Joana dos Santos Peixoto Gomes Expression of Megalin in the Central Nervous System

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Joana dos Santos Peixoto Gomes

Expression of Megalin in the Central

Nervous System

Universidade do Minho

Escola de Ciências

Joana dos Sant

os P eix o to Gomes Expression of Megalin in t he Central Ner v ous Sys tem 1 7

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Joana dos Santos Peixoto Gomes

Expression of Megalin in the Central

Nervous System

Tese de Mestrado

Mestrado em Bioquímica Aplicada

Trabalho efetuado sob a orientação de:

Doutora Maria João Saraiva

e

Doutora Sandra Paiva

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DECLARAÇÃO

Nome: Joana dos Santos Peixoto Gomes

Endereço eletrónico: juaninhspg@gmail.com

Telefone: 911 175 116

Número do Cartão de Cidadão: 13829752

Título da dissertação: Expression of Megalin in the Central Nervous System

Orientadoras:

Doutora Maria João Gameiro de Mascarenhas Saraiva Doutora Sandra Cristina Almeida Paiva

Ano de Conclusão: 2017

Mestrado em Bioquímica Aplicada

Área de Especialização: Biomedicina

DE ACORDO COM A LEGISLAÇÃO EM VIGOR, NÃO É PERMITIDA A REPRODUÇÃO DE QUALQUER PARTE DESTA TESE.

Universidade do Minho, 30 de Outubro de 2017

___________________________________________ (Joana dos Santos Peixoto Gomes)

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AGRADECIMENTOS

Mais uma etapa concluída, mais um objetivo traçado. Não foi fácil, houve momentos de desespero, houve momentos cansativos, mas no final de contas, valeu a pena. Houve momentos em que a minha auto-estima se ia completamente a baixo, mas depois obtinha aquele resultado e parecia que simplesmente tudo se compunha.

No entanto, a maior força para que não entrasse em colapso mental deveu-se a algumas pessoas que foram essenciais para conseguir terminar esta etapa da minha vida.

Em primeiro lugar, quero agradecer à minha orientadora Maria João Saraiva, por me ter acolhido e por me ter proporcionado todos os meios necessários à realização deste trabalho. Por me ter transmitido muito do seu vasto conhecimento nesta área. Por me ter ensinado a ser mais proativa. Muito do que cresci durante este ano deveu-se ao fato de ter de racionalizar as coisas por mim mesma e não estar dependente dos outros.

Em segundo lugar, quero agradecer aos membros do laboratório 207.S2, especialmente à Sofia, à Paula e à Susete, que tiveram crucial importância na minha aprendizagem e evolução durante todo o percurso laboratorial, desde ao manuseamento dos animais no biotério até aos protocolos mais básicos de imuno.

Saindo do Porto e chegando a Braga, tenho de agradecer aos meus diferentes grupos de amigos: ao Striclas e à Sara por sempre me mostrarem o lado positivo, por me tentarem sempre animar e mostrar que a minha vida não é o caos que eu pinto que é; aos membros femininos do “já fostes” por mesmo estando longe se mostraram sempre perto, em especial à Marta, a minha fiel companheira, que mesmo a 400km nunca me falhou nas palavras e no apoio; ao “menos bé” por estar sempre disponível para dúvidas científicas mas sobretudo existenciais, em especial à Cata, por ter estado presente em todos os meus dramas e por me ter conseguido acalmar sempre; ao Bruno por ser o meu tradutor ambulante e se ter mostrado sempre disponível para tudo o que precisasse e às “gatas de bq”, especialmente à Renata por me ter salvo sempre dos meus dramas e me ter sempre levantado quando foi preciso.

Por último, mas definitivamente não menos importante, à minha família, aos meus bichinhos e à minha mãe. À minha família, especialmente aos cromos dos meus tios e à minha avó, por ser o meu anjo da guarda, por ser a pessoa mais carinhosa neste mundo, por sempre ter acreditado em mim e por muito ter rezado este ano para que “os meus trabalhos corressem bem”. Aos meus bichinhos por terem sido a maior companhia durante ao processo de escrita, por com eles conseguir ficar mais tranquila só com uma corrida por um pau e pelos seus olhares ternos me mostrarem o quão sou importante, pelo menos para eles. À minha mãe, ao meu porto seguro, à minha ancora e a todos os possíveis adjetivos que são sinónimos destes. A Ela, devo-lhe toda a minha pessoa, a minha personalidade, a minha educação, as minhas qualidades e também os meus defeitos! À minha melhor amiga e à única pessoa que sei que, quando o mundo estiver a acabar me vai salvar primeiro que a si mesma. Muita da força deste trabalho retirei-o de ti, és um

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orgulho de mãe, de pessoa e de mulher! Por isso uma grande obrigada por tudo a que te sujeitas e a tudo que fazes por mim, isto porque com uma mãe como tu as coisas tornam-se muito mais fáceis de fazer. Obrigada por todas as tuas palavras quer de incentivo quer de reprovação, a todos os “sins” e a todos os “nãos”, sem ti definitivamente esta tese não era possível.

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ABSTRACT

The incidence of neurodegenerative diseases is increasing today, so there is an increasing interest and need to better understand the mechanisms involved.

Transthyretin (TTR) is a protein that has several physiological functions, being highly involved in the homeostasis of the nervous system. Changes in TTR expression levels are associated with several neurodegenerative pathologies. In addition, TTR plays a crucial role in the preservation and survival of neurons since its internalization through the action of megalin, activates several signalling mechanisms.

Megalin has been extensively studied over the years; changes in the levels of this protein are also associated with neurodegenerative pathologies, thus functioning as a biomarker.

Furthermore, TTR is regulated by heat shock factor 1 (HSF-1). HSF-1 acts in response to thermal shock, as well as to environmental stress (including ROS presence) and in pathological conditions such as protein misfolding, thus being associated with neuronal stress conditions.

Thus, one of the aims of this work was to produce an antibody specific for soluble non membranar megalin and to characterize it so that one could quantify the levels of megalin in mice samples. The anti-megalin antibody was characterized by immunohistochemistry (IHC), by enzyme-linked immunosorbent assays (ELISA) and Western blot; it was used to study megalin expression in the brain of wild-type (WT), TTR-knockout (TTR-KO) and HSF-1-deficient mice (with and without TTR), with and without an oxidative diet. The results show that megalin is expressed in the brain, especially in the retrosplenial area of the cortex. Additionally, WT animals present higher levels of megalin in this region than other stains, namely TTR-KOs and HSF-1-deficients. Furthermore, untreated animals presented higher levels of megalin expression in comparison to treated animals.

The results suggest that i) the oxidative diet has an impact on the expression of megalin and ii) TTR influences the expression of megalin. The implication of megalin expression and its modulation in the retrosplenial area of the cortex deserves further investigation.

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RESUMO

A incidência das doenças neurodegenerativas é cada vez mais elevada nos dias de hoje, pelo que cada vez é maior o interesse e a necessidade de se compreender melhor os mecanismos envolventes.

A transtirretina (TTR) é uma proteína que apresenta diversas funções fisiológicas, estando altamente envolvida na homeostasia do sistema nervoso. Alterações nos níveis de expressão de TTR estão associadas a diversas patologias neurodegenerativas. Além disso, a TTR desempenha um papel crucial na preservação e sobrevivência dos neurônios uma vez que a sua internalização pela ação da megalina, ativa mecanismos de sinalização.

A megalina tem sido um alvo de estudo uma vez que alterações nos níveis desta estão também associadas a patologias neurodegenerativas, funcionando por isso como um biomarcador.

A TTR é também regulada pelo fator de transcrição de choque térmico 1 (HSF-1). HSF-1 para além de atuar na resposta ao choque térmico, atua como resposta ao stress (como na presença de espécies reativas de oxigénio) e em condições patológicas como no mau-enrolamento de proteínas, estando associado a condições de stress neuronal.

