated immune response also correlates with the activation of certain proinflammatory molecules such as TNF-α. Ontiveros-Torres, previously reported the expression of TNF-α in the triple transgenic mouse (Ontiveros-Torres et al., 2016). When the 3xTg-AD mice were treated with ethanol, it was also observed an increment in the expression of TNF-α. In patients with AD, there has been found an increase in the expression of TNFR1 (tumor necrosis factor receptor superfamily 1A) which is a TNF-α receptor. Its activation along with its adaptor proteins such as TRAF2 (TNF receptor-associated factor 2) is linked with the activation of caspase 1 as well as with the activation of cell death signaling.

The expression of two protein kinases involved in tau phosphorylation were also studied. GSK-3β (glycogen synthase kinase) and CDK5 (cyclin dependent kinase 5) are Ser/Thr, proline directed kinases involved in AD (Ojala, Sutinen, Salminen, &

Pirttilä, 2008). GSK-3β is regulated by Wnt signaling and insulin in a negative regulatory way (Hooper, Killick, & Lovestone, 2008). It is also known that CDK5 may be increase in an inflammatory state because of calpain activation which cleaves

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p35 to p25. Accumulation of p25 is correlated with a CDK5 activity increment(Pareek et al., 2007). CDK5/p25 complex is associated with Tau hyperphosphorylation, this event may precede the NFT formation (Patrick et al., 1999). In the 3xTg-AD transgenic mouse treated with ethanol, it was found a decrease of GSK-3β and an increase in CDK5 expression (Figures 12 and 13.) in relation with the control group.

Both events correlate with the presence of fibrillar deposits of Aβ positive to TR and the presence of activated astrocytes in the Subiculum area of the 3xTg-AD model.

Here we propose a model (Figure 21.) where there is an exacerbated inflammatory response triggered by the ethanol treatment in the 3xTg-AD transgenic mouse characterized by an increase in Aβ fibrillar deposits and astrocyte recruitment. In the presence of which there is an increase in TNF-α expression that may be activating certain signaling pathways in the pyramidal neurons of the hippocampus that could be implicated in the regulation of protein kinases GSK-3β and CDK5 leading to a sequential hyperphosphorylation of Tau and contributing to fibrillar Tau formation.

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Figure 21. Model of the possible mechanism by which Tau is sequentially hyperphosphorylated.

Accumulation of Aβ fibrillar deposits (red) and astrocyte recruitment (green) exacerbated by the ethanol treatment correlate with an increase of TNF-α expression that may be activating certain signaling pathways in the pyramidal neurons associated with protein kinase regulation such as GSK- 3β and CDK5 implicated in the hyperphosphorylation of protein Tau.

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5. Conclusions

1. The ethanol treatment on 3x-Tg-AD mice model induces an exacerbated inflammatory response characterized by an increase in the astrocyte count in the hippocampus of the 3xTg-AD mice model treated with ethanol compared with the control group.

2. In the presence of ethanol Caspase-3 is activated, but its activation did not lead to proteolysis on Tau Asp421 in the 3xTg-AD mice with ethanol treatment.

3. Only in the hippocampus of the 3x-Tg-AD mice (17 months) the positive staining of TR was observed, indicating the presence of fibrillar Tau.

4. The presence of fibrillar Tau in the hippocampus of the 3xTg-AD mice model treated with ethanol, seems to be independent from the proteolytic event on Asp421 of protein Tau.

5. It was observed an increase in the expression of protein kinase CDK5 associated with the phosphorylation of the C-terminal region of Tau in the hippocampus of the 3xTg-AD mice model treated with ethanol.

6. There was an increase in the expression of TNF-α associated with the increased fibrillar Aβ aggregation in the hippocampus of the 3xTg-AD mice model treated with ethanol.

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6. Perspectives

1. Characterize the signaling pathways in the pyramidal neurons as a response to an inflammatory state.

2. Standardize the quantification method for the immunoreactivity of Amyloid β.

3. Apply the quantification method on immunofluorescence samples.

4. Evaluate pharmacological targets in the 3xTg-AD with ethanol treatment.

5. Assess the 3xTg-AD mice model with ethanol treatment as an alcoholism model.

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7. Appendix A Abbreviations

Description

AD Alzheimer's Disease APP Amyloid Precursor Protein Aβ amyloid-β peptide

CDK5 Cyclin Dependent Kinase 5 DAB Diaminobenzidine

GFAP Glial Fibrillar Acidic Protein GSK3β Glycogen Synthase Kinase 3 β HRP Horseradish Peroxidase

IL Interleukin

MAP Microtubule Associated Protein MTBD Microtubule Binding Domain NFT Neurofibrillary Tangle

NP Neuritic Plaque

PBS Phosphate Buffer Saline

PBS-T Phosphate Buffer Saline with Triton X100 PFA Paraformaldehyde

PHF Paired Helical Filaments PK Protein Kinases

PSEN1 Presenilin1 PSEN2 Presenilin2

PTM Post-translational Modification TGF-β Transforming Growth Factor-β

TNFR1 Tumor Necrosis Factor Receptor Superfamily 1A TNF-α Tumor Necrosis Factor-α

TR Thiazine Red

TRAF2 TNF receptor-associated factor 2

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9. Curriculum Vitae

Pedro Ricardo Moreno Velez was born in Estado de Mexico, Mexico, on February 7^th, 1991. He earned the Biotechnology Engineer degree from the Instituto Tecnologico y de Estudios Superiores de Monterrey, Toluca Campus in May 2014.

He was accepted in the graduate program of Master of Science in Biotechnology in January 2019.

This document was typed in using Microsoft Word by Pedro Ricardo Moreno Velez.

polypeptides in the pyramidal neurons on the hippocampus of a triple transgenic murine model for the Alzheimer’s Disease.

Instituto Tecnológico y de Estudios Superiores de Monterrey

School of Engineering and Sciences

Effect of the inflammation on the aggregation of the Tau and amyloid-β

polypeptides in the pyramidal neurons on the hippocampus of a triple transgenic murine model for the Alzheimer’s Disease.

Pedro Ricardo Moreno Velez

Effect of the inflammation on the aggregation of the Tau and amyloid-β polypeptides in the pyramidal neurons on the hippocampus of a triple transgenic murine model for the

Alzheimer’s Disease.

by

Pedro Ricardo Moreno Velez.

Abstract

List of Figures

List of Tables

Contents

1. Introduction

2. Experimental Protocol

3. Results

4. Discussion

5. Conclusions

6. Perspectives

7. Appendix A Abbreviations

8. Bi bli o gr a p h y

9. Curriculum Vitae