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UNIVERSIDADE DE LISBOA
FACULDADE DE CIÊNCIAS
DEPARTAMENTO DE BIOLOGIA ANIMAL
Microglia demonstrate age-dependent performance and
interaction with beta-amyloid peptide
Gonçalo Manuel da Costa Lidónio
Dissertação
Mestrado em Biologia Humana e Ambiente
2013
UNIVERSIDADE DE LISBOA
FACULDADE DE CIÊNCIAS
DEPARTAMENTO DE BIOLOGIA ANIMAL
Microglia demonstrate age-dependent performance and
interaction with beta-amyloid peptide
Gonçalo Manuel da Costa Lidónio
Dissertação
Mestrado em Biologia Humana e Ambiente
Dissertação orientada por:
Professora Doutora Dora Brites (orientação externa)
iMed.UL – Faculdade de Farmácia da Universidade de Lisboa
Professora Doutora Deodália Dias (orientação interna)
Faculdade de Ciências da Universidade de Lisboa
2013
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NOTA PRÉVIA
A realização desta dissertação também contou com a co-orientação da Doutora
Ana Sofia Falcão, investigadora auxiliar da Faculdade de Farmácia da
Universidade de Lisboa.
Parte dos resultados inseridos nesta dissertação foram apresentados nos
seguintes encontros:
Lidónio G, Caldeira C, Vaz AR, Santos M, Moreira R, Falcão AS, Brites D. Exploring a
therapeutic approach to prevent Ab-induced dysfunctional microglia. 5th Postgraduate
iMed.UL Students Meeting, Lisboa, Portugal, 18 Julho, 2013 [Poster]
Caldeira C, Frederico A, Oliveira AF, Lidónio G, Vaz A, Fernandes A, Brites D. Cell
ageing effects on microglia response to Aβ.XIII Reunião da Sociedade Portuguesa de
Neurociências, Luso, Portugal, 31 de Maio - 2 de Junho, 2013 [Poster]
A formatação das referências usada nesta dissertação baseou-se no utilizado
pela revista Neurochemical Research da Springer, conceituada na área e na qual o
grupo de investigação tem vindo a publicar.
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AGRADECIMENTOS
Em primeiro lugar, naturalmente, quero agradecer à Prof. Doutora Dora Brites,
orientadora externa desta dissertação. Fico muito grato pela oportunidade de realizar a
minha dissertação de mestrado no seu grupo de investigação, Neuron Glia Biology in
Health and Disease, e por todos os conhecimentos que me transmitiu ao longo deste
ano. O seu rigor científico, espírito crítico, empenho e inteligência são admiráveis, e
uma fonte de inspiração.
À Doutra Sofia Falcão, co-orientadora deste trabalho, agradeço todo o apoio,
dedicação, disponibilidade e preocupação revelados ao longo deste projecto. Sem ti,
Sofia, este trabalho não seria possível. Agradeço, profundamente, toda a paciência e
compreensão que tiveste comigo e toda a motivação que me deste. Contigo, muito
aprendi, tanto a nível profissional como a nível pessoal.
À Prof. Doutora Deodália Dias, orientadora interna desta dissertação e
coordenadora do mestrado em Biologia Humana e Ambiente, agradeço por toda a
disponibilidade e pelo apoio e conselhos que me deu, não só ao longo deste ano, mas
durante toda a minha estadia neste mestrado.
Ao Prof. Doutor Rui Silva e à Prof. Doutora Alexandra Brito agradeço toda a
disponibilidade para esclarecer dúvidas que foram surgindo.
Quero também agradecer à Prof. Doutora Adelaide Fernandes e à Doutora Ana
Rita Vaz toda a disponibilidade que sempre demonstraram, o conhecimento que
partilharam comigo e, principalmente, a ajuda no ultrapassar de muitos dos desafios
que encontrei.
Quero também expressar o meu agradecimento para com a Doutora Teresa
Pais por gentilmente nos ter cedido a linha microglial N9, e ao Prof. Doutor Rui Moreira
por nos ter cedido o composto explorado neste trabalho.
Como não poderia deixar de ser, um grande agradecimento para todos os
restantes membros do grupo:
Inês Palmela, ao contrário do que contam, quero salientar a boa disposição que
sempre observei e agradecer toda a ajuda que me deste – mesmo pensando que
adormeci na tua aula. À Filipa agradeço os bons conselhos, o apoio e, claro, os
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conhecimentos para melhorar as minhas imunocitoquímicas. À Andreia Barateiro
agradeço toda a disponibilidade e a sua contribuição ao alegrar os nossos dias com os
seus bolinhos. Em ti, Cláudia, vejo um exemplo. Acho fascinante a tua força de
vontade. Agradeço-te todo o apoio, ajuda e maturidade que partilhaste comigo neste
trabalho que tão próximo está do teu. Cátia e Carolina, apesar das dores de cabeça
que vocês me deram, tenho muito que vos agradecer. Transmitiram-me
conhecimentos preciosos e, passo a passo, contribuíram para que me tornasse um
melhor cientista. A ti, Gisela, agradeço o apoio, a disponibilidade e todas as sugestões
que me deste em muitos dos problemas que encontrei. Agradeço ainda a simpatia, a
motivação que sempre me transmitiste e o ouvido para os meus desabafos nas
situações mais complicadas. E não me esqueço de ti, Inês Figueira, que mesmo que
não tendo os dois pezinhos no grupo, também contribuíste neste meu percurso.
Obrigado.
Um agradecimento também aos mais novos, aos que seguiram ao meu lado
nesta viagem e que mais força me deram para chegar ao destino. À Vera, um enorme
obrigado. Principalmente por todo o teu positivismo, por partilhares comigo os teus
conhecimentos laboratoriais e, acima de tudo, por todos os momentos de convívio que
contribuíram para tornar esta viagem menos difícil. A vocês as duas, Marta e Andreia,
temo que se deixasse passar tudo o que sinto para o papel, a minha dissertação seria
um agradecimento. Desde os dias de faculdade que seguem ao meu lado e que
preenchem as minhas boas memórias. Agradeço-vos por toda a amizade, por me
puxarem sempre por um sorriso e por todos os momentos de drama, comédia e Sci-Fi
com que comigo partilharam. Sem vocês, esta viagem não seria a mesma. Não posso
passar também sem agradecer ao telemóvel da Marta, que muitos desabafos partilhou
comigo e muito me animou nos dias de chuva.
Agradeço ao grupo, como um todo, pela forma como me receberam. Fizeram-
me sentir em casa.
Quero agradecer também aos constituintes do meu grupo de mestrado. Em
especial à Marília e ao Gonçalo, pelo seu companheirismo e amizade, que me
encheram as horas de risos e desabafos.
Um obrigado muito especial aos meus amigos da terrinha, que estão comigo
desde que lembro e me têm acompanhado nos momentos mais importantes da minha
vida. Em especial a vocês, Nuno, Miguel e Sandra. De perto ou de longe, tornaram
este trabalho não apenas meu, mas também vosso.
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Por último, mas acima de tudo, quero agradecer à minha família. Aos que cá
estão para presenciar esta etapa e aos que gostaria que cá estivessem. Sem vocês,
não seria o que sou hoje. Agradeço principalmente aos meus pais e à minha irmã, pelo
esforço que têm feito para chegar onde cheguei e por nunca desistirem de mim. Não
poderiam ser melhores. Vocês estão e estarão sempre no meu coração.
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ABSTRACT
Alzheimer’s disease (AD) is the most common neurodegenerative disorder in
the elderly and remains without a cure. One of its prominent features is the appearance
in the brain of amyloid-beta (Aβ) deposits forming amyloid plaques. Microglia, which
are the first line of defense in the brain, are activated by Aβ assuming a phagocytic
phenotype followed by the inflammatory state that is thought to play a role in the
progression of this disease. However, the important role of microglia in Aβ clearance
and neuronal support seems to be decreased with age, through an ensemble of
alterations still not clarified that render cells to function abnormally. Indeed, in these
circumstances, microglia lose responsiveness to their key regulating factors, thus
compromising their neuroprotective or inflammation solving properties. Therefore, in
this work we aimed to: a) understand how ageing influences microglia reactivity to Aβ
and microglia-neuron cross-talk dynamics, using an in vitro ageing model of primary
microglia (isolated or in mixed microglia-neurons cultures) and by evaluating the
release of glutamate, adenosine triphosphate (ATP) and matrix metalloproteinases
(MMPs); b) explore the therapeutical properties of a vinyl sulfone-based compound
(VS) as a modulator of Aβ-induced microglial activation, using the N9 cell line and
assessing cell viability, inflammation-related factors, activation receptors and
phagocytosis.
Our results demonstrate that microglia behave differently accordingly with age,
releasing less glutamate and MMP-9, but more MMP-2. Ageing also seems to render
microglia more irresponsive to neuronal-mediated regulation, particularly regarding
their ability to modulate the extracellular glutamate content. In addition, in our
experimental model of ageing in vitro, Aβ failed to induce any microglia reactivity. In
regard to VS testing, no toxicity was observed for this compound, and it successfully
diminished the Aβ-induced release of MMP-9 and MMP-2, as well as the expression of
high-mobility group box 1 protein, important inflammatory mediators. Moreover, Aβ
reduced the level of microglial toll-like receptor 4 and phagocytosis, effects that were
counteracted by VS.
Overall, these results deepen our knowledge about the role of ageing in
microglial biology and elect VS as a potential new therapeutic approach to modulate
Aβ-induced microglial activation and dysfunction in AD.
Keywords: Ageing, Alzheimer disease, Amyloid-beta peptide, Microglia reactivity,
Neuroinflammation, Neuron-microglia communication, Vinyl sulfone compound
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SUMÁRIO
A Doença de Alzheimer (DA) é considerada a doença neurodegenerativa mais
comum e a principal causa de demência a nível mundial, afectando cerca de 35
milhões de pessoas. Esta doença é caracterizada por uma perda progressiva das
capacidades cognitivas, o que leva a um completo estado de debilitação e, por último,
à morte. O principal factor de risco é o envelhecimento e permanece sem tratamento
até hoje.
No encéfalo dos pacientes com DA, para além de uma acentuada perda de
neurónios e sinapses, são ainda observadas duas características distintas da doença:
emaranhados neurofibrilares e placas amilóides. Os primeiros consistem em
agregados filamentosos intracelulares de proteína tau hiperfosforilada, enquanto que
as últimas se devem a depósitos extracelulares do péptido β-amilóide (βA), um
pequeno fragmento de 38-43 aminoácidos que resulta da cisão sequencial da proteína
precursora amilóide por duas secretases, a β-secretase e a γ-secretase.
A causa primordial da DA ainda está por desvendar; contudo, o βA, devido aos
seus variados efeitos neurotóxicos é apontado como um dos principais factores
envolvidos no desenvolvimento da doença. Este, principalmente na forma de 42
aminoácidos, possui uma grande tendência para se auto-agregar, formando estruturas
que vão desde oligómeros até fibrilhas, as quais originam as placas amilóides. Apesar
de inicialmente se julgarem ser os agregados fibrilares as formas mediadoras de
toxicidade, nos últimos anos, as formas oligoméricas têm sido apontadas como as
verdadeiramente tóxicas. Porém, além da oligomerização e deposição de βA, outros
mecanismos têm vindo a ser estudados pela sua associação com a patofisiologia da
DA, nomeadamente a hiperfosforilação da proteína tau, o stress oxidativo, a disfunção
mitocondrial e a neuroinflamação.
As células da microglia, consideradas as células imunes do sistema nervoso
central, desempenham um papel fundamental na resposta inflamatória. Estas células,
conhecidas por constantemente sondarem o tecido cerebral e responderem a vários
estímulos patológicos no sentido de reporem a homeostasia, são frequentemente
encontradas num estado activado e associadas às placas amilóides. Estudos já
realizados evidenciaram que o βA interage com a microglia por meio de vários
receptores, induzindo a libertação de diversas moléculas de carácter pro-inflamatório e
citotóxico. Neste sentido, a deposição de βA pode induzir uma permanente activação
da microglia, conduzindo a uma perpetuação da inflamação e, consequentemente,
contribuindo para neurodegeneração. Por outro lado, a microglia também é
responsável pela libertação de vários factores neurotróficos e pela degradação e
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fagocitose de βA, funções fundamentais na restrição da acumulação de βA e suporte
dos neurónios, impedindo a progressão da DA.
Vários estudos têm demonstrado que a microglia durante o envelhecimento e
em presença de diversos estímulos assume um fenótipo mais do tipo inflamatório, o
qual na presença de uma nova indução acaba por ocasionar uma resposta mais
exacerbada. Para tal, pode igualmente contribuir qualquer alteração da comunicação
entre a microglia e os neurónios, os quais desempenham um papel fundamental no
controlo do estado de activação da célula glial. Provavelmente na sequência destas
sucessivas activações a microglia pode tornar-se morfologicamente distrófica, sendo
assim frequentemente encontrada no cérebro envelhecido e, principalmente, na DA.
Tal indica que estas células sofrem senescência e degeneração com o avanço da
idade, predispondo à doença, que por sua vez intensifica a disfunção da célula,
possivelmente na sequência da exposição ao βA. Assim, com a idade e/ou à medida
que a doença progride, a microglia tende a degenerar, deixando de dar suporte aos
neurónios, o que contribui para a sua morte. Este ponto de vista também ajuda a
explicar a razão pela qual a abordagem terapêutica com fármacos anti-inflamatórios
não esteróides só produz efeitos benéficos nos estádios mais iniciais da doença,
enfatizando a importância do intervalo de actuação nas abordagens terapêuticas.
Com base no crescente trabalho que tem vindo a ser desenvolvido sobre o
papel da microglia na progressão da DA, esta dissertação teve como primeiro
objectivo compreender de que modo o envelhecimento microglial influencia a
reactividade da microglia ao βA e se a capacidade da microglia responder à
sinalização neuronal fica comprometida em resultado do envelhecimento. Para isto,
recorreu-se a um modelo de envelhecimento in vitro utilizando células microgliais
primárias. Foram escolhidas duas idades celulares diferentes, as de 2 e 15 dias de
cultura, de modo a mimetizar um fenótipo microglial jovem e envelhecido,
respectivamente. Estas células foram então colocadas na presença, ou não, de
neurónios e estimuladas com 50 nM e 1000 nM de βA. Depois da incubação,
determinou-se no meio extracelular a concentração de glutamato, de adenosina
trifosfato (ATP) e das metaloproteinases de matriz extracelular (MMP), factores que
desempenham um papel importante na mediação da resposta inflamatória.
Os resultados obtidos demonstraram que a microglia liberta uma menor
quantidade de glutamato e de MMP-9 à medida que envelhece. Verificou-se ainda
uma ausência de resposta da microglia envelhecida em termos de libertação de
glutamato, quando exposta ao βA na presença de neurónios. Em relação à MMP-2,
por outro lado, foi demonstrado um aumento na sua libertação pela microglia em
resultado do envelhecimento. Quanto à libertação de ATP, em qualquer uma das
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condições estudadas, não foram observadas alterações. Nesta abordagem, a
exposição ao βA também não provocou activação microglial, em qualquer uma das
idades avaliadas. Porém, é de salientar que observámos respostas diferentes em
termos de libertação de glutamato e MMP-9 e MMP-2 com a idade e, de forma geral,
estes resultados fortalecem a ideia de que a biologia microglial se altera como
resultado do envelhecimento. Apesar de o envelhecimento poder condicionar a
resposta da célula glial ao βA, tal não foi evidenciado pelos parâmetros analisados,
pelo que se optará pela determinação de indicadores directos de neuroinflamação no
futuro.
O segundo objectivo desta dissertação foi o de avaliar a capacidade de uma
vinil sulfona (VS) sintetizada pelo grupo de Química Medicinal como modulador da
activação microglial pelo βA. Para isto, a linha microglial N9 foi estimulada com as
duas concentrações de βA (50 nM e 1000 nM), na presença ou ausência de duas
concentrações de VS (10 µM e 20 µM), sendo posteriormente avaliados os seguintes
parâmetros: viabilidade celular, libertação de MMPs, capacidade fagocítica da
microglia e a expressão de proteínas como milk fat globule-EGF factor 8 protein (MFG-
E8), toll-like receptor 4 (TLR-4) e high-mobility group box 1 (HMGB1).
A nível de viabilidade, não foram observadas alterações significativas por parte
deste composto testado, nem pelas concentrações de βA usadas. Tanto quanto à
libertação de MMP-2 e MMP-9 como relativamente à expressão de HMGB1,
importantes mediadores da resposta inflamatória, observou-se uma notória indução
após exposição ao βA, sendo a mesma revertida após tratamento com VS. Quanto à
expressão do TLR-4, verificou-se uma redução na presença de βA, sendo este efeito
invertido na presença da maior concentração de VS usada. Em relação tanto à
capacidade fagocítica microglial como à expressão de MFG-E8, um factor com um
papel importante na fagocitose, foi observada uma redução após indução com βA;
porém, apenas a capacidade fagocítica retomou os valores do controlo após adição de
VS. Assim, de forma geral, o composto por nós estudado demonstrou ser capaz de
reverter alguns dos efeitos causados pelo βA, reduzindo de forma significativa a
expressão de factores inflamatórios. Porém, mais estudos ainda têm que ser
realizados para determinar a verdadeira extensão dos efeitos mediados por este
composto, assim como os mecanismos pelos quais actua.