Desta forma, um dos objetivos deste trabalho foi produzir um anticorpo específico para a megalina solúvel e caraterizá-lo para que com ele se pudesse analisar os níveis de megalina em amostras de ratinho. A caraterização do anticorpo foi feita por estudos de imunohistoquímica, por ensaios de imunoabsorção enzimática e Western blot. A expressão da megalina foi estuda em grupos de animais com TTR (WT), sem TTR (TTR-KO) e animais com ausência de HSF-1 (com e sem TTR), na presença e ausência de uma dieta oxidativa. Os resultados obtidos mostram que a megalina é expressa no cérebro nomeadamente na área retrosplenial do córtex. Além disso, verificou-se que os animais WT apresentam níveis mais altos de megalina nesta zona do córtex do que as outras estirpes, nomeadamente em animais com ausência de TTR e com falta de HSF-1 (quer com e sem TTR). Verificou-se também que os animais sujeitos à dieta apresentam menor expressão de megalina que os animais sem sujeitos à dieta.

Os resultados sugerem que: i) a dieta oxidativa tem impacto na expressão de megalina e ii) a TTR influencia a expressão de megalina. Estudos sobre a interferência da expressão de megalina e a sua modulação na área retrosplenial do córtex devem ser desenvolvidos.

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LIST OF ABBREVIATIONS

AD Alzheimer's disease Aβ peptide Alzheimer’s beta peptide APP Amyloid precursor protein ApoA-I Apolipoprotein A-I ApoE Apolipoprotein E CNS Central nervous system CSF Cerebrospinal fluid cDNA complementaryDNA Dab-2 Disabled-2

DNA Deoxyribonucleic acid DRG Dorsal root ganglia

ELISA Enzyme-linked Immunosorbent assay EGF Epidermal growth factor

FAP Familial amyloid polyneuropathy FBS Fetal bovine serum

GSK3 Glycogen synthase kinase 3 HSEs Heat shock elements HSF-1 Heat shock factor-1 Hsps Heat shock proteins Hsp25 Heat shock protein 25 Hsp27 Heat shock protein 27 Hsp40 (or Hdj-1) Heat shock protein 40 Hsp70 Heat shock protein 70 Hsp90 Heat shock protein 90 IHC Immunohistochemistry kDa kiloDalton

KO Knockout

LDL Low density lipoprotein LRP2 Lipoprotein-related protein 2

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MT Metallothionein NPY Neuropeptide Y NE Norepinephrine

PAM Peptidylglycine α-amidating mono-oxygenase PBS Phosphate buffer saline

PNS Peripheral nervous system ROS Reactive oxygen species RAP Receptor-associated protein RBP Retinol-binding protein RNA Ribonucleic acid

qRT-PCR Real-time reverse transcription polymerase chain reaction T4 Thyroxine

TMB Tetramthylbenzidine TTR Transthyretin WT Wild-type

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LIST OF FIGURES

Figure 1 - Ribbon structure of homotretameric Transthyretin (TTR) ... 2

Figure 2 - Signalling mechanism triggered by the interaction of TTR with megalin ... 7

Figure 3 - Conditions that induce heat shock response ... 8

Figure 4 - Regulation of heat shock factor 1 (HSF-1) ... 9

Figure 5 - Structural organization of core members of the LDL receptor gene family ... 13

Figure 6 - Structural organization of megalin ... 14

Figure 7 - Internalization of ligands by megalin ... 15

Figure 8 - Characterization of antibodies #33 and #34 by enzyme-linked immunosorbent assay (ELISA) ... 33

Figure 9 - Analysis of the results obtained by ELISA for the purified antibody #34 and megalin-sheep polyclonal antibody ... 34

Figure 10 - Analysis of the results obtained by sandwich ELISA ... 35

Figure 11 - Characterization of antibody #34 as anti-megalin antibody by Western blot analysis .. 35

Figure 12 - Analysis of ELISA results for megalin expression in supernatant cells and extract cells of SH-SY5Y cells ... 36

Figure 13 - Analysis of the results obtained by sandwich ELISA with purified antibody #34 as capture antibody and using megalin-sheep polyclonal antibody as antibody conjugate, for different concentrations of peptide ... 37

Figure 14 – Immunohistochemistry (IHC) analysis for different megalin antibodies (#33, #34, pre-bleed 33, pre-pre-bleed 34, megalin-sheep) in the kidney of WT mice with a magnification of x20 ... 38

Figure 15 - Immunohistochemistry analysis for different megalin antibodies (#33, #34, pre-bleed 33, pre-bleed 34, megalin-sheep) in the kidney of TTR-KO mice with a magnification of x20 ... 39

Figure 16 - Immunohistochemistry analysis for different megalin antibodies (#33, #34, pre-bleed 33, pre-bleed 34, megalin-sheep) in the kidney of Meg(+/-) mice with a magnification of x20 ... 40

Figure 17 - Representation in red of the retrosplenial area of the cortex, where the expression of megalin was identified ... 41

Figure 18 - Immunohistochemistry confocal analysis for megalin in WT and TTR-KO mice at 12 months of age ... 42

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Figure 20 - Immunohistochemistry analysis of retrosplenial area with antibody #33 in IW mice with and without treatment ... 43 Figure 21 - Immunohistochemistry analysis of retrosplenial area with antibody #33 in TTR-KO (M) mice with and without treatment ... 44 Figure 22 - Immunohistochemistry analysis of retrosplenial area with antibody #33 in HSF/IW mice with and without treatment ... 45 Figure 23 - Immunohistochemistry analysis of retrosplenial area with antibody #33 in HSF/M mice with and without treatment ... 46 Figure 24 - Analysis of normalized expression of megalin in IW and M mice ... 47 Figure 25 - Analysis of polymerase chain reaction (PCR) product by agarose electrophoresis ... 48

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LIST OF TABLES

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CHAPTER 1 - GENERAL INTRODUCTION

Nowadays, many are the diseases with no cures that affect the human being, making us susceptible to the surrounding environment. Neurological pathologies affect the nervous system, whose control is beyond our reach. In this way, it is essential to know more about them as well as to understand the beginning of the problem in order to create forms of treatment and/or prevention.

The nervous system is one of the most complex part of our body and is involved in many essential activities such as coordination of muscle activity, locomotion, organ activity, neuronal activity, senses and emotions. Thus, it is essential that the nervous system functions properly and does not suffer any kind of damage or disorder, as this would be reflected in the health of the organism.

1.1. Neurodegenerative Diseases

Neurodegenerative diseases are essentially characterized by the loss/destruction of the main cells of the nervous system, the neurons. The severity of the destruction varies widely, but it usually leads to the loss of cognitive, motor and/or physiological functions. These diseases are associated with many mechanisms that are strictly regulated. However, minimal variations on the regulation of one molecule can be crucial for the development of pathologies.

One protein immensely involved in the nervous system and in some pathologies is transthyretin (TTR). TTR shows alterations in expression levels in some neurodegenerative diseases, such as Guillain-Barré Syndrome, Frontotemporal Dementia, Lateral Amyotrophic Sclerosis, Parkinson's Disease, Alzheimer's disease (AD), Cerebral Ischemia, Schizophrenia, and Creutzfeldt-Jakob Disease, functioning for many of them as a biomarker in either disease identification, progression studies or as a route to possible treatment1.

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1.2. Transthyretin

1.2.1. Structure

Transthyretin is a homotetrameric protein (it has four identical subunits) with 54,980 kiloDaltons (kDa). Each subunit has 13,745 kDa, containing 127 amino acids. It has a globular shape of 70 Å × 55 Å × 50 Å dimensions2,3, consisting essentially of b-pleated sheet structure. By

the analysis of X-ray crystallography, it is known that each dimer is formed by the association of two monomers from two b-sheets composed of four b-strands, from each monomer, into two b-sheets of eight b-strands and the monomer assembly in dimers is stabilized by an extensive hydrogen bond. The interface of the two dimers forms a central hydrophobic channel (figure 1)4,5.