Em suma, estes resultados demonstraram contribuir para o conhecimento de
como o envelhecimento da microglia condiciona a funcionalidade da célula e
evidenciaram a VS como uma potencial terapêutica a ser explorada na modulação de
outros determinantes, quer da activação, quer da latência microglial, observada na DA.
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Palavras-chave: Beta-Amilóide, Comunicação neurónio-microglia, Doença de
Alzheimer, Envelhecimento celular, Neuroinflamação, Reactividade Microglial, Vinil
sulfona
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TABLE OF CONTENTS
NOTA PRÉVIA ................................................................................................................. I
AGRADECIMENTOS ....................................................................................................... III
ABSTRACT ................................................................................................................... VII
SUMÁRIO ...................................................................................................................... IX
INDEX OF FIGURES ................................................................................................... XVII
ABBREVIATIONS......................................................................................................... XIX
I. INTRODUCTION ................................................................................................... 1
1. Alzheimer’s disease (AD): An overview .............................................................. 1
1.1. Disease presentation................................................................................... 1
1.2. Pathophysiology .......................................................................................... 2
1.2.1. Amyloid beta peptide ............................................................................... 4
1.2.2. Tau .......................................................................................................... 7
1.2.3. Oxidative Stress ...................................................................................... 8
1.2.4. Mitochondrial Dysfunction ........................................................................ 8
1.2.5. Neuroinflammation................................................................................... 9
1.3. Diagnosis and treatment ........................................................................... 10
2. Microglia in AD: A closer look to the central player in neuroinflammation ......... 12
2.1. Microglia: origin and functions ................................................................... 12
2.2. Activation of microglia in AD ...................................................................... 14
2.2.1. Interaction of amyloid beta with microglia .............................................. 14
2.2.2. Production of inflammation-related factors by amyloid beta-stimulated
microglia .............................................................................................................. 16
2.2.3. Amyloid beta-induced microglial phagocytosis ....................................... 17
2.3. Microglial role in the progression of AD: activation vs. dysfunction ............ 18
2.4. Microglia-neuron interplay in AD ............................................................... 20
2.5. Vinyl sulfones and inflammatory diseases: a possible therapeutic approach
for microglial modulation in AD? .......................................................................... 23
2.6. Microglial in vitro models for studying AD .................................................. 24
3. Aims ................................................................................................................. 27
II. MATERIAL AND METHODS ............................................................................... 29
1. Material ............................................................................................................ 29
1.1. Reactives .................................................................................................. 29
1.1.1. Cell culture media .................................................................................. 29
1.1.2. Supplements and chemicals .................................................................. 29
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1.1.3. Antibodies .............................................................................................. 30
1.2. Equipment ................................................................................................. 30
1.3. Animals ..................................................................................................... 31
2. Methods ........................................................................................................... 31
2.1. Primary cultures ........................................................................................ 31
2.1.1. Isolation of primary cell cultures ............................................................. 31
2.1.2. In vitro treatment of primary cell cultures with amyloid beta ................... 32
2.1.3. Quantification of extracellular ATP ......................................................... 33
2.1.4. Measurement of extracellular glutamate ................................................ 34
2.1.5. Gelatin zymography ............................................................................... 34
2.2. Cell line ..................................................................................................... 35
2.2.1. N9 microglial cell line culture ................................................................. 35
2.2.2. Treatment of N9 microglial cell line ........................................................ 35
2.2.3. Determination of Cell viability ................................................................. 36
2.2.4. Microglia phagocytic capacity ................................................................ 37
2.2.5. Gelatin zymography ............................................................................... 37
2.2.6. Western blot analysis ............................................................................. 37
2.3. Statistical analysis ..................................................................................... 38
III. RESULTS .......................................................................................................... 39
1. Characterization of young and aged microglia reactivity to Aβ and of modulation
by neurons .............................................................................................................. 39
1.1. Young cells release higher levels of glutamate than older ones when in
monoculture, but not in mixed culture with neurons, and no alterations are
produced by Aβ in each differently aged cell ........................................................ 39
1.2. ATP release by microglia in response to Aβ is not affected by ageing or
neurons ............................................................................................................... 40
1.3. Release of MMP-9 and MMP-2 has opposite profiles in young and aged
microglia and is not altered by Aβ or by the presence of neurons ........................ 41
2. Exploring a new therapeutic approach to prevent Aβ-induced microglial
activation ................................................................................................................. 43
2.1. Loss of viability in Aβ-stimulated microglia is slightly prevented by VS ...... 43
2.2. Release of active MMPs by microglia is enhanced upon Aβ stimulation and
decrease by VS ................................................................................................... 45
2.3. Expression of TLR4 is reduced by the highest concentration of Aβ, an
effect that is counteracted by VS ......................................................................... 46
2.4. Aβ triggers a reduction in microglia phagocytosis, which is prevented by VS
47
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2.5. The expression of MFG-E8 is reduced by 50 nM Aβ and VS alone, while
increases when the highest concentrations of both compounds are concomitantly
used 49
2.6. Aβ induces an increase in the expression of HMGB1 and effect that is
prevented by VS .................................................................................................. 50
IV. DISCUSSION .................................................................................................... 51
Future perspectives ................................................................................................. 59
V. REFERENCES ................................................................................................... 61
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INDEX OF FIGURES
I. INTRODUCTION
Fig. I.1: Neuropathology of Alzheimer’s Disease ........................................................... 3
Fig. I.2: Proteolytic processing of amyloid precursor protein ......................................... 5
Fig. I.3: Amyloid beta assembly states .......................................................................... 6
Fig. I.4: Microglia activation states .............................................................................. 14
Fig. I.5: Amyloid beta interaction with microglial receptors .......................................... 16
Fig. I.6: Microglia-neuron communication .................................................................... 21
II. MATERIAL AND METHODS
Fig. II.1: Experimental procedure used in the primary cellular cultures and parameters
evaluated .................................................................................................................... 33
Fig. II.2: Analysis of Aβ aggregation forms present in our experimental conditions ..... 34
Fig. II.3: Experimental procedure used in the N9 microglial cell cultures and parameters
evaluated. .................................................................................................................. 36
III. RESULTS
Fig. III.1: The extracellular levels of glutamate are higher in young microglia than in old
microglia, being reduced in the first by the presence of neurons and no effect by the
presence of Aβ could be observed in any of the cases. ............................................. 40
Fig. III.2: Microglia release of ATP by Aβ stimulation does not change by age or by
incubation with neurons .............................................................................................. 41
Fig. III.3: MMP-2 release from microglia is up-regulated by ageing, while MMP-9 is
instead down-regulated and these features are not modulated by Aβ or neuronal
signaling ..................................................................................................................... 42
Fig. III.4: Cell viability shows a trend to be decreased by Aβ and was slightly prevented
by VS .......................................................................................................................... 44
Fig. III.5: Stimulation of microglia with Aβ increases the release of active MMP-2 and
MMP-9, which is prevent by VS .................................................................................. 46
Fig. III.6: Expression of TLR4 is reduced by both Aβ and VS, but revealed to be
induced by the highest VS concentration when in the presence of 1000 nM Aβ .......... 47
Fig. III.7: Microglia phagocytic ability is decreased upon exposure to Aβ and restored
by VS. ........................................................................................................................ 48
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Fig. III.8: The expression of MFG- E8 that is reduced by 50 nM and by VS alone, is
increased when the highest concentration of both compounds are concomitantly used.
................................................................................................................................... 49
Fig. III.9: Expression of HMGB1 greatly increases in microglia treated with Aβ, but VS
shows ability to prevent such effect from occurring ..................................................... 50
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ABBREVIATIONS
ABAD Amyloid-binding alcohol dehydrogenase
Ab/Am Antibiotic-antimycotic
AD Alzheimer’s disease
ADAM A desintegrin and metalloproteinase
APOE Apoliprotein-E
APP Amyloid precursor protein
ATP Adenosine triphosphate
Aβ Amyloid beta
Aβ40 Amyloid beta with 40 amino acids
Aβ42 Amyloid beta with 42 amino acids
BACE1 β-site APP cleaving enzyme 1
BBB Blood-brain barrier
BSA Bovine serum albumin
CaCl2 Calcium chloride
CD Cluster of differentiation
CNS Central nervous system
CSF Cerebrospinal fluid
DIV Days in vitro
DMEM Dulbecco’s modified Eagle’s medium
EDTA Ethylenediamine tetraacetic acid
fAβ Fibrillar amyloid beta
FBS Fetal bovine serum
GLT1 Glutamate transporter 1
HMGB1 High-mobility group box 1
HRP HorseRadish Peroxidase
Iba1 Ionized calcium-binding adaptor molecule 1
IDE Insulin-degrading enzyme
IL Interleukin
KOH Potassium hydroxide
LPS Lipopolysaccharide
MAP Microtubule associated protein
MCI Mild cognitive impairment
MEM Minimum essential medium
MFG-E8 Milk fat globule EGF-like factor 8
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MHC Major histocompatibility complex
MMP Matrix metalloproteinase
NEAA Non-essential amino acids
NEP Neprilysin
NF-κB Nuclear factor-κB
NGF Nerve growth factor
NMDA N-methyl-D-aspartate
NO Nitric oxide
NSAID Non-steroidal anti-inflammatory drugs
oAβ Oligomeric amyloid beta
PBS Phosphate buffered saline
PCR Polymerase Chain Reaction
PDL Poly-D-lysine
PET Positron emission tomography
PI Propidium iodide
PMSF Phenylmethylsulfonyl fluoride
PRR Pattern recognition receptors
PS Phosphatidylserine
PSEN1/2 Presinilin 1/2
RAGE Receptor for advanced glycation end-products
RNS Reactive nitrogen species
ROS Reactive oxygen species
RPMI Roswell Park Memorial Institute
SDS Sodium dodecyl sulphate
SDS-PAGE SDS- polyacrilamide gel electrophoresis
TLR Toll-like receptor
TNF Tumor necrosis factor
VR Vibronectin receptor αvβ3
VS Vinyl sulfone-based inhibitor
1
I. INTRODUCTION
1. Alzheimer’s disease (AD): An overview
1.1. Disease presentation
In 1901, Alois Alzheimer, a german physician-pathologist, faced the case of
August Deter, a 51 years-old woman who suffered from an intriguing set of symptoms,
namely, progressive memory loss, confusion, disorientation and delusions [1]. During
almost five years, her mental capacities continued to deteriorate rapidly, until she finally
died in a completely demented state [2]. After patient’s death, Alzheimer performed an
autopsy on her brain, finding significant cerebral atrophy and the presence of unusual
plaques and tangles [1]. His characterization and notes on the case led to the
discovery of a new neurodegenerative disease, a disorder that now bears his name –
Alzheimer’s disease (AD).
One hundred years after its identification, AD has become one of the most
serious preoccupations of our society. AD is considered to be the most common
neurodegenerative disorder and the leading cause of dementia, accounting for 50-60%
of all dementia cases [3]. Presently, the number of people affected by AD worldwide is
estimated to be around 35 million [4]; however, this number is expected to increase,
reaching more than 115 million by 2050 [5]. Like most dementias, AD’s clinical course
begins insidiously, with an unusual gradual decline in memory - classified as mild
cognitive impairment (MCI) - and then progresses to involve the deterioration of other
cognitive functions, such as thinking and reasoning, and also behavior abnormalities,
leaving the individual in a completely debilitated state and totally dependent on others.
[6]. Symptoms unroll from several years to a decade and mortality is frequently related
to resulting secondary issues, like multi-organ failure or opportunistic infections
[7]..There is still no effective treatment for the disease, which in association with the
huge need for patients’ care, makes AD an enormous financial burden to families and
healthcare systems [8].
The elderly population is the main group at risk of developing AD, since
advanced age is the factor most strongly associated with the disease [8] . Based on the
age of onset, AD can be classified in two types: late-onset AD and early-onset AD. The
late-onset AD is the most common form of the disease, accounting for around 90-95%
of cases, and has its onset after age 65. On the other hand, the other 5-10% of AD
2
cases occur between ages 30-65, being defined as early-onset AD [4]. Some of early-
onset AD cases, termed familial AD, are inherited in an autosomal dominant manner
and are caused by mutations in three genes: the amyloid precursor protein (APP) gene
on chromosome 21, the presinilin 1 (PSEN1) gene on chromosome 14 and the
presinilin 2 (PSEN2) gene on chromosome 1 [8]. Nonetheless, most of AD cases are
spontaneous - or within familial clusters that present no clear Mendelian inheritance
pattern -, representing a complex disorder likely caused by an interplay between
multiple susceptibility genes, environmental factors and ageing [5]. In the last years,
several genome-wide association studies have been done, leading to the identification
of different susceptibility genes for AD. Of the genes identified, Apoliprotein-E (APOE)
on chromosome 19 presents, by far, the strongest correlation with AD [5]. There are
three common variants of this gene: ε4, which is the high risk form, increasing the risk
in a dose-dependent manner; ε3, the most common in humans and the neutral allele;
and ε2, which appears to be of low risk [9].
1.2. Pathophysiology
On a macroscopic level, AD pathology can be characterized by a progressive
atrophy of the brain, affecting some regions more than others [10] (Fig. I.1). At a
microscopic level, it is observed that atrophy mainly reflects the loss of neurons and
synaptic contacts [11]. This happens in a defined pattern, with neurons in the
enterohinal cortex being first affected, followed by hippocampal neurons and,
ultimately, surrounding neurons in extra-hippocampal regions, such as neocortical
areas [12]. Nonetheless, not all neuronal populations are equally susceptible to AD. For
instance, neurons that are capable of projecting over long distance (projection
neurons), possessing disproportionately long in relation to cell body size and sparsely
myelinated axons seem to be particularly prone to the disease process [13]. It is not yet
fully elucidated why this happens; however, these neurons have a large cell surface, a
high energy requirement (due to the lack of myelinization) and are very reliant on
axonal transport (anterograde and retrograde) for sustained function and trophic
support, which might increase their susceptibility to damage [14]. In the case of AD,
damage is believed to occur owning to mechanisms outside as well as inside the
neuron and it is characterized by the appearance of intracellular filamentous
aggregates of tau protein, called neurofibrillary tangles, and by extracellular deposits
consisting largely of aggregated amyloid beta (Aβ) peptide, present typically between
neurons with dystrophic neurites, called amyloid plaques [15,16]. These changes that
where firstly described by Dr. Alzheimer are considered the major histopathological
3
hallmarks of the disease [1]. Besides these, gliosis, the accumulation of reactive
astrocytes and microglia near lesions, is also seen in AD [17]. Of the various
pathological features of AD, synaptic loss and selective neuronal death (in the limbic
system and neocortex) are the ones that correlate best with the progression of
cognitive decline, representing the main substrates of dementia [18].
Fig. I.1: Neuropathology of Alzheimer’s Disease. (A) Extensive atrophy is observed in later stages of
Alzheimer’s Disease (AD), which is accompanied by enlarged ventricles, narrowed cortical gyri and widened sulci. Temporal lobes are most severely affected, while occipital lobes and motor cortex are usually spared (adapted from Holtzman [19]). (B) High number of degenerated neurons containing neurofibrillary tangles are found in AD, as well as extracellular deposits of amyloid beta protein (amyloid plaques), which are frequently surrounded by reactive glia cells. (C) In healthy brain, although some characteristics of the disease may be found as a result of the ageing process, they are usually not only present or present in a lower extent.
One of the most important issues for unraveling the AD mystery is finding out
the mechanisms underlying the disease process. In this regard, several mechanisms
are under study, including Aβ aggregation, deposition and toxicity, tau
hyperphosphorylation with tangle formation, as well as oxidative stress, mitochondrial
dysfunction and neuroinflammation.
4
1.2.1. Amyloid beta peptide
Aβ, the principal component of amyloid plaques, is considered a central player
in AD and therefore it has been a major focus of research in the last 30 years. The
main source of Aβ is the processing of APP, a transmembrane protein located on the
plasmatic membrane or in intracellular compartments of diverse cells, like neurons and
glia cells, whose main function still remains unknown [20,21].
APP can undergo several different endoproteolytic cleavage events, which
culminate, or not, in the formation of the Aβ peptide (Fig. I.2). This proteolyic cleavage
is sequential, starting with the cleavage by α-secretase or β-secretase, followed in both
cases by cleavage by γ-secretase. Under normal conditions, APP is preferentially
processed by the so-called non-amyloidogenic pathway, which starts with cleavage by
α-secretase [22]. The enzyme is one of the ADAM9, 10 or 17, all a desintegrin and
metalloproteinase (ADAM) family members [18], and cleaves within the Aβ domain,
resulting in the production of a small peptide presumably non-pathogenic dominated
P3, instead of Aβ . Alternatively, APP may be a substrate of β-secretase, a role that
has been essentially attributed to the β-site APP cleaving enzyme 1, BACE1 , initiating
the amyloidogenic pathway, which results in the formation of Aβ [22]. α-secretase and
β-secretase cleave APP at single sites, whereas γ-secretase, an enzyme complex for
which PSEN1 and PSEN2 act as catalytic subunits, performs a sequential series of
intramembranous cuts, giving rise to products of varying length [23]. Aβ peptide length
varies between 38 to 43 amino acids. The most common isoforms within the brain are
the ones with 40 (Aβ40) and 42 (Aβ42) residues, representing ~90% and ~10%,
respectively of the total [24]. Both Aβ40 and Aβ42 can spontaneously self-aggregate into
higher order structures, ranging from low molecular oligomers (oAβ) to insoluble
aggregates of fibrils (fAβ) [25] ; however, A42 is more hydrophobic and therefore more
prone to aggregation [22] (Fig. I.3).
5
Fig. I.2: Proteolytic processing of amyloid precursor protein. There are two different pathways by
which amyldoid precursor protein (APP) can be processed: the non-amyloidogenic pathway and the amyloidogenic pathway. In the non-amyloidogenic pathway, APP is initially cleaved by α-secretase, originating a secreted fragment of APP (sAPPα) and a membrane-bound carboxy-terminal fragment of 83 amino acids (C83). Subsequently, C83 is further processed by γ-secretase, giving rise to P3, precluding amyloid beta (Aβ) formation. Amiloidogenic processing, alternatively, is initiated by β-secretase and results in the release of sAPPβ. The retained membrane-bound fragment of 99 amino acids (C99) is also a γ-secretase substrate, leading to the production of Aβ. APP intracellular domain (AICD) is an end-product in both pathways, being translocated into the nucleous to regulate gene transcription.
Several lines of investigation support the notion that the pathogenesis of AD is
directly related to a progressive accumulation of Aβ, resulting from an imbalance
between the levels of Aβ production, aggregation and clearance in the brain [26]. The
strongest evidence for this is seen in familial AD, where all cases identified carry
mutations in APP or presenilin genes, leading to an increase of total Aβ production or a
shift in the Aβ40/Aβ42 ratio toward Aβ42. In addition, individuals with Down’s syndrome,
who possess an extra APP gene, also develop an AD-like pathology [27]. In sporadic
form of AD, the Aβ accumulation is thought to be more related with a dysfunction of its
clearance mechanisms [26]. Indeed there is an association between APOE isoforms
and Aβ clearance, with the ε4 isoform showing to be the less efficient in clearing Aβ.
[11]. Furthermore, the activity of major Aβ-degrading enzymes, like neprilysin (NEP)
and insulin-degrading enzyme (IDE), has also been reported to be reduced with age
and in brain regions affected by AD [28]. The identification of these AD-related genes
has led to the creation of diverse animal models, which although not completely
mimicking AD, are a valuable tool to study the mechanisms involved in Aβ
accumulation [29].
6
How Aβ contributes to neurodegeneration in AD is still not fully understood, but
aggregation seems to be essential for A toxicity [27]. Since extracellular deposition of
A is a major hallmark of AD, attention was given to the insoluble fA, the main
aggregated forms in the amyloid plaques [22]. Some studies demonstrated that fAβ are
neurotoxic in vitro [30] and in vivo [31] and it was then hypothesized that they could
represent a primary cause of neurodegeneration in AD. Nevertheless, the number and
temporal progression of plaques do not correlate well with the local extent of the
neuronal death and synaptic loss, nor with the disease progression [22]. Moreover,
some individuals show high levels of plaques without revealing any cognitive
impairment [18]. On the other hand, the levels of extracellular soluble oA show a
robust correlation with the extent of synapse loss and cognitive impairment [27]. A great
number of different oA, natural or synthetic, possessing various sizes and shapes,
have been reported: dimers and trimers, A-derived diffusible ligands, A56* (56 kDA)
and others [32]. These intermediate forms are considerably more toxic than fA, and
are appointed as the main culprits for neurodegeneration in AD [33] .
Fig. I.3: Amyloid beta assembly states. Amyloid beta (Aβ) can exist in diverse assembly states, which
include monomers, oligomers or fibrils, with the latter being the state deposited as amyloid plaques. Formation of fibrils is a complex multi-step process that is preceded by the oligomerization and aggregation of monomeric Aβ. The mechanism driving this process is not fully understood, but may be related to protein misfolding
Besides extracellular accumulation, studies indicate that A also deposits inside
the cells and that this may be an early event in AD, since it precedes extracellular
accumulation and has been reported on brain regions that are more prone to the
development of early AD pathology, such as entorhinal cortex and hippocampus.
Moreover, it is inside the cells that oligomerization process is thought to begin [34]. The
intracellular localization of Aβ is a result of both APP cleavage inside the cell and new
uptake of extracellular Aβ through receptors and transporters, such as the receptor for
advanced glycation end-products (RAGE) [33].
The mechanisms by which Aβ induce neuron and synaptic degeneration are
complex and far from being resolved, but it seems to be influenced by differences in
aggregation forms and localization (intracellular or extracellular). Some of the proposed
mechanisms are: interaction with diverse cell-surface receptors, conducting to inhibition
Aβ monomers Aβ oligomers Aβ fibrils Amyloid plaques
7
or aberrant activation of signal transduction pathways; insertion in membrane and
formation of ion channels or pores, disrupting ionic homeostasis; disruption of
lysosomal membrane; proteasome impairment [35,33].
It is important to have in mind that Aβ is a physiological product of the organism
and as so it may have a physiological role. In this regard, some authors suggest a vital
function in modulation of synaptic plasticity, neuronal survival and neuronal
development, particularly at low concentrations, when in the absence of oligomerization
[36].
1.2.2. Tau Almost concomitantly with the identification of Aβ in amyloid plaques,
neurofibrillary tangles were demonstrated to be composed of abnormally
hyperphosphorylated tau protein. Tau is a member of the family of microtubule-
associated proteins (MAPs) that is particularly abundant in neuronal axons [37]. The
best-established functions of tau are the stabilization of microtubules and the regulation
of motor-driven axonal transport [38]. The biological activity of tau is dependent on its
degree of phosphorylation, which modulates the capacity to bind to microtubules [39].
Several kinases (e.g, glycogen synthase kinase 3 beta) and phosphatases (eg, protein
phosphatase 1) are responsible for the fine tuning of tau phosphorylation state [37].
During AD pathogenesis, tau becomes increasingly phosphorylated, which
causes its detachment from microtubules, resulting in a loss of its normal function [40].
Unbound hyperphosphorylated tau is able to sequester normal tau and other MAPs,
further compromising microtubule structure. Moreover, it possesses an inherent
capacity to self-assembly, forming paired helical filaments that will eventually give rise
to neurofibrillary tangles [39]. Tau aggregates have been reported to be toxic primarily
in the form of oligomers, like in the case of Aβ [41]. Therefore, it is though that the loss
of normal functions and the toxic gain of function of tau, resulting from its
hyperphosphorylation, might contribute to synaptic dysfunction and neurodegeneration
[42]. The causes of abnormal phosphorylation are unclear, but altered function of
kinases or phosphatases, or maladaptation by cellular and microenvironmental stress,
are thought to be implicated [43] .
The involvement of tau in neurodegenerative process is also supported by the
existing correlation between neurofibrillary tangles and the AD onset and progression
[39]. Nonetheless, tau mutations have not been connected to AD. Indeed they are used
to distinct phenotypic diseases, like frontotemporal dementia, that do not display
plaques [42]. This suggests that tau pathology in AD is downstream of Aβ
8
accumulation, and is supported by experimental evidence indicating that Aβ
accumulation precedes and drives tau aggregation. Some studies also indicate that the
presence of tau might be necessary for Aβ neurotoxicity [37].
1.2.3. Oxidative Stress
Oxidative stress derives from an imbalance between the production of reactive
oxygen/nitrogen species (ROS/RNS) and the cellular anti-oxidant mechanisms [44].
ROS and RNS, resulting from hydrogen peroxide and nitric oxide (NO) accumulation,
may damage nucleic acids, carbohydrates, lipids (lipid peroxidation) and proteins
(protein oxidation) [45]. Neurons are considered highly susceptible to an increase of
ROS/NOS due to their low levels of antioxidants mechanisms when compared with
other neural cells [44].
Extensive evidence of increased oxidative damage, including lipid peroxidation,
protein oxidation and nucleic acid oxidation, has been found in AD vulnerable neurons
and suggested to precede any other feature of the disease [46,47]. In addition, low
levels of naturally occurring anti-oxidants, such as α-tocopherol, excessive iron and
copper deposits, intense microglial activation and mitochondrial abnormalities, are all
known to be involved in the production of ROS/RNS and reported in AD [48,49].
Aβ is able to promote the oxidation of compounds like phospholipids and
cholesterol, if an intact methionine in position 35 is present [48]. Furthermore, Aβ also
promotes ROS production by directly associating with transition metals such as iron
and copper, by stimulating microglial activation or through its effects on mitochondria
dynamics [50,51]. Despite promoting oxidative stress, some authors suggest that
accumulation of Aβ might also be a protective response against oxidative stress. For
instance, Aβ has demonstrated to follow the appearance of oxidative stress markers in
AD and to have anti-oxidant properties in cerebrospinal fluid (CSF) and plasma,
protecting lipoproteins from oxidation [49].
Oxidative stress further potentiates Aβ oligomerization and aggregation, which
in turn produces neuroinflammation, mitochondrial damage, and thus, further ROS
generation, leading to a vicious cycle of more oxidative stress and more damage to the
cells [49]. Consequently, there is an enhanced cell dysfunction and demise.
1.2.4. Mitochondrial Dysfunction
Mitochondria are the main producers of both energy and free radicals, through
oxidative phosphorylation and endogenous ROS species, playing a key role in
neuronal function and survival in the brain, the third most energy-expensive organ in
9
the human body [52,53].
Increasing evidence in patients and disease models indicates that mitochondrial
dysfunction accompanies ageing and plays an important role in AD. Indeed, it seems to
be present at all stages of AD and worsens as the disease progresses [54].
Abnormalities on mitochondrial function that are observed in AD include a decreased in
activity of respiratory chain enzymes (e.g. cytochrome c oxidase) and some Krebs
cycle enzymes (eg. α-ketoglutarate dehydrogenase). Moreover, a reduction in the
mitochondrial membrane potential and in the levels of adenosine triphosphate (ATP),
together with increased oxidative modifications in mitochondrial DNA are observed in
the disease [55,56].
The recent intracellular existence of Aβ has led researchers to consider it as a
potential cause of mitochondrial dysfunction. Indeed, evidence suggests that Aβ
crosses mitochondrial membranes, accumulating in the interior of mitochondria [57],
thus influencing the activity of electron transport chain complexes and disturbing
calcium storage, leading to apoptotic pathways. Moreover, Aβ interacts with
mitochondrial matrix components, causing a decrease in the membrane potential and
impairing ATP formation [58]. amyloid-binding alcohol dehydrogenase (ABAD) is one of
Aβ targets. Interaction with ABAD leads to an inactivation of the enzyme activity and
promotes mitochondrial generation of free radical species [59]. It is also important to
refer that mitochondria-derived ROS seem to be able to trigger amyloidogenic APP-
processing, possibly by inducing BACE1 activation, which further implies an existence
of a vicious cycle that contributes to AD pathogenesis [60].
It is now accepted that mitochondria are dynamic organelles and maintain their
homeostasis and function by constantly undergoing fission (or splitting) and fusion (or
combining) [52]. In AD this dynamic equilibrium is disrupted in favor of fission, which
may compromise cellular integrity [61]. Recently, it has also been appointed that APP
overexpression, likely through Aβ-mediated effects, could be responsible for abnormal
mitochondrial dynamics in AD, including decreased mitochondrial mobility and
alterations in fission/fusion processes [54]
1.2.5. Neuroinflammation
The term neuroinflammation refers to the inflammatory-like process that occurs
in the central nervous system (CNS), which is usually a transient, well controlled
process, resulting from the response to a toxic insult [62]. In general, an acute
neuroinflammatory response is beneficial to the CNS, since it contributes to repair the
damaged tissue and to minimize further injury; in contrast, chronic neuroinflammation
10
that persists after an initial toxic insult may produce degenerative changes in neurons
and alter brain function [63,64].
Although contribution of chronic neuroinflammation to AD is a controversy
issue, namely in late AD stages [65], it may exacerbate the course of the disease, while
leading to microglia senescence [66]. This process seems to have a genetic relation
with the disease, since AD patients are more represented with pro-inflammatory
genotypes than anti-inflammatory genotypes [67]. Signs of inflammation are particularly
localized in brain areas exhibiting high levels of AD pathology. Furthermore, high
pathology controls (that is, individuals presenting Aβ and tau aggregates at levels
similar to AD patients, but that do not develop dementia) show lower signs of
inflammation. In addition, the inflammatory mechanisms present in AD patients are
comparable to those present in peripheral inflammatory reactions, which are
established to be cytotoxic, and therefore are likely to have cytotoxic effects on
neurons [68]. AD patients who develop a short-term peripheral infection present a
sudden decline in cognitive state, rarely returning to previous level, even after the
eradication of the infection [69]. It is worthwhile to mention that although some studies
demonstrate that individuals treated (+ 24 months of cumulative use) with non-steroidal
anti-inflammatory drugs (NSAIDs) present a 60-80% reduction in the risk of developing
AD [70,71], anti-inflammatory drugs are ineffective or even harmful when used in
advanced stages of the disease [72,73].
The neuroinflammatory process involves primarily the activation of microglia,
the immune cells of the CNS, but also astrocytes, and even neurons. [74]. All these
cells are able to produce/release several inflammatory mediators, which include pro-
inflammatory cytokines, mainly tumor necrosis factor (TNF)-α, interleukin (IL)-1β and
IL-6, as well as ROS/NOS, complement proteins and various proteolytic enzymes [62].
Some of these inflammatory factors by enhancing Aβ deposition and tau
phosphorylation, concur to further activation and proliferation of glial cells and to the
chronic inflammatory scenario seen in AD, at least in the earlier stages [75,76]. All
these factors, alone or in concert, can then contribute to the neuronal dysfunction seen
in the disease.
1.3. Diagnosis and treatment
Diagnosis of AD can reach about 95% confidence when realized by highly
experienced clinicians, but a full confirmation can only be obtained at autopsy [77,78].
Until recently, the criteria for the diagnosis of AD only considered the late stages of the
disease, when dementia is already present [79]. In 2011, however, a new form for
11
diagnosis was proposed [80]. This new diagnostic criteria expanded the definition of
AD into 3 phases: a pre-symptomatic phase; a symptomatic, pre-dementia phase (or
MCI); and a dementia phase. Moreover, it also incorporated the use of biomarkers to
improve diagnosis, which are mainly used for research purposes.
These biomarkers are physiological, biochemical or anatomical parameters that
can be measured in vivo and reflect specific features of disease-related
pathophysiologic processes [81]. Five biomarkers were incorporated in the new
criterion proposed, being divided in two classes. The first set is constituted by
biomarkers reflecting Aβ deposition in brain, comprising both CSF evaluation of Aβ42
and positron emission tomography (PET) with the amyloid binding tracer N-methyl-
11C-2-(4-methylaminophenyl)-6-hydroxybenzothiazole. The second group reflects
neuronal degeneration or injury and includes tau (total and phosphorylated) level in
CSF, fluorodeoxyglucose uptake on PET (used to measure brain metabolic energy)
and regional brain atrophy (hippocampus, medial, basal and lateral lobes, and the
parietal lobe) on structural magnetic resonance imaging [81]. Besides these, other
promising biomarkers under investigation are the level of BACE1 [82] and isoprostanes
(markers of oxidative stress) in CSF [83]. Plasma or serum biomarkers have also been
explored, but none has shown the diagnostic accuracy of CSF biomarkers [84].
Biomarkers might certainly improve diagnosis of AD, principally at early stages.
Nonetheless, deficient standardization or their limited access still needs to be solved
[80].
After diagnosed, there is still no way to effectively deal with AD. Currently
available treatments include cholinesterase inhibitors (donepezil, galanthamine,
rivastigmine), which target the impairment of cholinergic function due to loss of basal
forebrain cholinergic neurons, and memantine, a N-methyl-D-aspartate (NMDA)
receptor antagonist, that aims to reduce excessive glutamate activation [85]. However,
although these treatments show some clinical benefit, they are just symptomatic, not
stopping disease progression [3]. Therefore, several studies for the development of
new treatments are under way. Drugs aiming to prevent Aβ aggregation by decreasing
Aβ generation and stimulating Aβ clearance are under investigation, and some of them
are even in phases II and III of clinical trials [86]. Treatments to target tauopathy in AD
have received less attention than amyloid therapies, but drugs aiming to inhibit tau
hyperphosphorylation, tau oligomerization or to promote the degradation of
hyperphosphorylated tau are also under study [87]. Additional approaches to treatment
intend to improve neuronal function by using trophic factors like nerve growth factor
(NGF). Moreover, due to the importance of oxidative stress and inflammation in AD,
neuroprotective approaches may also include anti-oxidants (e.g. vitamin E) and anti-
12
inflammatory drugs (e.g. NSAID), although clear positive effects are still missing. In
addition, nondrug approaches for cognitive rehabilitation such as cognitive training and
cognitive stimulation have shown modest, but promising results [88].