1.2.2. Function

Initially, TTR was designated as prealbumin. However since 1981, its designation derived from the main functions that it presents: transport of thyroid hormone thyroxine (T4) and retinol (or

vitamin A)2,6.

TTR is mainly synthetized in the liver and in the choroid plexus of the brain, which are the sources of TTR in plasma (3-7µM) and cerebrospinal fluid (CSF, 0.1-0.4µM), respectively2,4. Besides

Figure 1 - Ribbon structure of homotretameric Transthyretin (TTR). In red is represented Leucine 82 and in blue is represented Leucine 110. From Yang et al. 4.

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the liver and choroid plexus, TTR is also synthesized in the retina, skeletal muscle, heart, spleen7,

pineal gland, visceral endoderm of the amniotic sac6, pancreatic islet of Langerhans (α cells),

placental trophoblasts, and ghrelin cells of the stomach2,8.

The major sites of TTR degradation are the liver, muscle, and skin. However, the kidneys, adipose tissue, testes and gastrointestinal tract also appear to degrade TTR but in much smaller amounts. On the other hand, no brain site seems to be associated with TTR degradation. The liver and kidneys are the most active organs in the TTR catabolism2. TTR uptake by the liver occurs via a

receptor member of low density lipoprotein (LDL) family-sensitive receptor-associated protein (RAP)9,

while in the kidneys it is mediated by megalin10.

Transport of T4 and retinol

One of the various functions of TTR is the transport of the hormone T4. T4 is the most abundant

thyroid hormone secreted by the thyroid gland, and can circulate in the plasma bound to three different proteins: thyroxin-binding globulin, TTR and albumin. In some cases, and in very low amounts, it circulates in the unbound form2,3. There are two binding sites for T

4 in double trumpet

shaped channel of TTR structure. However, only one T4 molecule is bound to TTR, as the binding of

the first T4 molecule decreases the binding affinity of the second T4 molecule to the other binding site,

in a process called negative cooperativity3. Despite being involved in the transport of T

4, TTR is not

involved in the uptake of T4. Studies suggest that in TTR-knockout (KO) mice there is a significant

decrease in T4 in the blood and CSF when compared to normal mice. On the other hand, the levels

of free T4 are not altered, indicating that the TTR is not involved in the uptake of T4 11–13.

One of the functions of TTR in CSF may be closely related to the accumulation of thyroid hormones (especially T4). T4 enters the epithelial cells of the choroid plexus and binds to the TTR,

forming a T4-TTR complex. The complex is secreted and distributed throughout the brain, keeping the

T4 concentrations in the nervous system adequate. Thus, TTR plays an important role in the

distribution of thyroid hormones in the central nervous system (CNS)3.

TTR also binds to another important molecule, the retinol-binding protein (RBP) which is responsible for the transport of retinol in circulation. Retinol, or vitamin A, is obtained from the diet and upon oxidation it generates retinoic acid. Retinoic acid has an important role in many physiological functions such as vision, reproduction, growth, development14, regulation of cell

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involved in the modulation of neuronal processes such as neurogenesis, neuronal survival, synaptic plasticity in different brain regions (hippocampus, olfactory bulb and hypothalamus)15 and is also

implicated in growth and proliferation of neurites2,16.

Furthermore, vitamin A appears to hold therapeutic features in some neurodegenerative diseases involving the Alzheimer’s beta peptide (Aβ peptide) as in AD17. The Aβ peptide is produced

by amyloidogenic processing of the amyloid precursor protein (APP) and deposited by forming amyloid fibrils. Retinoids have protective activity since they are involved in the suspension of induced Aβ deposition in cerebral blood vessels18; high levels of vitamin A suspends the formation of amyloid

fibril19 and greatly reduces the deposition of Aβ, which is reflected in an increase in spatial learning

and memory capacities17,20.

RBP is synthesized in the liver and is released into the plasma only after retinol binding and after binding of TTR to the RBP-retinol complex, by which the retinol is transported. The TTR-RBP complex is crucial to prevent degradation of RBP in the kidney. TTR contains four binding sites for RBP, two in each dimer of the TTR structure. However, only two molecules of RBP can bind because of the steric hindrance that is formed. Although TTR binds to either T4 or RBP, the binding of the first

one does not influence the binding of the other one. T4 binds to a hydrophobic channel and retinol

binds at the surface of the TTR7.

Proteolytic Activity of TTR

Another important role of TTR is its proteolytic activity, which is involved in cleavage of apolipoprotein A-I (ApoA-I), Neuropeptide Y (NPY) and also Aβ peptide21,22.

TTR cleaves ApoA-I after a phenylalanine residue. ApoA-I is the major protein component of HDL particles in plasma and therefore the action of TTR will be crucial in the metabolism of lipoproteins: high density lipoprotein (HDL) loses the ability to promote cholesterol flow and ApoA-I increases the aggregation capacity (amyloidogenicity) 22.

NPY is a 36 amino acid peptide, highly distributed in CNS and peripheral nervous system (PNS) and is implicated in many biological effects and brain disorders.The TTR acts directly on NPY cleavage after Arginine 33 and Arginine 3. Thus animals with TTR (wild-type animals (WT)) have lower NPY levels than TTR-KO animals. Furthermore, neuropeptides to be activated need to be amidated by PAM (peptidylglycine α-amidating mono-oxygenase). PAM is a rate limiting enzyme in

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neuropeptide maturation and is overexpressed in the CNS and in PNS in the absence of TTR i.e. the TTR is involved in the regulation of PAM messenger ribonucleic acid (mRNA) expression. Thus, TTR-KO animals exhibit high levels of PAM, reflected by the high levels of activated NPY. However, the proteolytic activity of TTR is not related to the regulatory affinity of TTR in PAM and therefore the cleavage of NPY by TTR is not related to the action of TTR in PAM22.

Neuroprotective Action of TTR

TTR is the main Aβ-binding protein and is capable of inhibiting its aggregation and toxicity. TTR also facilitates Aβ clearance23. Thus, in the case of failure of Aβ TTR binding, amyloid formation

occurs24. The binding nature of TTR to Aβ is still an open discussion among researchers, however it

is believed that TTR can bind to distinct forms of Aβ in both soluble form, oligomers and fibrils. However, Yang et al. proposed a new mechanism: in the presence of toxic soluble Aβ oligomers, the tetrameric structure of the TTR becomes destabilized and exposes the inner hydrophobic sheet, leading to the elimination of the oligomers4.

The cleaved form of Aβ peptide exhibits lower toxicity than when complete, corroborating the protective action of TTR on the degradation of Aβ aggregates, decrease of Aβ fibril formation24 and

reduction of the levels of aggregation17,25. Studies have corroborated these findings, as in situations of

high TTR levels, Aβ levels and amyloid deposits decrease significantly in the hippocampus and cortex, as well as cytotoxicity. Thus, TTR has a neuroprotective action26.

Amyloidogenicity is the origin of several diseases in which the conformational alteration of proteins and the aggregation in amyloid fibrils occurs. The accumulation of these amyloid fibrils in tissues can cause damage in tissues and crucial changes in its performance/function. Mutations/alterations at the TTR level are also the basis of some dominant autosomal diseases, where TTR extracellular deposition occurs in various organs and tissues. Familial amyloid polyneuropathy (FAP) and familial amyloid cardiomyopathy are characterized by the deposition of amyloid fibrils in the peripheral nerve and in the heart, respectively4. It is believed that the tendency

for altered TTR to form amyloid fibrils is related to a decrease in its tetrameric stability and the dissociation of the tetramer into monomers may be the basis for amyloid formation24. Therefore,

stabilization of the tetrameric structure of TTR will be critical to inhibit the formation of fibrils, as well as to maintain binding with Aβ and prevent its aggregation and toxicity5.