2. Microglia in AD: A closer look to the central player in
neuroinflammation
The inflammatory process is unlikely to be the cause of AD [89]; nevertheless,
this does not mean that it is not important. In fact, like it was appointed before, several
evidences indicate that it may play a major role in the early disease stage. Of the
different cells involved in the inflammatory process, microglia by their immune nature
are certainly the most important ones.
2.1. Microglia: origin and functions
Microglia comprise about 12% of all the cells in the brain, occupying both grey
and white matter, with a higher density in hippocampus, substantia nigra, basal ganglia
and olfactory telencephalon [90]. Although still in debate, the general consensus is that
microglia are of hematopoietic origin, being derived from the myeloid precursor cells
from yolk sac that enter the developing CNS during embryogenesis [91].
Usually, under normal conditions, microglia are in a surveying state,
characterized by a ramified morphology with long and thin processes [92]. These
processes are highly mobile, being constantly extending and retracting to monitor
microglia’s surroundings [93]. Each microglia covers a defined territory, non-
overlapping with neighboring microglial cells [94]. Microglia are considered to be long-
lived cells, but might be replaced by low level of local self-renewal [95].
Microglia play numerous important roles through life. In the developing brain,
microglia have seen to be involved in the removal of the massive number of cells that
undergo apoptosis during developmental programmed cell death [96], in the control of
the number of synapses through the process of synaptic pruning [97] and also in CNS
vascularization [96]. Nonetheless, within the adult CNS, the primary role of microglia is
similar to that of macrophages in other tissues, representing the first line of defense
against injury or pathogens[98]. In order to detect and interpret potential insults that
disturb CNS homeostasis, a plethora of different surface receptors are expressed by
microglia, including neurotransmitter receptors and pattern recognition receptors
(PRRs), which represent a vast array of conserved receptors with ability to recognize
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and bind small molecular motifs present in pathogens (pathogen-associated molecular
patterns) or factors associated with tissue damage (alarmins or damage-associated
molecular patterns) [99,94]. These signals evoke the activation of microglia, leading to
a change of their ramified morphology to an amoeboid shape. Microglia became highly
mobile and able to migrate to site of the signal. Furthermore, the activation process is
accompanied by the up-regulation of several surface receptors, as well as by the
production of a plethora of bioactive molecules. [94]. The type of activation is
dependent on factors like the intrinsic properties of the stimulus, duration and exposure
to a prior or another existing stimulus [98].
Microglia are highly plastic cells and while displaying similar morphology they
may show distinct activated phenotypes. Inspired by studies in peripheral
macrophages, the activation of microglia has been generally classified in i) classical
activation, ii) alternative activation and iii) acquired deactivation [100]. Classical
activation (commonly called M1 phenotype) is induced by pro-inflammatory mediators
such as lipopolysaccharide (LPS) or interferon-γ and is characterized by the production
of numerous pro-inflammatory cytokines (e.g. TNF-α, IL-1β and IL-6), proteases and
ROS/NOS. These molecules play an important role in the defense of the organism to
pathogens, but can also damage neurons and glial cells [101]. Alternative activation
(M2a phenotype) is induced by IL-4 or IL-13 and is associated with the production of
anti-inflammatory cytokines and trophic factors such as insulin-like growth factor,
playing a role in tissue repair. [100]. Acquired deactivation (M2c phenotype) is
produced by inducing agents like transforming growth factor β and IL-10, and it is
associated with a robust suppression of the innate immune system, mainly through an
elevated production of the anti-inflammatory cytokine IL-10. [102,100]. Furthermore, the
acquired deactivation is also elicited by the exposure to apoptotic cells, permitting the
phagocytosis of these cells without triggering an immune response, which is important
to tissue maintenance [102]. Both alternative activation and acquired deactivation
down-regulate innate immune responses and demonstrate similar, but not identical,
gene profiles; therefore, some authors treat them as subgroups of a single category,
commonly called M2 or alternative activation [100] . It is also important to refer that
these phenotypes are not static. In fact, microglia is able to switch between
phenotypes, and may exist in many intermediate states (Fig. I.4).
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Fig. I.4: Microglia activation states. Depending on the stimulus, surveying microglia might respond with
different activation profiles. Lipopolysaccharide (LPS) is responsible for inducing a classical activation of microglia, mainly through toll-like-receptor (TLR) 4, resulting in a pro-inflammatory profile characterized by an increase in the production of interleukin (IL) -6, 1β, tumor necrosis factor (TNF)-α and nitric oxide (NO). Although playing an important role in organism defense, the production of these cytokines is commonly related to neurotoxicity. In contrast, alternative activation and acquired deactivation are both associated with an anti-inflammatory, neuroprotective profile, with the former being characterized by the up-regulation of insulin-like growth factor (IGF)-1, arginine 1 and Ym1 and related to tissue repair and extracellular matrix reconstruction, while the later through the production of high levels of IL-10, is more related to a robust suppression of the immune system. Described inducer agents include IL-4 or IL-3 and IL-10 or TGF-β, respectively, which act through different cytokine receptors (CRs).
2.2. Activation of microglia in AD
2.2.1. Interaction of amyloid beta with microglia
Studies evidence the presence of activated microglia clustered near amyloid
plaques in AD patients [103], as well as in AD mouse models [104]. This clustering
might be explained by chemotactic signaling mediated by A, or by other molecules
found to be associated with plaques, such as complement factors or chemokines [105].
Recently, it was visualized by in vivo imaging techniques that microglia migrate to
newly formed plaques within 1-2 days [106]. The number and the dimension of
15
microglia around amyloid plaques increase in proportion to the size of plaques, and it
was suggested that plaque-associated microglia can regulate plaque dynamics [107].
Accordingly, microglia activation has been considered an early event in the
pathogenesis of AD [108].
Several triggers for microglial activation are present in AD, which might include
either molecules released from damaged neurons or even Aβ [109]. Indeed, Aβ was
demonstrated to induce microglial activation and to be one of the main triggers of
microglial response in AD [110]. Both oA and fA are able to bind and activate
microglia through several receptors, including a number of different PPRs [111] (Fig.
I.5). In this context, toll-like receptors (TLR) appear to be particularly important. Among
the cell-surface TLRs present in microglia, TLR2 and TLR4 were shown to be involved
in Aβ-induced microglial activation [111]. Furthermore, several studies in vitro and in
vivo have confirmed that TLR4 mediates the neurotoxicity induced by microglia
[112,113]. Besides TLRs, it was demonstrated that receptors like RAGE, scavenger
receptors (e.g. cluster of differentiation 36, CD36) or formyl peptide receptors (FPR; eg.
FPR-like 1) are also involved in Aβ-induced microglial activation[111]. The binding of
Aβ to these receptors is not only responsible for the secretion of inflammatory
molecules through the activation of signaling cascades such as nuclear factor-κB (NF-
κB), but also for inducing phagocytosis [114,111]. Worth to note that, although they
both stimulate a pro-inflammatory activation, oAβ and fAβ present different activation
profiles [115], and oAβ revealed to be a stronger M1-inducter than fAβ [116].
16
Fig. I.5: Amyloid beta interaction with microglial receptors. Microglia cells express a panoply of
surface receptors with which they are able to sense their environment and respond to stimuli. Amyloid beta (Aβ) is able to interact with several of these receptors, inducing microglial activation. Among the array of receptors described to be involved in Aβ binding are: toll-like receptors (TLR), like TLR2 and TLR4; scavenger receptors (SRs), like CD36; formyl peptide receptors (FPR); and receptor for advanced glycation endproducts (RAGE). The binding of Aβ to microglial receptors elicits a pro-inflammatory response, with the release of inflammatory cytokines like Interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α, probably through activation of the nuclear factor- κB (NF-κB) signaling pathway, but also seems to induce phagocytosis, which is mediated, at least in some cases, by initiating the Src-Vav-Rac signaling cascade
2.2.2. Production of inflammation-related factors by amyloid beta-stimulated microglia
As aforementioned, challenging of microglia with A results in a pro-
inflammatory response. Inflammatory cytokines like IL-1β, IL-6 and TNF-α have all
been reported to be released by A-induced microglia [117]. IL-1β and TNF-α, for
instance, can directly injure neurons when at elevated levels [118]. These cytokines
may act in an autocrine manner further inducing cytokine production, while also
activating astrocytes [17]. Indeed, inflammatory cytokines have been reported to be
increased in vulnerable brain regions during AD and where found to be associated with
plaques [62]. Interaction of microglia with Aβ also leads to the secretion of chemokines,
such as monocyte chemotactic protein-1 (MCP-1, also known as CCL2) and
macrophage inflammatory protein-1α (MIP-1α, also known as CCL3) [119,120].
Chemokines play an important role in the recruitment of microglia and astrocytes, and
an increased expression of the chemokines receptors CCR3 and CCR5 was found in
microglia associated with Aβ deposits [121].
Other inflammatory-related molecules produced by microglia are ROS and
NOS. In this regard, stimulation with Aβ, for instance, was demonstrated to lead to the
17
release of NO and superoxide radical [90]. Although these molecules have an
important role in the defense against pathogens, high levels are detrimental and might
contribute to AD, as it was discussed before. Derivatives of APP, including Aβ, were
also reported to induce the release of glutamate from microglia cells [122,123].
Glutamate is an excitatory amino acid that plays an important role in synaptic plasticity,
memory and learning, but it can also be highly toxic to neurons, inducing excitotoxicity,
which consists on overstimulation of glutamate receptors like NMDA receptors, leading
to a massive influx of extracellular Ca2+ that damages cell structures and components
[124,125]. However, these reports are questioned by recent findings indicating that Aβ
might not affect glutamate release [109]. Also an important molecule that was recently
reported to be released by Aβ-stimulated microglia is ATP, which was linked to ROS
production by microglia [126].
Several proteases are released by microglia, contributing to their functions.
MMPs, for example, which are essential to the maintenance and reconstruction of
extracellular matrix, were indicated to be involved in AD [127]. MMP-3, -12, -13 and -9
have all been reported to be released by Aβ-stimulated microglia [128,129]. Moreover,
both MMP-9 and MMP-2 seem to be able to degrade Aβ in vitro [130,131], and their
relevance to amyloid deposition was further corroborated by knockout mice [132];
however, these proteases also have a role as mediators of tissue degradation and
inflammation, damaging the blood-brain barrier (BBB) and processing pro-inflammatory
cytokines [127] .
2.2.3. Amyloid beta-induced microglial phagocytosis
Phagocytosis represents a central housekeeping function played by microglia
that is important to the elimination of neurotoxic compounds, cellular debris and
pathogens. Accordingly to the receptor that is stimulated, it may be or not coupled with
inflammation [133]. Aβ has been documented by several studies to be phagocytized by
microglia. For instance, examination of microglia at amyloid plaques using electron
microscopy has shown that they are able to engulf Aβ with their processes and indeed
Aβ was observed in endosome-like cellular compartments [134]. Moreover, in vitro
studies using labeled Aβ [135] in combination with direct injection of fAβ into rat brain
[136] further demonstrated the capability of microglia to phagocytize Aβ. The
mechanism by which Aβ is phagocytized upon interaction with microglia is dependent
on its physical and biochemical properties. Insoluble Aβ aggregates are internalized
through receptor mediated phagocytosis (e.g TLR2 and 4), while soluble Aβ has been
suggested to not be truly internalized by phagocytosis, but through macropinocytosis
18
[137,138,99]. However, the question on whether or not microglia restricts Aβ growth in
AD has remained controversial, since the recruitment of microglia to plaques does not
seem to result in their degradation [139,140]. In fact, other in vitro studies indicate that
although microglia is able to phagocytize Aβ the cells may have a limited capacity to
degrade the peptide [141]. Recently, it was even demonstrated that ablation of
microglia does not significantly affect the formation and maintenance of amyloid
plaques and that this might be mediated through the recruitment to the brain of
monocyte-derived macrophages [142], which present a better capacity for Aβ
degradation [143]. The infiltration of monocyte-derived macrophages is thought to only
occur under exceptional conditions, as in the case of CNS disorders such as AD
[144,139]. Nevertheless, this feature is still controversial and to that accounts the
difficulty in distinguishing macrophages from activated microglia based on known
immunophenotypic markers due to their similarity [139].
Recently, besides a possible role in Aβ clearance, microglia was demonstrated
to be able to phagocytize viable neurons and synapses when low (at the range of low
nM) concentrations of Aβ are present in the culture [145]. In line with these findings, Aβ
has also been shown to induce the extracellular exposure of phosphatidylserine (PS)
on neurons [146]. The PS is the best known of “eat-me” signals, which represent a set
of indicators recognized by microglia and evidenced by cells needing to be
phagocytized. Usually, under normal conditions, PS is found in the inner leaflet of cell
plasmatic membrane. However, as a result of several factors, PS may become
externalized and, therefore, it might bind to specific microglial receptors, either directly
or indirectly, inducing the phagocytosis of the cell [147]. One example is the binding to
vibronectin receptor αvβ3 (VR) through the bridging protein milk fat globule EGF-like
factor 8, also known as MFG-E8. [147]. Interestingly, both in AD and MCI it seems to
occur an increase in the number of PS-exposing neurons [148]. Furthermore, the level
of MFG-E8 was indicated to be reduced in AD [149], possibly due to phagocytosis and
degradation, while the β3 subunit of VR was shown to be up-regulated on active
microglia in AD [150]. Nonetheless, MFG-E8 also contributes to Aβ phagocytosis and
its reduction was found more accentuated near amyloid plaques [151]. Thus, it remains
to be determined whether in AD MFG-E8 mediates the phagocytosis of Aβ or of PS-
exposing neurons.
2.3. Microglial role in the progression of AD: activation vs. dysfunction
The involvement of microglia in AD progression is still enigmatic. Aβ is a pro-
inflammatory stimulator of microglial activation. This, in association with the presence
19
of activated microglia near amyloid plaques, led to the proposition that Aβ deposition
might act as a persistent activator for microglia [152]. As a result, microglia might then
become over-activated and produce pro-inflammatory mediators that act directly on
neurons, leading to their damage, while also activating more glial cells, which further
amplify inflammatory signals [153]. The uncontrolled inflammation may drive the
progression of AD, exacerbating and stimulating neuronal death [154]. However, it
might be that in early stages of the disease the role of microglia is protective and not
detrimental, since microglia assume a more phagocytic phenotype and might be able
mediate Aβ clearance by phagocytosis or by secreting enzymes involved in its
degradation, like IDE and NEP [155,62]. In another point of view proposed by Streit
and Xue [156], instead of a gain in inflammatory function, microglia, which above all
things are beneficial cells, might become dysfunctional. In such circumstances,
microglia lose their capacity to support neurons and do not produce trophic factors, like
bone-derived neurotrophic factor, NGF and glial-derived neurotrophic factor. Moreover,
they are not able to clear out neuronal debris and waste residues, or to eliminate toxic
substances, such as Aβ or glutamate in excess. All this findings together will trigger
neurodegeneration [156]. One possible explanation these alterations in normal
microglia function that contribute to the disease might result from ageing [157]. Indeed,
in many ways, the aged brain differs from the young one and this also includes
microglia.
Microglia in the aged brain have been described as presenting less and smaller
branches [158], with an increase in activation markers, such as major histocompatibility
complex (MHC) II and CD11b, as well as an elevation in the basal production of pro-
inflammatory cytokines [159]. Accumulating evidence indicate that microglial
responses to perturbations in CNS also seem to be dysregulated as a result of ageing.
For instance, studies using several animal models of inflammatory challenge
demonstrate that responses by aged animals are in general larger and increasingly
sustained, leading to more pronounced and deleterious effects when compared to
young animals [92]. It has been indicated that this is might be due to a microglial
priming by inflammatory stimuli along life, causing a phenotypic shift of these cells to a
more sensitized state, which results in prolonged and exaggerate responses to
subsequent inflammatory stimuli [159,160]. These features are paralleled in human
aged brain by the appearance of microglia presenting a “dystrophic” morphology, which
was characterized by deramification and abnormalities at cytoplasmic level, such as
atrophy and cytoplasmic fragmentation. Although dystrophic microglia may be
observed in aged brain, they are rarely found in young brain, what conducts to the
hypothesis of microglia senescence and, consequently, degeneration, impairing their
20
capacity to support neurons [161]. The finding that telomere shortening, which is a sign
of senescence, occurs in microglia overtime, both in vitro and in vivo further
corroborates such idea [162,163].
The discovery that microglia suffer alterations with ageing, although adding
another degree of complexity to the subject, might help to clarify their role in the
progression of AD. Dystrophic microglia have been reported to be more prevalent in
AD, accompanying the disease progression and to be associated to its pathological
features [164]. Such finding indicates that microglia senescence is related to AD and
that microglia suffers an accentuated deterioration during the disease process
[165,164]. Indeed, Aβ seems to be able of inducing microglia degeneration [166];
therefore, it may be that the loss of neuroprotection by microglia during ageing is
aggravated by the presence of Aβ, and that the lack of neuronal support leads to
neuronal demise [167]. On the other hand, due to lifelong induced priming, some
microglia might also respond abnormally to the pathogenic stimuli that arise during AD,
over-expressing neurotoxic factors, further contributing to neuron degeneration [165].
These age-related alterations might also help to elucidate why the use of NSAIDs,
which have microglia as one their targets, only produce significant effects at early
stages of AD[168]. It may be then expected that the microglia already primed and
producing an exacerbated response to stimuli, may be restrained by the use of
NSAIDs, while the senescent microglial and cell latency during disease progression will
lead to a point where therapeutical intervention has no benefits.
2.4. Microglia-neuron interplay in AD
In the last years, the idea that neurons are just passive targets of microglia
uncontrolled actions suffered an important change. In fact, studies indicate that
neurons play a very active role in microglial regulation by releasing “on” and “off”
signals [169]. Furthermore, it was proposed that an imbalance of this regulation might
be a possible mechanism for the age-related derangements and vulnerabilities to
neurodegenerative processes [159] (Fig. I.6).
21
Fig. I.6: Microglia-neuron communication. (A) Under steady-state conditions, microglia are maintained
in a surveillant state, presenting an extensively ramified morphology. This phenotype is maintained, in part, through “off” signals constitutively expressed by neurons, such as CX3CL1 and CD200, which act by interacting with their correspondent receptors expressed by microglia. In case of damage, these signaling pathways might become disrupted, inducing microglial activation. (B) Damaged neurons are also responsible for secreting several factors to signal microglia on the need of assistance, and inducing their recruitment and activation. Signals like high-mobility group box 1 (HMGB1) act by binding to toll-like receptors (TLR)2 and 4 or receptor for advanced glycation products (RAGE) to induce the release of pro-inflammatory compounds. Adenosine triphosphate (ATP) is another released factor that may bind to receptors like P2X7 and induce the transcription of pro-inflammatory cytokines. Glutamate, on the other hand, acts on ionotropic and metabotropic glutamate receptors (mGluR) in microglia, leading to a neurotoxic or neuroprotective role, which is dependent on the receptor stimulated.
“On” signals are released by damaged neurons and are responsible for
inducing a defined microglial activation program [169]. One of the most interesting “on”
signals is the high-mobility group box 1 (HMGB1). This alarmin is usually present in the
nucleus, where it functions to stabilize DNA structure and modulate transcriptional
activity. Nevertheless, during activation or cell necrosis, HMGB1 elevates in the
cytoplasm and is released to the extracellular space, mediating inflammatory
responses through binding to receptors such as RAGE and TLR2-4, which seems to
conduct to memory impairment [170,171]. In AD, the levels of HMGB1 are increased,
primarily associating with amyloid plaques [172]. In addition, this protein was reported
22
to inhibit microglial phagocytosis of Aβ [173], suggesting that it may play a role in
preventing Aβ clearance. Other signals also released by damaged neurons are purines
and neurotransmitters [169]. Purines, such as ATP, are detected by the nucleotide
receptors P2Y and P2X in microglia [174]. Different studies indicate that purines are
able to trigger various microglial responses, playing an important role in the activation
of microglia and inducing their migration to the lesion [175,176]. Moreover, it was
recently demonstrated that microglia reacts to extracellular ATP by releasing ATP
themselves, thus providing a positive feedback mechanism to attract more microglia to
the site of injury [177]. The neurotransmitter glutamate also seems to play an important
role in neuron-microglia communication. In fact, although glutamate directly leads to
neuronal death, it also activates microglia, which express both ionotropic and
metabotropic glutamate receptors [94]. Depending on the receptor stimulated in
microglia, glutamate binding might result in neurotoxicity or neuroprotection [169]. Both
ATP and glutamate are intimately connected to neuroinflammation and, therefore,
might also play an important role in AD.
However, probably more important to the context of microglial ageing are the
“off” signals. This type of signals is constitutively expressed in the healthy, normal
brain, being responsible for down-regulating microglial activation [169]. Two “off”
signals that have received great attention in the literature are CD200 and CX3CL1
(also known as fractalkine). CD200 is a neuronal membrane protein that interacts with
its cognate receptor CD200R which is expressed by microglia to deliver regulatory
signals that result in the inhibition of microglia inflammatory phenotype [178].
Interestingly, the axis CD200-CD200R seems to be decreased in the AD brain [179].
Similarly to the CD200-CD200R pair, CX3CL1, a transmembrane chemokine
expressed by neurons in the brain, is also able to interact with its specific
correspondent receptor CX3CR1, which is expressed mainly by microglia in the CNS
and also able to restraining microglial activation [178]. CX3CL1 can be cleaved from
the cell membrane by the ADAM10 and ADAM17, as well as by cathepsin S [180]. Its
soluble form is of crucial importance for chemotaxis, being involved in the recruitment
of microglia [178]. In relation to AD, the role of CX3CL1-CX3CR1 signaling is still
controversial. For instance, some studies in AD mice indicate that the disruption of this
signaling pathway might reduce amyloid deposition, probably by altering microglial
activation and their phagocytic capability [181].On the other hand, it was also reported
that the absence of CX3CR1 protects against neuronal loss [182]. Still, other studies
indicate that the disruption of this signaling enhances tau pathology [183], as well as
inflammation and plaque-independent neuronal dysfunction [184]. Therefore, the
precise contribution of this signaling to AD is yet to be determined. Noteworthy, CD200
23
and CX3CL1, as well as CX3CR1, are all decreased by ageing, representing a loss of
communication between neurons and microglia and may be associated with the
exaggerated immune responses observed in the elderly [159].
2.5. Vinyl sulfones and inflammatory diseases: a possible therapeutic
approach for microglial modulation in AD?
Sulfones are a core functional group of interest in both organic and medicinal
chemistry because of their versatile synthetic utility and due to their role as inhibitors of
various types of enzymatic processes. More specifically, vinyl sulfones are well known
for their ability to irreversible inhibit cysteine proteases, including cysteine cathepsins
like cathepsin B and S [185,186]. Cathepsins are essentially endosomal/lysosomal
proteases involved in physiological protein turnover. Nonetheless, increasing evidence
indicates that some cathepsins also possess other functions [187]. For instance,
cathepsins S is preferentially expressed by cells of mononuclear phagocytic origin, like
microglia, and plays a role in MHCII antigen presentation [188]. Moreover, some
members, such as cathepsins S and cathepsins B, are also secreted upon activation of
microglia with inflammatory mediators and seem to be involved in extracellular matrix
degradation and neuronal death [189]. Notably, cathepsin S is particularly stable at
neutral pH [180].
Increasing evidence indicates that disturbance of normal balance and
extralysosomal localization of cathepsins contributes to neurodegenerative diseases,
like AD [190]. Cathepsin S that plays an important role in microglial activation
maintenance was found overexpressed in AD [191]. Furthermore, cathepsins such as
cathepsins S and cathepsins B have also been suggested to play a role in Aβ
formation, since specific inhibitors for these cathpesins were shown to reduce the level
of Aβ in cells and animals [192], connecting these proteases with AD. Cathepsin S was
recently reported to be directly associated with the ageing process [188], which may be
relate with the exacerbated inflammatory state observed in early stages of AD,
resulting from its role in modulating CX3CL1-CX3CR1 signaling. Taken these concepts
together, cathepsins deserve further evaluation as therapeutic targets to develop
disease modifying drugs to treat AD.
24
2.6. Microglial in vitro models for studying AD
Cell cultures systems have been an appealing method for studying microglial
biology. These systems present a valuable tool to study the activation state, releasable
factors, mobility and other important characteristics of microglia functionally, which
cannot be appropriately examined in vivo [193]. Between the most used cell culture
systems, are the primary microglial cultures and microglial cell lines like BV2 and N9.
The primary cultures are a common choice in inflammatory research due to
their similarity with in vivo cells. They are often derived from the cortex of mouse or rat,
either before or shortly after birth [193]. This type of experimental model is the one that
most closely mimics the endogenous cells and has been greatly used to study
microglial function in AD, mainly at the level of Aβ-related effects [194,195]. However,
due to the time consuming techniques and reduced yield, many studies have utilized
microglia cell lines instead of primary cells to investigate the role of microglia in
physiological and pathological conditions [193]. Interestingly, studies in our lab have
evidenced that cells undergo different characteristics, from young to mature and
senescent, accordingly to the time in culture (in vitro ageing) [196].These findings were
recently observed by us in regard to microglia [197] and reveal to be of high relevance
to evaluate age-related microglial changes. Indeed, up to now there is no suitable
model to obtain reproducible age-related microglia, even from murine models at
different ages, due to the isolation processes limitations
The microglial cell line N9 is derived from mouse and was immortalized using
an oncogene-carrying retrovirus [198]. The usage of a cell line is less expensive and
less time consuming than the use of primary microglia. In addition, there is also less
variations across different cultures [199]. Nonetheless, microglial cell lines are
genetically modified, which determines changes in their adhesion and proliferative
capabilities, and thus inflammatory responses not truly reproducing that of primary
cultures [200]. Still, these cultures represent a useful advantage to investigate
microglia-related phenomena in AD.
Another important approach in the study of microglia dynamics is the use of co-
culture systems, which allows the evaluation of the interactions between microglia and
other cell types. The understanding of the interaction between microglia and neurons is
of prime interest to establish the link between neuroinflammation and
neurodegenerative diseases. Therefore, combined cultures of primary neuronal and
microglial cells have been useful tools for assessing the effect of an inflammatory
response at the level of neurons from diverse brain regions. In fact, there are numerous
published studies using different co-culture approaches [201,202]. Mixed co-culture
25
systems are probably one of the approaches that more closely mimic what happens in
vivo, permitting the study of both physical and soluble cell-to-cell communication
pathways [203].
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3. Aims
The main goals of the present work are a) to understand the role of ageing in
microglial responses and b) to explore the use of a vinyl sulfone-based compound (VS)
as a modulator for microglial activation - both in the context of AD.
More specifically, the aims are to:
a) Evaluate if young and aged microglia are differently stimulated by Aβ and if ageing
also results on a differential response to neuronal derived signals. For this, we will
use an in vitro model of microglial ageing in which primary microglial cell cultures will
be cultivated for 2 and 15 days in vitro (DIV), representing respectively young and
aged phenotypes. Behavior of these different aged microglia in the presence of Aβ
will be assessed by using monocultures and mixed cultures with 17 DIV
hippocampal neurons. The parameters to be determined will be the release of
glutamate, ATP and MMPs.
b) Explore the benefits of VS to modulate microglial activation by Aβ. This will be done
by stimulating the N9 microglial cell line with Aβ, in the presence or absence of VS,
and by evaluating cell viability, the release of inflammation-related factors,
phagocytic capacity and the expression of activation-related receptors.
Collectively, the results to be obtained in this project aim to give further insights
to microglial biology in the context of AD, and hopefully, find a possible new therapeutic
approach for this still untreatable neurodegenerative disorder.
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II. MATERIAL AND METHODS
1. Material
1.1. Reactives
1.1.1. Cell culture media
Dulbecco’s modified Eagle’s medium (DMEM)–Ham’s F12 and Minimum
Essential Medium (MEM) with Earle’s salts were acquired from Biochrom AG (Berlin,
Germany). Neurobasal medium was from Invitrogen Corporation™ (Carlsbad, CA,
USA). Roswell Park Memorial Institute (RPMI) 1640 medium was purchased from
Sigma Aldrich (St. Louis, MO, USA).
1.1.2. Supplements and chemicals
Fetal bovine serum (FBS), L-glutamine, sodium pyruvate, non-essential
aminoacids (NEAA), penicillin/streptomycin and HEPES (4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid) were purchased from Biochrom AG (Berlin, Germany).
Trypsin-EDTA (Ethylenediamine tetraacetic acid) solution (1x) Hoechst 33258, bovine
serum albumin (fraction V, fatty acid free) (BSA), tris-base, β- nicotinamide adenine
dinucleotide 2’-phosphate, ATP, phenylmethylsulfonyl fluoride (PMSF), poly-D-lysine
(PDL), fluorescent latex beads 1 µm (2.5%), Coomassie Brilliant Blue R-250, propidium
iodide [PI; 3,8-diamino-5-(3-(diethylmethylamino) propyl)-6-phenyl phenanthridinium
diiodide], antibiotic-antimycotic (Ab/Am) and Dimethyl sulfoxide (DMSO) were from
Sigma Aldrich (St. Louis, MO, USA). Trypsin-EDTA solution (0.25% trypsin and 1 nM
EDTA in Hank’s balanced salt solution), laminin and B-27 supplement were acquired
from Invitrogen Corportation™ (Carlsbad, CA, USA). L-glutamic acid kit, Triton X-100,
6-phosphateglucose dehydrogenase and hexokinase were from Roche Diagnostics
(Indianapolis, USA). Aβ42 was from EZBiolab (Carmel, IN, USA). DPX mountant for
microscopy was obtained from BDH Prolabo (Bangkok, Thailand). Cell lysis buffer®
and LumiGLO® were from Cell Signaling (Beverly, MA, USA). Sodium dodecyl
sulphate (SDS) was acquired from VWR-Prolabo. Nitrocellulose membrane was
obtained from Amersham Biosciences (Piscataway, NJ, USA). Acrylamide, bis-
acrylamide, Tween 20, glycerol, absolute ethanol, acetic acid, potassium hydroxide
(KOH), gelatin, calcium chloride (CaCl2) and perchloric acid (70%) were obtained from
30
Merck (Darmstadt, Germany). BioRad’s Proteins Assay Reagent was obtained from
BioRad Laboratories (Hercules, CA, USA). All the other common chemicals used were
purchased either from Sigma-Aldrich or Merck. The VS was synthetized in Rui
Moreira’s Medicinal Chemistry unit of iMed.UL.
1.1.3. Antibodies
Primary antibodies: Rabbit anti-ionized calcium-binding adaptor molecule 1
(Iba1) was purchased from Wako (Japan). Rabbit polyclonal anti-MFG-E8 and rabbit
polyclonal anti-TLR4 were from Santa Cruz Biotechonlogy® (Santa Cruz, CA, USA).
Mouse monoclonal anti-HMGB1 was acquired from BioLegend® (San Diego, CA,
USA). Mouse anti-β-actin was obtained from Sigma-Aldrich (St. Louis, MO, USA).
Secondary antibodies: Alexa Fluor® 594 goat anti-rabbit was obtained from
Invitrogen Corporation™ (Carlsbad, CA, USA). HorseRadish Peroxidase (HRP)-labeled
goat anti-rabbit IgG was from Santa Cruz Biotechonlogy® (Santa Cruz, CA, USA).
HRP-labeled goat anti-mouse IgG was from Amersham Biosciences (Piscataway, NJ,
USA).
1.2. Equipment
Basic equipment included an Axio Scope A1 fluorescent microscope with and
adapted camera Leica DFC4900 (both from Zeiss, Gottingen, Germany). For Western
Blot and Zymography assays it was used the Mini-Protean P3 Multi-Casting Chamber
from BioRad (Hercules, CA, USA). Microplate reader (PR 2100) was also from BioRad
and it was used for spectrophotometric measurements of protein and glutamate
content. GloMax® Multi Detection System (Sunnyvale, CA, USA) was used for ATP
quantification. For MMPs gels photos and immunodetection in Western Blot
nitrocellulose membranes it was used the Chemidoc™ system, also from BioRad
Laboratories. Samples sonication for a well homogenization was performed in the
Ultrasonic Processor UP100H (Hielscher-Ultrasound Technology, Teltow, Germany).
To ensure a stable environment to optimal cell growth (37ºC and 5% CO2), cell cultures
were maintained in HERAcell 150 incubators (Thermo Scientific, Waltham, MA, USA).
The work was performed in sterile conditions in a Holten Lamin Air HVR 2460 (Allerod,
Denmark). Eppendorf 580R (Eppendorf, Hamburg, Germany) and Sigma 3K30
centrifuges were used for different experimental procedures.
31
1.3. Animals
The animals used were CD1 mice. Animal care followed the recommendations
of European Convention for the Protection of Vertebrate Animals Used for
Experimental and other Scientific Purposes (Council Directive 86/609/EEC) and
National Law 1005/92 (rules for protection of experimental animals). All animal
procedures were approved by the Institutional Animal Care and Use Committee. The
best efforts were made to minimize the number of animals used and their suffering.