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1.2.3. Transthyretin in the Central Nervous System

TTR holds a fundamental role in several functions like behaviour, cognition, 14-3-3z metabolism and proteolytic activity. Furthermore, it is involved in the nervous system homeostasis, as it participates in neuropeptide maturation, nerve regeneration, axonal growth, myelinisation, neurite outgrowth, neurogenesis, etc22.

Studies in TTR null mice revealed a less depressive behaviour than normal mice27, which may

be related to increased levels of norepinephrine (NE) in these animals. These changes in NE levels are dependent on the metabolic activity of the TTR-KO animals since the activity of tyrosine hydroxylase enzyme (rate-limiting enzyme in the biosynthetic pathway of catecholamines) and monoamine oxidase A enzyme (the major NE catabolic enzyme) is altered in these animals. In addition, they also present a higher levels of NPY, a known antidepressant neurotransmitter, which enhances the importance of TTR in the modulation of depressive behaviour28. Furthermore, TTR is

related to memory, studies show that TTR-KO mice exhibit impairment in memory compared to normal mice. Both may indicate that the absence of TTR accelerates cognitive deficits associated with aging28.

The absence of TTR also influence motor behaviour: TTR-KO mice present a motor impairment. Fleming et al. studied the locomotor activity of WT and KO mice, showing that TTR-KO mice have a more active behaviour at 3 and 6 months of age. However, in older mice (12 months) the same does not happen, as WT mice present greater activity compared to TTR-KO mice. Such, may be a consequence of the motor discordance related to TTR-KO animals27.

TTR is also involved in nerve regeneration. Studies on PC12 cells and WT and TTR-KO animals show that TTR influences neurite proliferation: WT animals show greater nerve regeneration than TTR-KO animals. Thus, the absence of the proteolytic activity of TTR is responsible for decreased growth/length of neurites. So, the proteolytic activity of TTR in nerve regeneration and neurite proliferation is essential22,29. The absence of TTR also compromises retrograde transport and axonal

growth, and if retrograde transport is compromised, signals from the site of damage to the cell body will also be compromised, as well as the rate of regeneration29.

Fleming et al. described that TTR has an effect on neurite proliferation and nerve regeneration, mediated by TTR internalization by clathrin-dependent endocytosis with megalin as the receptor29. Furthermore, it is known that, TTR promotes neurites outgrowth in PNS specially in dorsal

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mechanism, Gomes JR et al. studied cultured hippocampal neurons in WT and TTR-KO mice and found that TTR promotes neurite outgrowth through megalin. Consequently, TTR has neurogenic activity in PNS and CNS, and this activity is mediated by megalin. However, the involvement of TTR in neurite growth is independent of its ligands30. Thus, TTR plays a key role in the survival and

preservation of neurons, with megalin playing a key role in the activation of TTR neuroprotective signaling mechanisms: when TTR interacts with megalin, it activates mitogen activated protein (MAP) kinase, extracellular-signal regulated kinase (ERK), protein kinase B (Akt) and proto-oncogene tyrosine-protein kinase (Src) which promotes the upregulation of the cyclic adenosine 3',5'-monophosphate (cAMP) response element-binding protein (CREB) transcription factor. In addition, TTR has influence in the intracellular levels of Ca2+ through NMDA receptors that are regulated by

Src, further promoting Ca2+ influx to neurons which may be involved in the proliferation of neurites

(figure 2)30.

Figure 2 – Signalling mechanism triggered by the interaction of TTR with megalin. Activation of mitogen activated protein (MAP) kinase, extracellular-signal regulated kinase (ERK), protein kinase B (Akt), and proto-oncogene tyrosine-protein kinase (Src). Src promotes the upregulation of the adenosine 3',5'-monophosphate (cAMP) response element-binding protein (CREB) transcription factor. TTR also contributes to a significant rise in intracellular calcium by NMDA receptors regulated by Src that allow more calcium influx to neurons. Adapted from Gomes JR et al. 30.

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1.3. Heat-shock Factor 1

In situations of environmental stress, the proteins tend to unfold, refold and aggregate. Many neurodegenerative diseases are also associated with the unfolding of proteins that tend to form aggregates. These aggregates that normally form with a common highly stable b-sheet structure which has high toxicity31. Thus, under these conditions the cell must be able to respond quickly and

efficiently to the demands and control the changes caused by the environment to the structure and metabolism of proteins. The cellular response to this stress situations is mainly controlled by the activation of the heat-shock factor 1 (HSF-1)31.

HSF-1 belongs to the family of transcription factors that respond to heat shock, promoting the activation of heat shock proteins (Hsps) by gene transcription. The heat shock response leads to increased levels of Hsps such as heat shock protein 27 (Hsp27), heat shock protein 40 (Hsp40), heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90); these proteins have as main function to avoid the misfolding of proteins and their aggregation. HSF-1 is one of the most relevant transcription factors since it responds to other types of environmental stress, such as stress caused by reactive oxygen species (ROS) and heavy metals, besides responding to stress caused by thermal shock32. It also presents protective effects in several pathological conditions, namely in

neurohormonal stress, inflammation, ischemia, aging, injury, and others as represented in figure 3. In addition to providing an adaptive response to stress is also triggered in non-stress conditions such as during the cellular cycle, factors of growth, development and differentiation, as demonstrated in figure 333.

Figure 3 - Conditions that induce heat shock response. Environmental stress, pathophysiological state and non-stress conditions that induce heat shock response. From Xiao et al. 33.

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HSF-1 exists in cytosol or nucleus in the monomer form, usually bound to an inhibitor31,34. The

interaction with some specific chaperones as Hsp70 and Hsp90 maintains the monomeric state of HSF-131.

When HSF-1 is activated in response to cellular stress, it is released, homotrimerized and translocated to the nucleus where it binds to heat shock elements (HSEs) present in the target gene promoters31,33,35. After responding to cell stress, and in order to attenuate the action of HSF-1, Hsp70

and Hsp40 (or Hdj-1) bind to HSF-1 to repress its transcriptional activity. Moreover, HSF binding protein 1 binds to dissociate the trimer and restore the inert monomer state of HSF-1 (figure 4)35.

HSF-1 targets many genes, increasing the expression of some and decreasing the expression of others. However, it has been found to have different effects on the expression of the TTR gene, in the same conditions but in different cell types (neurons compared to hepatocytes). The promoter of the TTR gene in mouse and in humans present HSEs, HSF-1 binds to the potential HSEs in the TTR promoter and increase its expression. HSF-1 binds to the TTR promoter of neuron-derived cells and this is evidenced by the high levels of TTR expression in SH-SYSH cells (human neuroblastoma cells)26.

Figure 4 - Regulation of heat shock factor 1 (HSF-1). Activated HSF-1 is translocated to the nucleus and homotrimerizes. It binds to heat shock elements (HSE) present in the deoxyribonucleic acid (DNA), increasing the transcription of genes of interest. To be inactivated, heat shock protein 70 (Hsp70) or heat shock protein 40 (Hsp40 or Hdj-1) bind by repressing the transcriptional activity of HSF-1 and bind to heat shock binding protein 1 to promote dissociation of the trimer. From Xiao et al. 33.

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In this way, in the absence of HSF-1 TTR expression decrease but this will not be the only regulator of neuronal TTR. For HSF-1 to act at TTR level, it must be first exposed to cytoplasmic stress such as formation of aggregates of Aβ, presence of ROS or others that induce cytoplasmic stress to promote the trimerization process of HSF-1 and its transport to the nucleus. Thus, TTR has a neuroprotective capacity in AD because with significant increases in its expression, it will be possible to inhibit/diminish Aβ aggregates and reduce their toxicity26.

Xiao et al. generated HSF-1 null mice and described them as viable until the adulthood. However, they present some deficits in their organism: they have defects in the placenta and prenatal lethality, females present infertility, absence of stress response by thermal shock and reduced expression of Hsp27 and B-crystallin33,36. They also present cerebral morphological

alterations: the lateral ventricles were increased and the white matter reduced.

In FAP, the aggregation of TTR in the PNS leads to inflammatory and oxidative stress and lastly to neurodegeneration. In this way, S.D. Santos et al. hypothesized by studies in HSF-1-KO animals with the most common TTR variant (substitution of a valine for a methionine at position 30 (TTR V30M))37 (labelled TTR/HSF1 mice), that HSF-1 could be involved in a defense mechanism

against TTR aggregates: by regulating the expression of Hsps, proteins that interact with aberrantly folded proteins to prevent aggregate formation. TTR/HSF1 mice showed an increase in TTR deposition in various tissues including in the PNS compared to animals with HSF-1. This suggests that animals with absence of HSF-1 have a lower response of Hsps to triggers from the external environment and further compromises the interaction with proteins aberrantly folded, promoting the formation of aggregates38.

In addition, with aging, the difficulty of activating HSF-1 and Hsps increases. This may explain why diseases involving problems in protein generation and formation of aggregates have a higher incidence with age like AD, Parkinson's disease and others diseases38.

In addition, HSF-1 action as a chaperone, it has antioxidant activity in situations of cellular oxidative stress. Oxidative stress occurs when cellular antioxidant defenses are insufficient to maintain ROS levels below a toxic threshold and thus cause problems in membrane dysfunction, deoxyribonucleic acid (DNA) and macromolecule damage, impaired protein function, and lipid peroxidation and may cause pathological consequences such as cancer, arthritis, neurodegenerative diseases, aging among others39,40.

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In response to oxidative stress, several known mechanisms have an antioxidant response, one of which is glutathione metabolism involving enzymes such as glutathione peroxidase, glutathione S-transferase and others39.

In addition, it has been found that HSF-1 and Hsps exhibit antioxidant activity against oxidative damage and play a role in the regulation of ROS levels during T cell activation41.

Increased hydrogen peroxide levels, promotes the activation of HSFs such as HSF-1 by promoting their translocation to the nucleus. Zhang et al. showed that overexpression of HSF-1 significantly suppresses intracellular ROS levels and apoptosis under oxidative stress. In addition, it induces the activity of Hsp27 and heat shock protein 25 (Hsp25) playing a crucial role in the response to oxidative stress. Hsp27 acts at the level of reduced glutathione and consequently decreases the levels of intracellular ROS and Hsp25 acts at the level of cellular redox homeostasis in the heart and kidney. Thus, HSF-1 has an important antioxidant activity at the level of the reduction of the levels of ROS levels and it also induces the activity of Hsp27 and Hsp25 at the antioxidant level41.

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1.4. Megalin

Since the discovery of megalin as an endocytic receptor involved in processes of neurodegeneration and nerve regeneration, as well as in the activation of neuroprotective mechanisms involving TTR, it has been the basis of many studies mainly regarding its mechanisms of regulation and expression at the level of the nervous system.

1.4.1. Low-Density Lipoprotein Receptor Gene Family

Megalin (also known as gp330 or lipoprotein-related protein 2 (LRP2)) belongs to the LDL receptor gene family which also contains low-density lipoprotein receptors, very-low-density lipoprotein receptors, lipoprotein-related protein 8 (LRP8 or apolipoprotein E (ApoE) receptor), lipoprotein-related protein 4 (LRP4 or multiple epidermal growth factor-repeat-containing protein 7 (MEGF7)), lipoprotein-related protein 1 (LRP1) and lipoprotein-related protein 1b (LRP1b)42.

The receptors from this family have seven common features: the fact that they are expressed in the cell surface, the complement-type repeats extracellular binding domains, the dependence on calcium to establish a connection with the ligand and the ability to recognize the RAP and ApoE. Furthermore, they all contain a homologous precursor epidermal growth factor (EGF)-domain comprising YWTD repeats and single membrane-spanning regions and lastly, they all function as an endocytosis means for several ligands43.

This family is responsible for recognizing extracellular ligands, bind and endocytose them for degradation in lysossomes44. Initially these endocytic receptors were related to the metabolism of

lipoproteins. However, over the years many other functions have been described in the regulation of many signalling processes42.

All members of the family are transmembrane glycoproteins with a much larger extracellular domain than the intracellular domain. The extracellular domain consists of a variable number of ligand-binding-type repeats (or complement-type repeat). The complement-type repeats are comprised of about 40 amino acids with six cysteine residues per replicate, forming a ligand binding motif. What specifies the recognition of the ligands is the set of several of these repeats for a ligand binding domain, and the differential grouping of these repeats within a domain43. These repeats are

separated from each other by β-propeller domains, structured by repeats of YWTD flanked by EGF-type repeats, that are generally important for the proper receptor folding in this family of proteins.

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EGF-type repeats are necessary for the dissociation of ligands from the receptor in endosomes. Most receptors in this family have an O-linked glycosylation domain before the transmembrane domain, and are characterized by preponderance of serine and threonine residues. These are not essential for receptor function. However, they contribute to the maintenance of the distance between the cell surface and the ligand binding domains. One to four of these regions are present in the extracellular domains of each receptor in various combinations. The transmembrane domain consists of hydrophobic residues that are anchored to the membrane. The cytoplasmic tails contain from one to three NPxY motifs that mediate endocytosis and adaptor-protein binding (figure 5)43–46.

1.4.2. General considerations about Megalin

Megalin is a 600kDa membrane glycoprotein (and with a non-glycosylated molecular weight of 517kDa45,46) containing an extracellular domain (containing 4400 amino acids, that recognizes

ligands and binds them), a transmembrane domain (containing 22 amino acids, that targets it to membrane domains rich in cholesterol and glycosphingolipids46) and a cytoplasmic domain

(containing 213 amino acids)45,47,48. However, megalin may also arise in the extracellular medium with

Figure 5 - Structural organization of core members of the low density lipoprotein (LDL) receptor gene family. These receptors containing ligand-binding-repeats, represented in green, epidermal growth factor (EGF)-precursor homology domains structured by repeats of YWTD (represented by stars) flanked by EGF-type repeats (represented in blue pentagons). The cytoplasmic tails contain between one and three NPxY motifs. From Howell & Herz43.

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soluble fragments of about 200-220kDa. This soluble form of megalin may be constituted by truncated forms of the extracellular domain of megalin bound to the membrane49.

The extracellular domain consisting of four clusters of ligand-binding-type repeats. Each repeat contains approximately 40 amino acids and contains a 36 cysteine-rich acidic repeats that are responsible for ligand binding, similar to the mentioned above. Among the ligand-binding-type repeats are the 16 EGF-type repeats and 40YWTD repeats, which function as alternative substrates for the ligand-binding repeats and are required for the release of the pH dependent ligands in the endosomal compartments (figure 6)43,45,46,50,51.

The cytoplasmic tail of megalin contains two NPxY motifs and one NPxY-like motif, a dileucine motif, a binding motif for endocytosis and two PDZ domain binding sites where several intracellular adaptor proteins bind to regulate the signalling, sorting45,48,51 and endocytosis processes (contains

target sites for binding of cytosolic adapter proteins and for kinases that control the movement from the membrane to the cytosolic compartments (figure 6)43,51.

Megalin is known endocytic receptor that acts mainly at the cell surface, where it efficiently binds and internalizes several physiologically relevant molecules50 including nutrients, hormones and

their carrier proteins, signalling molecules, morphogens, and extracellular matrix proteins49. Because

of this, it is involved in many regulatory processes and, therefore, a failure in the levels of this protein may imply crucial failures in the organism.

Figure 6 - Structural organization of megalin. The extracellular domain contains four clusters of ligand-binding repeats, EGF-precursor homology domains structured by 40 repeats of YWTD flanked by 16 EGF-type repeats. The cytoplasmic tail contains two NPxY motifs and one NPxY-like motif, a dileucine motif, a binding motif for endocytosis and two PDZ domain binding sites. From Willnow & Christ 50.