2. Methods
2.1. Primary cultures
2.1.1. Isolation of primary cell cultures
Primary cultures of hippocampal neurons: Primary hippocampal neurons were
isolated from E16 CD1 mice fetuses, as previously described by Lanier and colleagues
[204]. Cells (approximately 1x105 cells/ml) were plated on 24-well tissue plates coated
with PDL (100 µg/mL) and laminin (4µg/mL) in plating medium (MEM with Earle’s salts
supplemented with 10 mM HEPES, 10 mM sodium pyruvate, 0.5 mM L-glutamine, 12.5
µM glutamate, 10% FBS and 0.6% glucose). After 3 hours, the media was replaced
with neuronal growth medium (Neurobasal media supplemented with B-27 supplement
and 0.5 mM L-glutamine). Cultured hippocampal neurons were maintained under
stable growth conditions and the first replacement of the culture media was performed
after 4 DIV, with the procedure being repeated every 7 days until neurons achieve 17
DIV.
Microglia primary cultures: Mixed glial cultures, containing microglia and
astrocytes, were prepared from 1-to-2 day-old CD1 mice as previously described by us
[205]. Cells (4x104 cells/cm2) were plated on uncoated 6-well tissue culture plates in
culture medium (DMEM-Ham’s F-12 medium supplemented with 1% L-glutamine, 1%
sodium pyruvate, 1% NEAA, 10% FBS and 1% Ab/Am solution) and maintained at
37ºC in a humidified atmosphere of 5% CO2. The old medium was removed every 7
days and replaced with fresh culture medium. Pure microglia cells were then isolated
as previously described by us [206]. In brief, after 21 days in culture, microglia were
obtained by mild trypsinization with trypsin-EDTA solution diluted 1:3 in DMEM-Ham’s
F-12 medium for 45 to 60 minutes. The trypsinization resulted in a detachment of an
upper layer of cells containing all the astrocytes, whereas the microglia remained
32
attached to the bottom of the well. The medium containing detached cells was removed
and replaced with the initial mixed glial-conditioned medium. After 4 days, the mixed
glial-conditioned medium was removed and replaced with fresh culture medium, being
again replaced with fresh media at every 4 days. At 2 or 15 DIV post isolation, cells
were removed by total trypsinization with trypsin-EDTA solution for 10 minutes and
seeded on 24-well tissue plates, either alone or on top of 17 DIV neurons, at a density
of 4.0x104 cells/ml. Cultures were kept for 24h before being treated.
The intent of maintaining cells for 21 days in culture before isolation is to
achieve the maximal yield and purity of microglial cultures. In fact, the astrocyte
contamination was previously verified to be less than 2%, as assessed by
immunocytochemical staining with a primary antibody against glial fibrillary acidic
protein, a specific astrocyte marker. Furthermore, neuron contamination was also
excluded, as it was previously assessed by immunocytochemical staining with a
primary antibody against MAP2, a specific marker for neurons [206]. The choose of 2
DIV and 15 DIV as time points intends to mimic a young and aged microglial
phenotype, respectively, and it is based on observations obtained in our group that
indicate that microglia experiences age-related alterations along time in culture [197].
2.1.2. In vitro treatment of primary cell cultures with amyloid beta
Microglia cells, alone or in the presence of 17 DIV primary hippocampal
neurons, were incubated at 2 or 15 DIV in the absence (control) or in the presence of
50 nM or 1000 nM of Aβ42, during 24 h at 37ºC (Fig. II.1). Same procedure was
performed on 17 DIV hippocampal neurons alone, for control purposes. We chose 50
nM since it was the lowest concentration value demonstrated to be sufficient to elicit
microglial activation [194]. The 1000 nM, on the other hand, corresponds to a
concentration range reported in the brain of AD patients [145]. The Aβ42 stock solution
(111 µM) was prepared dissolving Aβ42 to 0.5 mg/ml in DMEM-Ham’s F12 medium.
Before cell treatment, Aβ42 peptides were incubated for 24 h at 37°C to form
aggregates (Fig.II.2).
.
33
Fig. II.1: Experimental procedure used in the primary cellular cultures and parameters evaluated.
Mixed glial cultures were isolated from newborn CD1 mice and maintained for 21 days in culture. After this time, microglia cells were isolated by mild trypsinization and maintained until 2 or 15 DIV. To obtain the mixed neuronal-microglial cultures, hippocampal neurons were isolated from E16 CD1 mice fetuses and maintained to 17 DIV, time when they were co-cultured with microglia, either with 2 or 15DIV. Neuronal hippocampal 17 DIV cultures were also used for control purposes. Microglial cultures, neuronal cultures and neuron-microglial cultures were maintained for 24 h before treatment. During this time, amyloid beta 1-42 (Aβ42) peptides were also allowed to aggregate. Treatment was done with 50 nM or 1000 nM Aβ42, or no addition (control), for 24h. After this period, cell media was removed and evaluated for the release of glutamate, adenosine triphosphate (ATP) and active matrix metalloproteinase (MMP)-2 and MMP-9. ATP was quantified by an enzymatic assay, glutamate by L-glutamic acid kit and MMP-2 and MMP-9 activities by gelatin zymography.
2.1.3. Quantification of extracellular ATP
The assessment of extracellular ATP levels was performed as previously
described by us [207]. The incubation media was collected and treated with 2 M
perchloric acid for 10 minutes, followed by centrifugation for 5 minutes at 10000 g, at
4ºC, to remove cellular debris. Subsequently, 4 M KOH was added for 10 minutes to
neutralize pH value and the centrifugation step was repeated. All the procedure was
performed on ice to prevent ATP degradation. ATP levels were measured indirectly by
the production of NADPH using an enzymatic assay, which consisted in the addition of
a solution containing NADP+, glucose and 6P-glucose dehydrogenase, followed by
hexokinase, the reaction starting enzyme. Fluorescence intensity was quantified using
34
a fluorimeter at ʎem = 450 nm and ʎex = 340 nm. A calibration curve of ATP was used
for each assay.
Fig. II.2: Analysis of Aβ aggregation forms present in our experimental conditions. We have
previously performed this study by western blot using a monoclonal anti-amyloid beta (Aβ) antibody 6E10 and separation on 4-16% Tris-Sodium dodecyl sulphate (SDS)-polyacrilamide gel electrophoresis (PAGE). It was observed that after the aggregation procedure and incubation time our Aβ solution contains a mixture of both oligomeric and fibrillar forms[197].
2.1.4. Measurement of extracellular glutamate
The glutamate content in the incubation media of primary cultures was
determined using the L-glutamic acid kit, as previously described [205]. The reaction
was performed in a 96-well microplate and the absorbance measured at 490 nm. A
calibration curve of glutamic acid was used for each assay.
2.1.5. Gelatin zymography
The quantification of the release of active MMP-9 and MMP-2 in cell media of
primary cell cultures was performed through gelatin zymography method, in which the
protease activity is directly observed in the running gel based in the absence of color
(white bands) at the particular site of protease action. For this determination, aliquots of
the treated culture supernatant were analyzed by SDS-polyacrilamide gel
electrophoresis (PAGE) zymography in 0.1% gelatin/10% acrylamide gels under non-
reducing conditions, at 30 mA/gel. After electrophoresis, gels were washed for one
hour with 2.5% Triton X-100 (in 5 mM Tris pH 7.4; 5 mM CaCl2; 1 µM ZnCl2) to remove
SDS and renature the MMP species in gel. Posteriorly, gels were incubated overnight
in developing buffer (5 mM Tris pH 7.4; 5 mM CaCl2; 1 µM ZnCl2) to induce gelatin lysis.
For enzyme activity analysis, the gels were stained with 0.5% Coomassie Brilliant Blue
R-250 and distained with 30% ethanol/ 10% acetic acid/ H2O. Gelatinase activity,
35
detected as a white band on a blue background, was photographed using Chemidoc
and analyzed using Image LabTM analysis software (BioRad laboratories, version
3.0.1). The digested bands promoted by the different MMPs are distinguished based on
the different molecular weights presented by active MMP-9 (82 kDa) and active MMP-2
(62 kDa).
2.2. Cell line
2.2.1. N9 microglial cell line culture
The N9 microglial cell results from immortalization of microglial cells obtained
from CD1 mouse cortex [198] and it was a kindly gift from Teresa Pais, Institute of
Molecular Medicine (IMM), Lisboa, Portugal. Cells were cultured (4x104 cell/ml) in
RPMI supplemented with 10% FBS, 1% L-Glutamine, 1% penicillin/streptomycin and
grown to confluence. The cells were splitted every 2 to 3 days. For each new
experiment, cells were plated (4x104 cell/ml) on uncoated 6-well or 24-well (with 12 mm
coverslips) tissue plates in the referred RPMI supplemented medium and were
maintained at 37ºC in a humidified atmosphere of 5% CO2 for 2 days before being
treated.
2.2.2. Treatment of N9 microglial cell line
Cells at 2 DIV (platting was considered day 0) were treated without or with Aβ42,
50 nM or 1000 nM for 24 hours at 37ºC, in the absence or presence of VS that was
synthetized at Rui Moreira lab from the Medicinal Chemistry unit at iMed.UL, and which
is currently indicated as a cathepsins S inhibitor (Fig.II.3). Aβ42 was aggregated as
previously mentioned for primary cell cultures. A 20 mM VS stock solution was
prepared by dissolving VS in DMSO. For treatment, VS was diluted in RPMI medium to
10 µM or 20 µM concentrations and added 10 minutes before incubation with Aβ42. We
based our concentrations choice in the values used for a known vinyl sulfone-based
cathepsin S inhibitor, termed LHVS (morpholinurea-leucine-homophenylalanine-
vinylsulfone phenyl) [208].
36
Fig. II.3: Experimental procedure used in the N9 microglial cell cultures and parameters evaluated.
N9 microglia cells were grown in supplemented RPMI medium and incubations were performed at 2 days in vitro (DIV) after passage with 50 nM or 1000 nM aggregated amyloid beta 1-42 (Aβ42) peptides , or no addition (control), in the absence or presence of 10 μM or 20 μM of a vinyl sulfone-based compound (VS). After 24 h of incubation period, cells were fixed or lysed and the respective cell media was also collected. Cell viability was evaluated by propidium idodide incorporation. Phagocytic capacity was evaluated by the incorporation of fluorescent latex beads followed by immunocytochemistry with an anti-Iba1 antibody. The activity of matrix metalloproteinases (MMP)-2 and -9 was accessed by gelatin zymography. Toll-like-receptor 4 (TLR4), milk fat globule-EGF factor 8 (MFG-E8) and high-mobility group box 1 (HMGB1) were evaluated by western blot using specific counter antibodies.
2.2.3. Determination of Cell viability
Necrotic-like cell death was assessed by monitoring the cellular uptake of the
fluorescent dye PI. PI readily enters and stains non-viable cells, but leaves viable cells
unstained, since it cannot cross their cellular membrane. This dye binds to double-
stranded DNA and emits red fluorescence (emission: 630 nm; excitation 493 nm).
After the incubation period, unpermeabilized adherent cultured cells on
coverslips were incubated with 75 µM PI solution diluted in RPMI for 15 minutes at
37ºC, in the absence of light. Subsequently, the medium was removed and the cells
were fixed with freshly prepared 4% (w/v) paraformaldehyde in phosphate buffered
saline (PBS). The nuclei were stained with Hoechst dye 33258 (1:1000, in PBS) during
2 minutes and coverslips were mounted on DPX. Fluorescence was visualized using
an Axio Scope A1 fluorescent microscope with and adapted camera Leica DFC4900.
Red-fluorescence and U.V. images of ten random microscopic fields were acquired per
sample and the percentage of PI positive cells was calculated.
37
2.2.4. Microglia phagocytic capacity
After incubation, to evaluate the phagocytic ability of N9 microglia, adherent
cultured cells on coverslips were incubated with 0.0025% (w/w) 1 µm fluorescent latex
beads for 75 minutes at 37ºC, in the absence of light. Subsequently, the medium was
removed and the cells were fixed with freshly prepared 4% (w/v) paraformaldehyde in
PBS. Cells were immunostained with rabbit anti-Iba1 followed by Alexa Fluor 594 and
the nuclei stained with Hoechst dye 33258. Fluorescence was visualized using an Axio
Scope A1 fluorescent microscope with and adapted camera Leica DFC4900. U.V,
green and red-fluorescence images of ten random microscopic fields were acquired.
Total number of beads internalized was counted to determine the mean number of
ingested beads per cell.
For immunostaining, cells were first permeabilized with 0.1% TritonX-100 in
PBS for 30 minutes and then incubated with blocking solution (PBS with 1% BSA, 0.4%
Triton X-100 and 4% FBS) for 30 minutes. Cells were incubated overnight at 4ºC with
rabbit anti-Iba1 (1:250) diluted in blocking solution. The secondary antibody used was
the goat anti-rabbit Alexa Fluor 594 (1:1000), which was diluted in blocking solution
and incubated for 2 h at room temperature. For nuclei staining, cells were stained with
Hoechst dye 33258 (1:1000, in PBS) during 2 minutes.
2.2.5. Gelatin zymography
The evaluation of the release of active MMP-2 and MMP-9 in N9 microglial cell
line was performed by gelatin zymography as previously described to the primary cell
cultures (section 2.1.5).
2.2.6. Western blot analysis
Western blot was carried out as usual in our lab [209]. Briefly, total cell extracts
from the 6-well tissue plates were obtained by lysing the cells with ice-cold Cell Lysis
Buffer plus 1 mM PMSF for 10 minutes on ice and with shaking, followed by sonication
during 40 seconds. The lysate was centrifuged at 14,000g for 10 minutes at 4ºC and
the supernatants were collected and stored at -80ºC. Protein concentration was
determined using the Bradford method [210], with the BioRad’s protein assay reagent.
Equal amounts of protein content were subjected to 12% SDS-PAGE, running with
fixed amperage of 30 mA/gel. After running the gel, proteins were transferred to a
nitrocellulose membrane, and blotted membranes were rinsed once with TBS-T (10 nM
Tris-HCl (pH 7.6), 150 mM NaCl and 0.1% Tween 20) and blocked for 1 hour at room
temperature with 5% milk in TBS-T. Membranes were then incubated overnight at 4ºC
38
with the following primary antibodies diluted in TBS-T with 5% BSA: anti-HMGB1
mouse antibody (1:500); anti-TLR4 rabbit antibody (1:200); anti-MFG-E8 rabbit
antibody (1:200). After washing with TBS-T, the membranes were incubated for 1 hour
at room temperature with HRP-labeled anti-rabbit or anti-mouse antibody (both
1:5000), as appropriate, diluted in blocking buffer. After washing membranes with TBS-
T, chemiluminescent detection was performed by LumiGLO® and bands were
visualized using Chemidoc. The relative intensities of protein bands were analyzed
using the Image LabTM analysis software (BioRad laboratories, version 3.0.1). All the
results obtained were normalized to β-actin, which was determined using an anti-β-
actin mouse antibody (1:5000 in TBS-T with 5% BSA) followed by a HRP-labeled anti-
mouse antibody (1:5000 in blocking buffer).
2.3. Statistical analysis
Results from at least two different experiments were expressed as mean ± SEM.
Statistical analysis was performed using two-tailed Student’s t test, on the basis of
equal or unequal variance, or one-way ANOVA using GraphPad Prism 5 (GraphPad
Software inc, version 5.00), as appropriate. It was considered p<0.05 as statistically
significant.
39
III. RESULTS
1. Characterization of young and aged microglia reactivity to Aβ
and of modulation by neurons
Microglia have several fundamental functions in the CNS and may mediate
either beneficial or detrimental effects [101]. Changes in normal microglial function
have been vastly implied in AD. As advanced age is the largest risk factor for the
disease, it has been hypothesized that microglia age-related changes may drive its
pathogenic progression through a diminution of neuroprotective functions and
dysregulated responses to signals and perturbations that increase neurotoxicity
[156,165,167]. To dissect whether microglial ageing might mediate an altered reactivity
to Aβ, we developed an in vitro model using cultures of primary microglial cells grown
till 2 and 15 DIV, in order to mimic young and aged cells, respectively. Since neurons
are major players in modulating the microglial reactivity, we decided to give another
dimension to our model and we also studied how microglia behaves in their presence.
The alterations in microglial reactivity were addressed by determining the release of
glutamate, ATP and MMPs
1.1. Young cells release higher levels of glutamate than older ones when in
monoculture, but not in mixed culture with neurons, and no alterations
are produced by Aβ in each differently aged cell
Glutamate release by activated microglia is a well described event in the
literature, being of special importance to the context of neurodegenerative diseases,
since the excess of glutamate may contribute to the neuronal death observed in these
disorders [109]. As evidenced in Figure III.1, we observed that glutamate released by
aged microglia in control conditions was significantly lower than by young microglia
(0.43-fold, p<0.01). Interestingly, when young microglia was in the presence of neurons
there was a remarkable reduction in glutamate levels (0.54-fold, p<0.01), a feature that
was not observed in mixed cultures of aged microglia with neurons. In all the cultures,
the exposure to Aβ did not lead to significant alterations.
40
1.2. ATP release by microglia in response to Aβ is not affected by ageing or
neurons
ATP has been reported to be released from damaged neuronal cells, being a
major factor in the migration of microglia to sites of injury [177]. Microglia have also
been reported to release ATP when stimulated with Aβ [211], which may be involved in
mediating the recruitment of more microglia to amyloid deposits. Therefore, next it was
evaluated the release of ATP. Once again, no clear effects were observed by cells
exposed to Aβ (Fig. III.2). Moreover, there were no significant alterations caused by the
presence of neurons in both young and aged microglia and no differences in the ATP
release between differently aged cells.