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1.4.3. Megalin in the endocytic process

In the endocytosis process, megalin is involved in three distinct pathways. In the first step, similar in all pathways, the ligand binds to megalin at the surface, and the internalization of megalin from the cell surface requires interaction with the clathrin adaptor related protein complex 2 (AP2) and is promoted by the Disabled-2 (Dab-2)51. Both binding to the first NPxY motif of the cytoplasmic

tail of megalin48. The megalin-ligand complex is internalized and moves to early endosomes. When

the luminal pH of the endosomes drops, there is a break in the megalin-ligand binding: megalin is recycled to the cell surface while ligands are transferred to lysosomes for protein degradation. A second pathway occurs when the ligands are resistant to the pH decrease of the initial endosomes and are recycled back to the surface, occurring a secretion of the ligand. The efficiency of ligand recycling can be stimulated or delayed depending on the presence of different molecules: GIPC PDZ Domain containing family member 1 promotes recycling of the ligand and binds to the carboxyl terminal PDZ domain binding site in the cytoplasmic tail of megalin while autosomal recessive hypercholesterolemia (ARH) delays recycling and binds to the first NPxY motif of the cytoplasmic tail of megalin. A third pathway involves transport of the ligand to early endosomes and secretion by basolateral membrane in a process called transcytosis. The ligand is secreted back to the outside and the megalin receptor is recycled in the cell surface (figure 7)48,51.

Figure 7 - Internalization of ligands by megalin. Endocytosis begins with ligand binding to megalin at the target cell surface. Receptor and cargo complexes internalize from the plasma membrane and move to early endosomes. Endocytosis begins with binding of the ligand to megalin on the surface of the target cell. The complex megalin-ligand internalizes from the plasma membrane and moves to the early endosomes. A decrease in pH breaks the binding between megalin-ligand and promotes recycling of the receptors back to the membrane while the ligand goes to lysosomes where it is degraded. Complex megalin-ligand that resist to pH decrease and are recycled back to the cell surface. Alternatively, there may be transcytosis in which the ligands are secreted by the basolateral membrane. From Willnow & Christ 50.

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1.4.4. Expression of Megalin

Expression of megalin at the mRNA level is positively regulated by retinoic acid, vitamin D, bile acids and ligands of peroxisome proliferator-activated receptor (PPAR) α and γ. Its presence on the cell surface is controlled by mechanisms regulated by chaperones RAP and mesoderm development LRP chaperone (MESD), at the level of the endoplasmic reticulum and by mechanisms of internalization and recycling of megalin at the membrane level, which are regulated by adapter proteins, such as Dab-2 and kinases like glycogen synthase kinase 3 (GSK3).

RAP is a 40kDa protein, which functions as a chaperone during the biosynthesis of some of the members of the LDL-R family and in their delivery to the cell surface9. It is necessary for folding

of megalin, acting at the level of ligand-binding type repeats, preventing the early binding of ligands. The lack of RAP influences the presence of megalin in the plasma membrane, also decreasing its quantity. RAP binds to megalin and inhibits binding of all other ligands45. MESD is another chaperone

that influences folding of megalin and acts at the level of the β-propeller/EGF domains.

For internalization and recycling of megalin, the adapter protein Dab-2 acts by binding to the cytosolic tail of megalin, and it is important for the expression and function of megalin during development and in adult tissues. The lack of Dab-2 causes a loss of polarity of the cell surface of megalin. GSK3 is a kinase that phosphorylates the cytoplasmic domain of megalin and decreases its expression to the cell surface by negatively regulating the recycling of megalin50.

Megalin is a multiligand endocytic receptor expressed in clathrin-coated pits9 at apical

surfaces of epithelia cells of the glomerulus and proximal tubule of the kidney and in visceral yolk sac, epididymis, female reproductive tracts, and in the type I pneumocytes and Clara cells in the lung. It may also be expressed in some sensory organs such as the inner ear and eye45,46,49,50. In the

proximal tubules of kidney and in the visceral yolk sac, megalin is co-localized with cubilin, another endocytic membrane receptor49.

In the brain, megalin is expressed in epithelial cells of the CNS, specifically at the apical surface51. It is also expressed in choroid plexus52,53, capillaries, ependymal cells lining the ventricular

wall and to the spinal cord46,49,51,54

Megalin is also found in non-epithelial cells: in a subpopulation of neural progenitors in the mouse embryonic lineage, oligodendrocytes, ganglion cells retina, cortical neurons, cerebellar granule neurons and in astrocytes29,41,45,50,51,52. Recent studies also support that megalin is present in

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astrocytes and is necessary for albumin binding and internalization into astrocytes, inducing the synthesis of neurotrophic factors for neighbouring neurons46. In addition, megalin is expressed early

in the development of the neural tube and plays a role in the formation of brain structures50, such as

forebrain and spinal cord29.

1.4.5. Megalin and its interactions

Megalin ligands include molecules of various categories, such as vitamin carriers like transcobalamin (vitamin B12-binding protein), vitamin D-binding protein and RBP9,44; proteins involved

in lipoprotein metabolism: apolipoproteinB 100, ApoE, lipoprotein lipase and β-very low density lipoprotein; proteases and protease/inhibitor complexes: aprotinin, α1-chymotrypsin/cathepsin G, plasminogen activator inhibitor-1, protease/plasminogen activator inhibitor-1 complexes, protease/protease nexin-1 complexes, pro-urokinase-type plasminogen activator and tissue plasminogen activator. Megalin also binds to albumin, clusterin (or apolipoprotein J), apolipoprotein J/Aβ peptide complexes, TTR, cubilin, gentamicin, lactoferrin, polymyxcin B, RAP, thyroglobulin43,

cystatin C50 and lipocalin56.

It is also involved in the regulation of leptin transport in the choroid plexus. Leptin is involved in the decrease of β-secretase activity, a limiting step in the formation of the Aβ peptide from APP. A decrease in megalin with age and in AD patients reduces the neuroprotective effect of megalin, reflecting on the brain functions of these individuals and patients 50.

Megalin is the receptor for metallothionein (MT) 46,57 and is intimately related to the signalling

pathway underlying the neuroprotective effect of MT58. MT belong to a family of low-molecular-weight

(6–7kDa) intracellular metal-binding proteins. There are four isoforms of MT (I, II, III, IV) in mammals, which are expressed in different tissues. MT I and II are co-expressed in all tissues and are involved in promoting repair of injured neurons. MT-III is only expressed in the brain and MT-IV is mostly expressed in the epithelium58. This way, megalin is involved in the activation of signalling

pathways with a neuroprotective function and in regeneration processes46,50,57.

Megalin plays a role in the uptake of molecules in the intestine, kidney and across the blood-brain barrier. It is involved in renal reabsorption of Ca2+ and contributes to Ca2+ homeostasis in

cytotrophoblasts and parathyroid. Furthermore, it is involved in the transport of cubilin and it is internalization43,50. Cubilin is a membrane receptor responsible for the absorption of intrinsic

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to its apical expression in the proximal tubules of the kidneys, avoiding the loss of cobalamin through the urine. However, it appears not to be involved in plasma cobalamin transport60.

Sousa MM et al. revealed that megalin is the receptor responsible for tubular uptake of TTR that it is be important to prevent TTR filtration through the glomerulus. In addition, megalin is responsible for the uptake of T4 from TTR. As mentioned above, TTR is responsible for the circulation

of T4 and retinol, via RBP in the latter case. However, RBP is also a ligand of megalin, so TTR does

not have crucial importance in RBP retinol uptake9. Thus, megalin is involved in tubular reabsorption

of RBP. The absence of megalin in the proximal tubules causes excessive loss of retinol and RBP, reflecting the crucial role that megalin plays in the uptake of RBP, avoiding RBP losses through urine. Uptaken RBP is degraded in the lysosomes, and retinol binds to RBP that is newly synthesized or retransported to the circulation61. Furthermore, due to its binding to TTR, megalin has recently

been implicated in the regeneration and development of neuritis of the PNS29.

Megalin as a receptor for TTR also plays a crucial role in nerve regeneration and neurite outgrowth since the neurogenic activity of TTR depends on the internalization of TTR by megalin. Fleming et al. shows that heterozygotic animals for megalin (Meg(+/-)) have a decrease in megalin

levels of about 30% in DRG neurons and that this decrease in megalin implies a decrease in nerve regeneration29.

The importance of megalin is also evident in megalin-deficient mice because they develop defects in pulmonary inflation and alveolar development; in holoprosencephalic syndrome, that there is an abnormal development of the prosencephalon where the hemispheres are fused; in absence of the olfactory apparatus, cranio-facial malformations and loss of hearing29,38,39,41. Mutations in LRP2

gene causes an enlarged cortex, abnormalities in the dorsal diencephalon and hypertrophy of the choroid plexus of the third ventricle46. In addition, megalin knockout mice show high mortality,

developmental abnormalities, and tubular reabsorption deficiency with excretion of low molecular weight plasma proteins in the urine like transferrin9,29. In addition, mice that lack megalin expression

in the brain present problems in the development of ventral telencephalon, as megalin is the receptor for signalling and morphogen proteins as sonic hedgehog protein and bone morphogenetic protein 4. Megalin also controls bone morphogenetic protein 4 levels50.

The expression of megalin in the CNS seems to play a key role in neuronal survival and regeneration, as well as in the control of neurogenesis in adulthood. It also plays a protective role against neurodegenerative conditions due to the involvement in clearance of the Aβ peptide to inhibit

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the formation of complexes with some megalin ligands, such as clusterin and apoE. In addition, it is involved in the uptake of insulin growth factor 1, a neurotrophic factor, from serum to brain50.

In this way, as megalin is the receptor for several molecules and complexes that are fundamental to the nervous system, it is crucial to understand which regulatory mechanisms it is involved in order to better understand its action and its importance in neurodegenerative pathologies.

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CHAPTER 2 –

RATIONALE

AND AIMS

(I) Megalin is a receptor that plays a key role in nerve regeneration and a protective role in neurodegenerative conditions. TTR is one of the ligands of megalin in the nervous system, however the binding site are still unknown.

Thus, it is pertinent to study the levels of megalin (both membranar and soluble) in TTR-KO mouse models, in order to understand the involvement of TTR in megalin expression levels. However, antibodies capable of detecting megalin are not yet optimized. In this sense, an antibody specific for megalin was developed by Cambridge Research Biochemicals.

One of the aims of this study is the characterization of this antibody.

(II) After the antibody has been characterized, WT, TTR-KO and Meg(+/-) mice will be study by

semi-quantitative IHC and confocal microscopy studies as well as in ELISA, to assess variations in megalin expression levels in the brain and in plasma samples.

(III) In addition, recent studies have shown that TTR behaves as neuronal stressed protein regulated by HSF-1, probably having pathological consequences. Therefore, one of the aims of this work is to study the expression of megalin at neuronal level and in plasma samples from HSF(+/-)/WT and HSF(+/-)/TTR-KO mice when subjected to an oxidative diet,

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CHAPTER 3 - MATERIALS AND METHODS

3.1. Animals and diet treatment

In this study, were used WT (labelled WT or IW) mice and TTR null mice (TTR(-/-) (labelled

TTR-KO or M))11 (in the 129/Sv background), as well as HSF(+/-)/WT (labelled HSF/IW) and HSF(+/)

/TTR-KO mice (labelled HSF/M) (in the 129/Sv background)38 and Megalin heterozygotic (Meg(+/-)) mice54,

with different ages. The studies were based on comparison of groups of 5 to 12 animals per genotype with 9-11months (considered old animals) and combined with sex (females and males). IW, M, HSF/IW and HSF/M mice of 10 months were subjected to an oxidative diet (Folate, B6, B12

Deficient Diet (excess methionine) MD.97345, Envigo) during 6/7 weeks. During that time, weight was checked weekly as well as behaviour was monitored daily so that no problem affected the animals. Studies with these animals are based on non-diet animals (controls) of the same strain and age. All experiments were performed with the observer blinded to the genotype of the animal which were determined from tail extracted genomic DNA.

Mice were handled according to the European Union and National rules. The macro-environment conditions to which the animals were subjected, were in accordance to the standards required by Directive 2010/63/EU of the European Parliament and of the Council of 22nd September

2010 on the protection of animals for scientific purposes. The animals were housed in a pathogen-free conditions, maintained at 22 ± 2°C and 45-65% humidity, with periods of 12hours of light followed by 12hours of darkness. These light/dark cycles are fundamental for the mice to perform their natural behaviours as they are nocturnal animals. The ventilation of the room was controlled, which is in accordance with the standard values of 15 to 20 air changes per hour. The cages had a protective filter on top to serve as a protective barrier for both animals and caretakers and also a metal grid with sufficient space to place the food in the form of pellets. Food and water were available ad libitum. The cages were enriched with corn husks, toilet paper strips and paper rolls, so that the mice could build nests and also function as bedding in order to contribute to their well-being and comfort and resemble as much as possible as their natural environment. All efforts were made to minimize pain and distress. To euthanize the animals, mice were anesthetized with an intraperitoneal injection of a mixture of ketamine (75 mg/kg) and medetomidine (1 mg/kg). CSF was collected from the cisterna magna using a stereotaxic apparatus and immediately preserved on dry ice for storage at -80°C. The blood was then collected from the inferior vena cava with syringes

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containing ethylenediamine tetraacetic acid (EDTA). Blood samples were centrifuged at 14,000xg for 10 minutes at room temperature, the supernatant removed and stored at -80°C. After the blood was collected, mice were perfused with cold PBS (Phosphate buffer saline 1X (1.5mM KH2PO4, 2.7mM

Na2HPO4, 150mM NaCl, pH 7.4) Organs and samples of interest were quickly collected in

well-identified eppendorfs and stored at -80°C.

3.2. Characterization of antibodies

Since no soluble mouse megalin compatible antibody was described so far 49one of the aims

of this study was to test the specificity of two antibodies that were specially ordered from Cambridge Research Biochemicals. For this purpose, two rabbits were immunized with a peptide specific for soluble megalin (protected information) and the immunized serum were stored, functioning as an antibody to the peptide which were labelled #33 and #34.

Optimization of Enzyme-linked Immunosorbent assay (ELISA) of antibodies #33 and #34

The specifity of these two antibodies (#33 and #34) was tested by ELISA. A fixed amount of the peptide used in the immunization (10µg/mL) was coated to a maxisort plate (ThermoFisher Scientific, Waltham, MA, USA) during 1hour at room temperature, in coating buffer (0.1M carbonate buffer pH 9.6). As blocking Universal Blocking Reagent 1X (HK085-5KE, BioGenex). was used, incubated 1 hour at room temperature. After blocking, the antibodies were diluted in different concentrations: 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600 and 1:51200, in order to create a dilution curve to optimize the ELISA protocol and to obtain the best dilution for future studies. As a negative control a serum sample from both rabbits before immunization (pre-bleed 33 and pre-bleed 34) was used. The secondary antibody Sheep anti-rabbit IgG (peroxidase conjugate) antibody (AP311, Binding Site) was incubated during 1hour at room temperature with a dilution of 1:2500; 3,3',5,5' Tetramthylbenzidine (TMB) Liquid Substrate System Super Sensitive Form for ELISA (product number T4444 SIGMA) was used as detection reagent and the reaction product was read at 450nm. All the washes steps were performed with PBS-T (PBS with 0,1% Tween (Tween® 20 596470, SigmaAldrich) and dilutions were performed in Universal Blocking Reagent 1X.