Fig. III.1: The extracellular levels of glutamate are higher in young microglia than in old microglia, being reduced in the first by the presence of neurons and no effect by the presence of Aβ could be observed in any of the cases. Microglia (MG) with 2 and 15 days in vitro (DIV) were cultured in the
absence or presence of 17 DIV neurons (N) and incubated with 50 nM and 1000 nM of amyloid beta (Aβ), or no addition (control). Aliquots of the culture media were collected at 24 h after incubation, and glutamate content was assessed by the L-glutamic acid kit. Results are mean ± SEM from at least two independent experiments performed in duplicate, and presented as fold change relatively to the control of MG (2 DIV), assumed as 1.
$$p<0.01 and
$p<0.05 vs. 2 DIV MG at the same experimental condition.
41
Fig. III.2: Microglia release of ATP by Aβ stimulation does not change by age or by incubation with neurons. Microglia (MG) with 2 and 15 days in vitro (DIV) were cultured in the absence or presence of 17
DIV neurons (N) and incubated with 50 nM and 1000 nM of amyloid beta (Aβ), or no addition (control). Aliquots of the culture media were collected at 24 h after incubation, and adenosine triphosphate (ATP) release was measured by an enzymatic assay. Results are expressed as mean ± SEM from at least two independent experiments performed in duplicate, and presented as fold change relatively to the control of 2 DIV MG, which was assumed as 1.
1.3. Release of MMP-9 and MMP-2 has opposite profiles in young and aged
microglia and is not altered by Aβ or by the presence of neurons
Increasing evidence indicates a link between the activation of MMPs and AD
pathophysiology, in which both detrimental and beneficial roles have been suggested
[212]. We decided to evaluate if the release of active MMP-2 and MMP-9 was altered
with microglial ageing in response to Aβ and neuronal signals. By gelatin zymography,
we were able to detect two different bands, at 62 kDa and 82 kDa, corresponding to
MMP-2 and MMP-9, respectively (Fig. III.3). We observed an age-dependent increase
in the activity of MMP-2 (2.9-fold, p<0.05); however, neither young nor aged microglia
presented any responsiveness to Aβ or to neurons. Conversely to MMP-2, the activity
of MMP-9 presented a marked reduction with age (0.4-fold, p<0.01). Nevertheless, no
alterations were produced by the exposure to Aβ or by the presence of neurons.
42
Fig. III.3: MMP-2 release from microglia is up-regulated by ageing, while MMP-9 is instead down-
regulated and these features are not modulated by Aβ or neuronal signaling. Microglia (MG) with 2 and 15 days in vitro (DIV) were cultured in the absence or presence of 17 DIV neurons (N) and incubated with 50 nM and 1000 nM of amyloid beta (Aβ), or no addition (control). Aliquots of the culture media were collected at 24 h after incubation and used to assess matrix metalloproteinases (MMPs) activity by gelatin zymography assay. (A) Active forms of MMP-2 and MMP-9 were identified by their apparent molecular mass of 62 kDa and 82 kDa, respectively. Representative results from one experiment are shown. (B) and (C) Graph bars represent the intensity of the bands for each MMP quantified by scanning densitometry. Results are expressed as mean ± SEM from at least two independent experiments performed in duplicate, and presented as fold change relatively to the control of 2 DIV MG, which was assumed as 1.
$$p<0.01
and $p<0.05 vs. 2 DIV MG at same the experimental condition.
A
B
C
43
2. Exploring a new therapeutic approach to prevent Aβ -induced
microglial activation
Microglia react to diverse stimuli by becoming morphologically and functionally
activated, which might lead to more protective or detrimental responses, depending on
the molecules involved [94]. In AD, one of the main microglial activators is Aβ, which
induces a classical, more inflammatory phenotype, that is thought to pave the way to
the chronical inflammatory state observed in the disease [213]. Therefore, in an
exploratory approach, we decided to evaluate the suitability of VS, which is currently
indicated as a cathepsin S inhibitor, to modulate Aβ-induced microglial activation. For
that, N9 microglial cell line was used. This cell line is obtained by immortalization of
CD1 mice cortex microglia and shows diverse features similar to microglia in primary
cultures, such as migration, phagocytosis and inflammation-related features. [214-216].
2.1. Loss of viability in Aβ-stimulated microglia is slightly prevented by VS
Since a new compound will be tested, the evaluation of toxicity is of special
importance. Toxicity of VS was determined by the number of necrotic cells, through
measurement of PI uptake. As evidenced in Figure III.4, we observed a tendency to a
concentration-dependent decrease in the viability of microglia cells exposed to Aβ. The
presence of VS did not increase toxicity and, interestingly, slightly reduced the
neurotoxic effects of the highest concentration of Aβ used. Nevertheless, no statistical
significance was obtained.
44
Fig. III.4: Cell viability shows a trend to be decreased by Aβ and was slightly prevented by VS. N9
cells were treated for 24 h with 50 nM and 1000 nM amyloid beta (Aβ), as in previous experiments, but now in the presence or absence of 10 μM and 20 μM vinyl sulfone (VS). Cells not-treated with Aβ served as controls. After incubation, cells were treated with 75 µM of the fluorescent dye propidium iodide (PI) for 15 min. (A) Uptake of PI was assessed by fluorescent microscopy and the percentage of PI-positive cells was counted. Results are mean ± SEM from at least four independent experiments. (B) Nuclei were stained with Hoechst dye (blue) and necrotic cells were labeled for PI (red). (C) Representative results of one experiment are shown. Scale bar represents 40 μm.
A
B
C
45
2.2. Release of active MMPs by microglia is enhanced upon Aβ stimulation
and decrease by VS
MMPs are important inflammatory mediators released by activated microglia
and might play an important role in AD onset and progression, as previously
mentioned. Thus, we evaluated if the release of MMP-2 and MMP-9 were induced by
Aβ and modulated by VS (Fig.III.5). We observed that the release of active MMP-2
increased significantly upon Aβ exposure, in a concentration-dependent manner (1.1-
and 1.4-fold for Aβ 50 and 1000 nM; p<0.05 and p<0.01, respectively). In regard to VS
alone, none of the two concentrations used evidenced to produce significant
alterations. However, the effects caused by 1000 nM of Aβ on microglia were reverted
to near control values in the presence of 20 µM VS (0.9-fold, p<0.05). The same
pattern of results was observed for the release of active MMP-9. The exposure to Aβ
induced a significant increase in the MMP-9 release by microglia in a dose-dependent
manner (1.1- and 1.6-fold for Aβ 50 and 1000 nM, respectively, p<0.01). No alterations
were induced by both concentrations of VS. In addition, considering the promising
modulatory effects by VS on Aβ, the highest dose of 20 µM caused a significant
decrease in MMP-9 (1.1-fold, p<0.05) release in response to 1000 nM Aβ.
46
Fig. III.5: Stimulation of microglia with Aβ increases the release of active MMP-2 and MMP-9, which is prevent by VS. N9 cells were treated for 24 h with 50 nM and 1000 nM amyloid beta (Aβ), as in
previous experiments, but now in the presence or absence of 10 μM and 20 μM vinyl sulfone (VS). Cells not-treated with Aβ served as controls. (A) Matrix metalloproteinases (MMP)-2 and MMP-9 were identified by their apparent molecular mass of 62 kDa and 82 kDa, respectively. Representative results from one experiment are shown. (B) and (C) Graph bars represent the intensity of the bands quantified by scanning densitometry. Results are expressed as mean ± SEM from at least five independent experiments performed in duplicate, and presented as fold change relatively to control, which was assumed as1. **p<0.01 and *p<0.05 vs. respective control;
$p<0.05 vs. same experimental condition in the absence of
VS.
2.3. Expression of TLR4 is reduced by the highest concentration of Aβ, an
effect that is counteracted by VS
The TLR4 receptor is one of the best described receptors and referred to be
involved in Aβ-induced microglial activation [111]. Therefore, its expression was an
important point to be here addressed. The determination of TLR4 expression was
A
B
C
47
performed by western blot analysis of N9 cell lysates. We observed a significant
decrease (0.7-fold, p<0.01) in the expression of TLR4 after exposure to Aβ 1000 nM
(Fig. III.6). Surprisingly, also VS revealed to decrease the expression of TLR4,
although only the highest concentration produced significant effect (0.5-fold, p<0.01).
To note, however, that this same concentration was able to even significantly increase
the expression of TLR4 when in the presence of 1000 nM (1.4-fold, p<0.05).
Fig. III.6: Expression of TLR4 is reduced by both Aβ and VS, but revealed to be induced by the highest VS concentration when in the presence of 1000 nM Aβ. N9 cells were treated for 24 h with 50
nM and 1000 nM amyloid beta (Aβ), as in previous experiments, but now in the presence or absence of 10 μM and 20 μM vinyl sulfone (VS). Cells not-treated with Aβ served as controls. After treatment, total cell lysates were analyzed by western blot with an antibody specific for toll-like receptor 4 (TLR4). (A) Representative results from one experiment are shown. (B) The intensity of the bands was quantified by scanning densitometry, standardized with respect to β-actin protein. Results are expressed as mean ± SEM from at least two independents experiments, and presented as fold change relatively to control, which was assumed as 1. **p<0.01 vs. respective control;
$$p<0.01 and
$p<0.05 vs. same experimental
condition in the absence of VS.
2.4. Aβ triggers a reduction in microglia phagocytosis, which is prevented by
VS
Phagocytosis is a fundamental feature of functional microglia, being essential to
the removal of dead cells and microbes, as well as misfolded protein aggregates [217].
To evaluate if the phagocytic ability of microglia cells was modified in the presence of
Aβ and modulated by VS, we determined the capacity of microglia in ingesting latex
beads. Interestingly, as depicted in Figure III.7, the number of latex beads per cell was
A
B
48
significantly reduced by Aβ, mainly at the highest concentration (0.7-fold, p<0.01). In
regard to VS, although not significantly, the compound revealed ability to reverse the
effects induced by Aβ to close control values
Fig. III.7: Microglia phagocytic ability is decreased upon exposure to Aβ and restored by VS. N9
cells were treated for 24 h with 50 nM and 1000 nM amyloid beta (Aβ), as in previous experiments, but now in the presence or absence of 10 μM and 20 μM vinyl sulfone (VS). Cells not-treated with Aβ served as controls. At the end of incubation, cells were treated with 1 μm green latex beads as described in methods. (A) Microglial cells were counterstained with an antibody raised against Iba-1(red) and nuclei were stained for Hoechst dye (blue). Latex beads (green) ingested are pointed out by a white arrow. Scale bar represents 40 μm. (B) Results are the mean number of ingested beads per cell ± SEM from at least four independent experiments, presented as fold change relatively to control, which was assumed as 1. **p<0.01 and *p<0.05 vs. respective control.
A
B
49
2.5. The expression of MFG-E8 is reduced by 50 nM Aβ and VS alone, while
increases when the highest concentrations of both compounds are
concomitantly used
The MFG-E8 is a glycoprotein secreted by microglia that has been shown to be
a key factor in the phagocytic removal of apoptotic cells [149] and suggested to be
involved in Aβ clearance [151]. Next we explored if MFG-E8 expression was affected
by Aβ and the potential modulation by VS. As shown in Figure III.8, MFG-E8
expression was significantly reduced by 50 nM of Aβ (0.9-fold, p<0.05). Although not
statistically significant, VS alone resulted in a concentration-dependent decrease in the
expression of MFG-E8. In conjunction with Aβ, although the lower VS concentration did
not lead to alterations, the highest concentrations of both compounds induced MFG-E8
expression; however, also not significantly.
Fig. III.8: The expression of MFG- E8 that is reduced by 50 nM and by VS alone, is increased when the highest concentration of both compounds are concomitantly used. N9 cells were treated for 24 h
with 50 nM and 1000 nM amyloid beta (Aβ), as in previous experiments, but now in the presence or absence of 10 μM and 20 μM vinyl sulfone (VS). Cells not-treated with Aβ served as controls. After treatment, total cell lysates were analyzed by western blot with an antibody specific for milk fat globule-EGF factor 8 protein (MFG-E8). (A) Representative results from one experiment are shown. (B) The intensity of the bands was quantified by scanning densitometry, standardized with respect to β-actin protein. Results are expressed as mean ± SEM from at least one experiment, and presented as fold change relatively to control, which was assumed as 1. *p<0.05 vs. respective control.
A
B
50
2.6. Aβ induces an increase in the expression of HMGB1 and effect that is
prevented by VS
Finally, a particular attention was given to HMGB1, an ubiquous nuclear protein
released by necrotic cells or stimulated monocytes, which mediates the activation of
immune response, including migration and the release of cytokines [170]. Recent
evidence points to HMGB1 as a risk factor for the progression of neuroinflammation
and chronic degeneration in AD[171]. Therefore, we decided to acquire more insight
into this topic. To study the expression of HMGB1 by N9, western blot assay was
performed. We observed a marked increase in the expression of HMGB1 by microglia
after exposition to Aβ (Fig. III.9; 3.5- and 2.7-fold increase for Aβ 50 nM and 1000 nM,
respectively, p<0.05). We verified, additionally, that VS alone did not change the
expression of HMGB1. However, when microglia cells were exposed to Aβ in the
presence of VS, Aβ-induced effects were reversed in a dose-dependent manner, which
was mainly statistically significant with 20μM VS to both Aβ concentrations (0.8- and
0.6-fold for Aβ 50 and 1000 nM; p<0.01 and p<0.05 respectively).
Fig. III.9: Expression of HMGB1 greatly increases in microglia treated with Aβ, but VS shows ability to prevent such effect from occurring. N9 cells were treated for 24 h with 50 nM and 1000 nM amyloid
beta (Aβ), as in previous experiments, but now in the presence or absence of 10 μM and 20 μM vinyl sulfone (VS). Cells not-treated with Aβ served as controls. After treatment, total cell lysates were analyzed by western blot with an antibody specific for high-mobility group box 1 (HMGB1). (A) Representative results from one experiment are shown. (B) The intensity of the bands was quantified by scanning densitometry, standardized with respect to β-actin protein. Results are expressed as mean ± SEM from at least four independents experiments, and presented as fold change relatively to control, which was assumed as 1. *p<0.05 vs. respective control;
$$p<0.01 and
$p<0.05 vs. same experimental condition in the
absence of VS.
A
B
51
IV. DISCUSSION
Current research indicates that microglia age-dependent neuroinflammatory
changes play an important role in neurodegenerative diseases such as AD [165].
Microglia are able to respond to different kinds of stimuli, being the primary mediators
of the inflammatory response. Several studies demonstrate that Aβ is able to activate
microglia, inducing the production of several pro-inflammatory and neurotoxic factors
[90]. Moreover, with ageing, microglia has been described to present an exaggerated
response to stimuli that subsequently shift to a loss in responsiveness to the same
stimuli and failure in sustaining the regulatory systems that keep the cell under control
[92]. In the first part of this thesis, we used an in vitro ageing model of primary microglia
cultures to study the differences between young and aged microglia when treated with
Aβ and the responsiveness to neuronal regulation. This model was previous
established by characterizing a pure primary microglial cell culture along different DIV,
which demonstrated that cells present typical alterations of ageing with the progression
of time in vitro. Thus, our preliminary data with aged (senescent/dormant) and young
(reactive microglia) cultures of microglia isolated from mice have evidenced that the
first show a diminished NF-κB activation and reactivity, less migration ability and
decreased phagocytic capacity as compared to the second [197]. Corroborating these
results, several aspects of microglial biology were already reported to be altered with
ageing by other studies, including morphology, migratory function and release of
inflammatory cytokines upon microglial activation [218,219]. Here we have further
deepened this topic by evaluating the release of glutamate, ATP and MMPs after Aβ-
stimulation. These inflammatory mediators released by microglia present important
roles in the homeostasis of brain tissue and disease. We were able to show that the
levels of glutamate and MMPs are altered with microglial ageing, and that the release
of glutamate is age-dependently modulated by neuronal signals, results that further
point to the idea that ageing leads to altered microglial reactivity.
It is known that microglia cells release high quantities of glutamate when
activated [220], which has been demonstrated to be an important factor in microglia
neurotoxicity by NMDA receptor signaling [221]. In our work, the presence of Aβ did
not lead to significant differences in the level of released glutamate at both ages. These
findings are in accordance with recent reports indicating that sAPP, but not Aβ, is able
to induce glutamate release from microglia [109]. On the other hand, we observed a
reduction on the basal level of extracellular glutamate with microglial ageing. It is
known that microglia possesses glutamate transporters [94] and, therefore, microglia
52
are both involved in glutamate release and glutamate uptake, being the extracellular
pool a reflection of this balance. Interestingly, it was reported that microglia ability to
uptake glutamate is increased with age [218], which might have a connection with the
decrease we observed in the extracellular media. Furthermore, the levels of glutamate
are also reduced in mice hippocampus as a result of ageing [222]. Although microglia
may not be the primary responsible, our results point these cells as possible
contributors to the observed decrease. In regard to co-cultures with neurons, the
extracellular level of glutamate in young microglia culture presents a marked reduction.