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Immunoglobulin Purification of bleed #34

As the serum obtained by immunization with the peptide contain many contaminants, purification of bleed #34 was performed using the Protein G GraviTrap column (GE Healthcare), based on the protocol suggested by the company. As binding buffer 50mM Tris pH 8.0 was used to equilibrate the column and to apply the sample (bleed #34) and was used as elution buffer Gentle buffer pH 6.6 (ThermoFisher Scientific, Waltham, MA, USA).

In order to evaluate the purification of bleed #34, an ELISA was performed according to the above protocol using as coat different concentrations of the peptide of immunization in coating buffer: 5µg/mL, 2.5µg/mL, 125µg/mL, 625ng/mL, 312ng/mL and 150ng/mL. Purified antibody #34 was used as primary antibody with a concentration of 10µg/mL in Universal Blocking Reagent 1X. As secondary antibody Sheep anti-rabbit IgG (peroxidase conjugate) antibody was employed with a dilution of 1:2500 in Universal Blocking Reagent 1X.

Optimization of a Sandwich ELISA protocol to detect soluble megalin in plasma

A sandwich ELISA was performed in order to optimize the detection of megalin in samples. First, it was necessary to test the specificity of the antibodies used. The capture antibody was antibody #34, which was tested above and for detection antibody megalin-sheep polyclonal antibody (kindly offered by Dr. Renata Kozyraki from French Institute of Health and Medical Research, Paris, France) was used and tested by ELISA as described above with a dilution of 1:250 and using as coat different concentrations of the peptide of immunization in coating buffer: 5µg/mL, 2.5µg/mL, 125µg/mL, 625ng/mL, 312ng/mL and 150ng/mL and using as secondary antibody Donkey anti-sheep/goat IgG (peroxidase conjugate) antibody (AP360, Binding Site) with dilution of 1:2000.

After antibodies being tested, a sandwich ELISA was performed using as the capture the antibody purified antibody #34 with a final concentration of 10µg/mL in coating, incubated ON at 4°C. As blocking Universal Blocking Reagent 1X was used, incubated 1 hour at room temperature. Peptide was tested in different concentrations: 5µg/mL, 1µg/mL, 500ng/mL, 100ng/mL, 50ng/mL, 10ng/mL and 1ng/mL and Universal Blocking Reagent 1X was used as negative control, incubated 1 hour at room temperature. The specific antibody conjugate used was a megalin-sheep polyclonal antibody at a dilution of 1:250, incubated 1hour at 4°C, and as the secondary antibody the Donkey anti-sheep/goat IgG (peroxidase conjugate) antibody at a 1:2000 dilution, incubated 1

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hour, at room temperature. The associated detection reagent used it was TMB. All the washes stapes were realized with PBS-T and all dilutions were performed in Universal Blocking Reagent 1X.

Qualitative analysis by Western Blot of antibody #34

In order to make a qualitative analysis of the antibody, a western blot was performed to analyse the molecular size of the bands that the antibody labelled. Thus, a western blot was performed with kidney samples from WT mice previously homogenized and having different protein concentrations.

To homogenize the kidney (stored at -80°C) kinexus lysis buffer pH 7.2 (20mM MOPS, pH 7.0, 2mM EGTA, 5mM EDTA, 30mM sodium fluoride, 60mM b-glycerophosphate pH 7.2, 20mM sodium pyrophosphate, 1mM sodium orthovanadate, 1% Triton X-100, 1mM phenylmethylsulphonyl fluoride and 1X protease inhibitors mixture (GE Healthcare)) was used. Total protein concentration was determined using the Bradford method (Bio-Rad, Hercules, CA, USA).

For Western blot, a homogenized kidney samples (n=2, old WT mice) were applied and resolved on gradient polyacrylamide gel (4-15%) and transferred onto PVDF transfer membrane (GE Healthcare) using a wet system, with Tris/Glycine/SDS buffer (Bio-Rad, Hercules, CA, USA). Membrane was blocked overnight at 4°C in 5% skimmed-milk in PBS-T. The membrane was incubated with antibody #34 diluted 1:200 in PBS for 1h at room temperature and washed in PBS-T for 1hour and then incubated with Sheep anti-rabbit IgG (peroxidase conjugate) at a dilution of 1:5000. After washing in PBS-T, the blot was developed using a chemiluminescence method by exposing in Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA) and visualized by ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA).

Study of the expression of megalin in SH-SY5Y cell culture

It is known that human neuroblastoma cell line SH-SY5Y express TTR23,26, and being TTR a

megalin ligand, the expression and secretion of megalin in these cells was analysed by ELISA of the extract and supernatant.

These cells were cultured in DMEM/F12 (1:1) medium (Invitrogen), supplemented with 10% (v/v) fetal bovine serum (FBS), 2mM L-glutamine, 1000 U/ml penicillin, 100U/ml streptomycin and

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1x of non-essential amino acids. Cells were maintained at 37°C in a humidified atmosphere of 5% CO2/95% air and were grown to 70–80% confluency.

- Preparation of cell lysates

For the preparation of cell lysates, cells in conditioned medium (medium without FBS) were washed twice in PBS. On ice, kinexus lysis buffer pH 7.2 was added and the cells were scraped and centrifuged for 20 minutes 10.000g at 4°C and cell pellet discarded.

- Preparation of supernatant of cells

The cells conditioned medium was removed and centrifuged at 3.000rpm for 10minutes at 4°C and the supernatant was concentrated with a Vivaspin ultrafiltration (10kDa MWCO, GE Healthcare).

Then, an ELISA was performed in the same manner as above, using the cell lysates and the cell supernatant as coat, with two different dilutions: 1:5 and 1:10 diluted in a coating buffer and using as positive control the peptide with a final concentration of 5µg/mL, overnight at 4°C. As blocking was used Universal Blocking Reagent 1X, incubated 1 hour at room temperature. The specific antibody used was anti rabbit-polyclonal #33 antibody at a dilution of 1:200, during 1hour at room temperature and as secondary antibody the Sheep anti-rabbit IgG (peroxidase conjugate) antibody with a dilution of 1/2500, incubated 1 hour, at room temperature. The associated detection reagent used it was TMB. All antibodies were diluted in Universal Blocking Reagent 1X and all the washes stapes were realized with PBS-T.

3.3. Analysis of the presence of megalin in plasma samples

To evaluate the presence of megalin in plasma samples, a sandwich ELISA was performed as described above, using as coat the purified antibody #34 with a final concentration of 10µg/mL. Plasma samples of IW, M, HSF/IW and HSF/M animals with and without treatment (n=6) was used diluted 1:5 in Universal Blocking Reagent 1X. The specific antibody conjugate used was rabbit-polyclonal #33 antibody diluted 1:200 and the secondary antibody used were Sheep anti-rabbit IgG (peroxidase conjugate) antibody with a dilution of 1:2500.

Joana dos Santos Peixoto Gomes Expression of Megalin in the Central Nervous System

Joana dos Santos Peixoto Gomes

Expression of Megalin in the Central

Nervous System

Universidade do Minho

Escola de Ciências

Joana dos Santos Peixoto Gomes

Expression of Megalin in the Central

Nervous System

Tese de Mestrado

Mestrado em Bioquímica Aplicada

Trabalho efetuado sob a orientação de:

Doutora Maria João Saraiva

e

Doutora Sandra Paiva

AGRADECIMENTOS

ABSTRACT

RESUMO

TABLE OF CONTENTS

LIST OF ABBREVIATIONS

LIST OF FIGURES

LIST OF TABLES

CHAPTER 1 - GENERAL INTRODUCTION

1.1. Neurodegenerative Diseases

1.2. Transthyretin

1.3. Heat-shock Factor 1

1.4. Megalin

CHAPTER 2 –

RATIONALE

AND AIMS

CHAPTER 3 - MATERIALS AND METHODS

3.1. Animals and diet treatment

3.2. Characterization of antibodies

3.3. Analysis of the presence of megalin in plasma samples