Indeed, Nakajima and colleagues [223] have already demonstrated that neuronal
stimulation induces an increase in glutamate uptake by microglia, primarily through the
up-regulation of the GLT1 (glutamate transporter 1) glutamate transporter.
Nevertheless, is important to note that the neuronal factors involved were not still
identified. In our case, this neuron-glia crosstalk seems to be only occurring in the
presence of young microglia, and to be absent in aged microglial cells, thus suggesting
a lower ability of these cells to react to neuronal signaling. This might be translated to a
loss of neuroprotection by microglia with ageing, which would lead to a reduced
capacity to cope with excessive glutamate, facilitating neurodegenerative events.
Extracellular ATP is a prominent signaling molecule usually present at low
levels in the extracellular space, but presenting a considerable increase in sites of
inflammation or intense cell stimulation [224]. It was already observed that Aβ induces
the release of ATP to the extracellular medium by microglia [211]. However, our results
are in contrast with these findings, since we did not observe differences regarding the
release of ATP in both isolated young and aged microglia, or even in the presence of
neurons, after exposure to Aβ. We think that this might be due to an increased
hydrolysis by ecto-ATPases or to CTFR inhibitors [225]. In addition, Kim and
colleagues [211] were able to detect an increase in ATP release after Aβ stimulation for
up to 2,5 hours, presenting its maximal peak at 10 minutes after stimulation. This
indicates that Aβ effects might be lost after our 24 h-incubation period. Unexpectedly,
no alterations were visible in the basal level of extracellular ATP between young and
aged microglia, suggesting that the energy metabolism is not impaired with ageing.
Nonetheless, these results refer to the level of extracellular ATP, and we cannot
exclude that differences might be occurring at the intracellular level.
Microglia cells can produce several MMPs that can degrade the extracellular
matrix and contribute to inflammation, and are thought to play a role in
neurodegenerative diseases, as in the case of AD [127,212]. In respect to the release
of active MMP-2 and MMP-9, the MMPs evaluated in this work, we observed no
significant variations after stimulation with Aβ, in either young or aged microglia,
53
isolated or in the presence of neurons. Nevertheless, notorious differences are
observed in the basal release of these two MMPs with age. Our results show that the
release of active MMP-2 is higher in aged microglia, which is in line with results
obtained in an in vitro model of senescence using keratocytes [226], as well as with our
previous work [197]. Although MMP-2 was reported to play a role in Aβ catabolism
[132], studies also indicate that this protease is involved in myelin degradation [227]. A
reduction of myelin proteins has been observed with ageing, being associated with an
increase in the activation state of microglia [228], pointing to a possible relation
between MMP-2 and myelin loss. This breakdown in myelin observed with ageing,
might contribute to the progressive nonrandom neuronal damage characteristic of AD
[229]. In regard to MMP-9, the results are the opposite, with isolated young microglia
releasing more active MMP-9 than aged cells, results that once more replicate our
previous findings [197]. Interestingly, MMP-9 presents α-secretase activity [230], and a
reduction in its levels with ageing might be linked with the appearance of β-amyloid
pathology. Moreover, MMP-9 is also able to degrade Aβ even in the form of fibrils and
plaques [131], which further suggests that a reduction of is level may have a role in
disease onset. In the presence of neurons, we observed no significant differences in
the release of active MMPs by young and aged cells, which suggests that the
expression of these proteases by microglia is not under neuronal modulation.
In the second part of this Master Thesis we followed a different approach and
aimed to explore the ability of VS to modulate the response of microglia cells to an
inflammatory stimulus. This aspect is particularly relevant since no therapy has to now
prove efficacy in halting AD progression. Once more, we used Aβ as a stimulant, since,
as it was already referred, it has been widely described that it activates microglia,
rendering them more inflammatory and cytotoxic [115]. Furthermore, this also helps to
give a better understanding of the role of Aβ in the immune processes in AD. Due to
the fact that this is an exploratory approach, we opted to use our N9 microglial cell line,
which was recently characterized in our group [231]. Here, we were able to
demonstrate that Aβ induces a more inflammatory microglial phenotype, characterized
by an increased release of the inflammatory mediators MMP-2, MMP-9 and HMGB1. In
addition, VS reveals to reduce these effects close to basal levels, presenting an anti-
inflammatory action. We also demonstrated that Aβ reduces TLR4 levels and
phagocytic capability of microglial cell line N9, and that again VS reveals aptitude to
counteract such effects.
We first evaluated cell viability after exposure to Aβ and in the presence of VS.
Our results indicate that VS does not present significant toxicity to the cells at both
concentrations tested, which points for a use in in vivo settings without expecting
54
significant direct side effects. Exposure to Aβ, although it also did not present
significant toxicity, demonstrated a slightly reduction in cell viability in a dose-
dependent manner. In fact, studies indicating Aβ-induced toxicity in microglia cells only
found significant reduction in cell viability at concentrations greater than 5 µM [232],
values higher than the ones here used. We also observed no significant protection by
VS at this level. However, it is worth to note that VS slightly increases the viability of
cells when exposed to the highest Aβ concentration. Thus, we cannot exclude that VS
may even have protective effects at concentrations of Aβ above the ones used in this
study.
To address the impact of VS in modulating microglial inflammatory properties,
we evaluated the release of MMPs, which are known to be involved in the modulation
of inflammatory processes [233]. In this study, Aβ was able to induce the release of
both active MMP-9 and MMP-2 in a dose-dependent manner. Indeed, the release of
MMP-9 [234] and MMP-2 [235] were already reported to be induced by Aβ,
corroborating our findings. The excessive production of these proteases might be
linked with various detrimental roles in neuroinflammatory-related diseases, as in the
case of AD. Both MMP-9 and MMP-2 were reported to be able to activate TNF-α in
vitro, indicating that they may contribute to the inflammatory state in AD [233].
Furthermore, MMP-2 is increased in AD mouse models and was found to correlate with
inflammatory injury [236]. On the other hand, MMP-9 was reported to be involved in
Aβ-induced cognitive impairment and neurotoxicity [237], suggesting that it may also
play a role in disease progression. In addition, MMP-2 and MMP-9 can regulate
endothelial permeability by cleaving occludin and may contribute to the opening of the
BBB [238], which has been reported to be impaired in AD [239]. In this regard,
inhibition of these proteases might represent a therapeutical approach to AD.
Consistent with this idea, in this study we have shown that VS, at 20 µM, is able to
counteract the release of MMP-9 and MMP-2 by the highest concentration of Aβ,
suggesting its utility for therapeutical intervention. Nonetheless, these results should be
carefully taken. As it was aforementioned, both MMP-9 and MMP-2 are involved in Aβ
degradation and may play different contributions in different states of the disease
development. Thus, further studies need to be done to understand the consequences
of inhibiting MMPs in the context of AD.
Several receptors have been reported to be responsible for the microglial Aβ
stimulation. Of these, TLR4 deserves special attention. It has been demonstrated that a
mutation in the TLR4 gene strongly inhibits microglial activation by Aβ, resulting in a
significant lower release of inflammatory products like IL-6, TNF-α and NO, and there is
an increased expression of TLR4 in brain tissue associated with amyloid plaques in AD
55
[113]. Interestingly, it was also recently reported that LPS, another pro-inflammatory
stimulant, induces the expression of MMP-9 through TLR4 [240]. However, our results
regarding this receptor were unexpected. We detected a decrease in the levels of
TLR4 after Aβ treatment, particularly at the highest concentration. Nevertheless, this
might be a result of the existence of two forms of TLR4. In our observations, we could
only detect a band of 93 kDa, which corresponds to the non-glycosylated form of TLR4,
the form that does not translocate to the membrane and that is not available for
binding. We could not detect the 130 kDa band, which is responsible for the
glycosylated form of TLR that is found on cell surface [241]. Thus, we speculate that
the decrease in the non-glycosylated form may be a consequence of an increase in the
glycosylated one, but that our detection method is not sensible enough to detect it. In
further studies we plan to use different methodology, such as immunocytochemistry or
Real Time-Polymerase Chain Reaction (PCR), to quantify TLR4 and better clarify the
involvement of this receptor in Aβ-induced activation. In respect to the effects of VS, we
observed that at 20 µM and in the presence of 1000 nM Aβ, VS increases the
expression of the non-glycosylated form. Moreover, VS alone appears to reduce the
non-glycosylated form of TLR4. Nevertheless, it is difficult to draw conclusions of these
results without further confirmation. Indeed, at the light of our speculation, these results
might indicate that the inflammatory effects of Aβ may be mediated through this
receptor, pointing for a positive role of 20 μM and a mechanism for its anti-inflammatory
effects. However, further studies need to be performed to obtain more conclusive
results, since other receptors beside TLR4 might also be involved in Aβ-induced
activation [111].
When activated, microglia can become highly phagocytic, a function critical for
the uptake and degradation of infectious agents and degenerated cells, contributing for
the homeostasis of the brain tissue [217]. Aβ has been described to not only incite an
inflammatory response, but also to be able to induce microglial phagocytosis [114].
Surprisingly, our results show a decrease in the phagocytic capacity of microglia in a
dose-dependent manner after exposure to Aβ. Although this is in discordance with
previous findings reporting a marked increase of phagocytic activity after Aβ-stimulation
[242], other studies indicate a frustrated phagocytosis [157]. To note that on one hand
microglia is able to phagocytize the aggregates of Aβ, but is not able to efficiently
perform their degradation [141], and that on the other hand it may result from an
increased formation of oligomers, which showed to be less capable of inducing
phagocytosis when compared with fibrils [243]. Decreased phagocytic capacity may
also derive from inflammation resulting from the Aβ-stimulation itself, since expression
of inflammatory cytokines has been demonstrated to attenuate phagocytosis induced
56
by Aβ [244,243]. In fact, it was previously demonstrated that microglial reactivity
changes from phagocytic to inflammatory along time of exposure to unconjugated
bilirubin, a well characterized pro-inflammatory stimulant, stressing the importance of
the time of incubation [206]. In regard to VS, both concentrations tested slightly
reversed the effects of microglia exposure to Aβ. Although this was not statistically
significant, it suggests that this compound may be an inducer of phagocytosis.
MFG-E8 is a bridging protein that is known to mediate the phagocytic removal
of apoptotic cells by binding to PS [147]. Due to its connection with phagocytosis, we
also evaluated how MFG-E8 is modulated by Aβ and VS. Here, we shown a significant
decrease in its content in microglia stimulated with the low nM concentration of Aβ. It
was previously reported that Aβ small concentrations (250 nM) is able to induce the
phagocytosis of neurons and that this is mediated by the release of MFG-E8, which
may account for the loss of neurons in AD [245]. Thus, we may assume that the
reduction we observed is related to a release of MFG-E8 to the extracellular space.
Unfortunately, we were not able to evaluate the extracellular content of this protein and
this still remains to be determined. To further add that VS, although not producing
significant changes, can be envisaged as a phagocytic inducer.
We also evaluated HMGB1, a nuclear protein that is released as an
inflammatory factor and related with several neurodegenerative diseases [171]. This
protein has been found in elevated levels in AD, co-localizing with amyloid plaques in
association with microglia [172]. Here we show, for the first time, that Aβ might present
a link to these associations. Our results demonstrate that Aβ is able to markedly induce
the expression of HMGB1 by microglial cells. Surprisingly, we also noticed that VS is
able to prevent the effects produced by Aβ, mainly at 20 µM. This is of particular
interest in the context of AD, since HMGB1 contributes to Aβ accumulation by
preventing its phagocytosis by microglia [173]. HMGB1 is also able to bind directly to
Aβ, stabilizing the oligomer conformations [172], and to interact with receptors like
RAGE and TLR4, leading to an increase in toxicity and inflammation [170].
Furthermore, HMGB1 was reported to mediate cognitive impairment [246], which raises
the possibility that it may be involved in cognitive deficit observed in AD. Therefore, a
reduction of the levels of HMGB1 might represent a valuable therapeutical approach to
AD, aiming to reduce the effects of Aβ accumulation and neuroinflammation.
Nonetheless, it is important to emphasize that only levels of HMGB1 inside the cells
were investigated and that further studies should search the existence of HMGB1 in the
extracellular milieu.
Overall, although VS presents some promising properties, it has to be referred
that further studies need to be done to better understand its effects, as well as its
57
mechanism of action, which is currently unknown. VS was indicated as a cathepsin S
inhibitor and indeed the inhibition of cathepsin S has been described to reduce de
activity of MMP-9 and MMP-2 [247]. Furthermore, it was also suggested that inhibition
of cathepsin S provides neuroprotective effects by suppressing the inflammatory
response [248]. Nevertheless, it is not safe to assume that all these results are just
mediated by the inhibition of cathepsin S at this point, since this same inhibitor might
be acting on other cathepsins as well. For instance, the inhibition of cathepsin L was
recently shown to be able to reduce the activation of NF-κB [249] which is involved in
the regulation of several genes linked to inflammation [250].
In conclusion, in the first part of this Master Thesis, we have shown that
microglia behaves differently with age, releasing lower levels of glutamate, MMP-9 and
higher levels of MMP-2. Furthermore, based on our glutamate results, we also
observed that aged microglia loses, at least to some extent, the ability to respond to
neuronal signaling. These results are indeed interesting in the context of AD, and in
age-related neurodegenerative disease in general, as well, since they converge to the
idea that microglia becomes dysfunctional with age, contributing to the progression of
the disease. Besides, these results also strength our model as an alternative approach
to study microglial senescence. In the second part of this Thesis, we showed that Aβ
activates microglia, inducing a more inflammatory phenotype, characterized by an
increase of the levels of MMP-2, MMP-9 and HMGB-1 and that VS, our promising test
compound, is able to at least partially prevent the Aβ-induced effects, which elects it to
further investigation as a possible modulator of microglial activity in inflammatory
diseases like AD.
58
[Escre
va um
59
Future perspectives
In his work, we have provided some important clues about the effect of
microglial ageing on these cells biology, using an innovative approach – an in vitro
model of microglial ageing. This model might be a valuable asset to further study
ageing in microglia and to evaluate at what extent it contributes to AD onset and
progression, which to date remains poorly understood. Nevertheless, some
considerations might still need to be taken in the future when evaluating the effects of
Aβ, like assessing the best time of incubation or the evaluation of effects using higher
concentrations. Furthermore, in regard to co-culture studies, cell viability should also be
assessed, since microglia might directly eliminate neurons in the presence of Aβ. In
this regard, the use of conditioned media studies, although not permitting the
evaluation of cell-to-cell signaling, might present a useful alternative. There are also
some parameters that may be interesting to assess in the future, to better characterize
this model and understand our results. Evaluation of the level of glutamate receptors
like GLutamate ASpartate Transporter and GLT1, might give some insight to the
differences observed in extracellular glutamate as a result of microglial ageing.
Moreover, evaluating the release of inflammatory cytokines would also help to better
characterized how ageing affects the immune state of microglial cells in our model. The
evaluation of CX3CL1-CX3CR1 was one of the initial aims of this Thesis, which we,
unfortunately, were not able to accomplish in due time. However, both CX3CL1-
CX3CR1 and CD200L-CD200R signaling are interesting parameters to be addressed
in order to understand how microglial ageing affects neuron-microglia communication.
In regard to our approach to explore VS as a modulator of microglia activation,
we presented some interesting preliminary results, which indicate that this compound
has anti-inflammatory properties. To further confirm this, it would be important to
directly measure the level of inflammatory cytokines. The receptor that mediates the
inflammatory effects of Aβ remains, however, to identify. Although we assessed the
expression of TLR4 by western blot we believe that it will be necessary its
determination by complementary methods, namely immunocytochemistry or Real
Time-PCR, to dissect the relevance that TLR4 and probably TLR2 represent in the
process of ageing, microglia irresponsiveness or over-activation, and susceptibility to
the appearance of age-related disorders. In respect to the phagocytic capability, some
unexpected results, in a form of a reduced phagocytosis of beads in response to Aβ,
were also observed. Nevertheless, repeating the phagocytic assay using fluorescently
labeled Aβ might represent a way to clarify the reason behind the results observed.
Another point of vital importance that we plan to address in the future is the evaluation
60
of extracellular MFG-E8 and HMGB1, since these molecules play most of their roles
outside the cell. Finally, to understand if the effects of VS are indeed mediated by a
specific inhibition of cathepsins S it would be important to perform specific tests in
regard to different cathepsin activity in the presence of this inhibitor. In case of
specificity to cathepsins S, this might also reveal new biological pathways in which this
cathepsin may be involved.
In a last note, although we did not address this question in the present study,
since oAβ and fAβ are the main forms of Aβ characterized in AD studies and might
possess different effects, the induction of microglia with purified oAβ and fAβ would
allow to identify if the effects we obtained may be appointed to any particular
aggregation form, giving further insight to the role of Aβ aggregation state in the onset
and progression of such a devastating disease.
61
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