Tau sensor, an approach based on FRET to study Tau protein in Alzheimer's disease: imaging and molecular characterization

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University of Pisa

Course of Molecular and Cellular Biology

Degree thesis

Tau sensor, an approach based on FRET to study Tau

protein in Alzheimer’s disease: imaging and

molecular characterization

Candidate: Giacomo Siano

Supervisor: Prof. Antonino Cattaneo

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ABSTRACT

Alzheimer’s disease is a neurodegenerative disorder which is very diffuse during the last two centuries. It’s characterized by progressive dementia and two main lesions lead to the beginning and development of the disease: neuritic plaques and neurofibrillary tangles formed due to aggregation of APP and Tau protein respectively.

Tau protein is a microtubule-associated protein (MAP) expressed mainly in neurons whose main function is to stabilize cytoskeleton by binding tubulin. During Alzheimer’s disease Tau undergoes many post-translational modifications and it’s not able to perform its function. In this condition Tau aggregates forming toxic tangles which cause neurodegeneration. Because of the importance of this protein in Alzheimer’s disease, many groups are working to elucidate the physiological and pathological role of Tau and to develop new therapies for this severe disorder.

In order to study the role of Tau in Alzheimer’s disease in the laboratory has been developed a chimeric protein (Tau sensor) made of a Tau protein linked to a Cfp and a Yfp at the N- and C-terminals. The aim of my thesis is to characterize Tau sensor by imaging employing FRET and FRAP techniques and by molecular analysis using Western blot technique. As a previous study the cellular system employed has been HeLa cells.

The study has revealed that Tau sensor binds microtubules and it’s in part cleaved and secreted in physiological conditions. The response to microtubules-destabilizing agents is the same of wild type Tau with the sensor diffusing into the cytoplasm. The effect of taxol, a microtubules-stabilizing agent, causes the displacement of Tau sensor from the microtubules and remarkably it determines the formation of aggregates. Staurosporine, a kinase inhibitor, stabilizes Tau sensor on the cytoskeleton.

These data suggest that the sensor shares the same function of Tau protein and allow to employ it in neuronal cells, a system nearer to that in which Alzheimer’s disease develops.

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INTRODUCTION

1. Alzheimer’s disease

Alzheimer’s disease (AD) is clinically defined by a gradual loss of memory and other cognitive functions and neuropathologically by atrophy of the brain, neurons death, accumulation of amyloid plaques and fibrillary tangles (Karch et al., 2014). The interest of the researchers for this disease is increased during the 20th century because of the rise in life expectation from about 50 years to more 75 years. This elongation in lifespan of individuals has brought to a condition in which neurodegenerative disorders become common. Among these, AD is one of the principal forms of late-life mental failure. This disease causes different symptoms which become more and more severe with the progression of AD. We can distinguish 4 different stages:

 The preclinical stage, the most difficult to diagnose, is characterized by mild symptoms in particular cognitive impairment, inability to plan and alterations in behaviour (Forstl et al, 1999; Jost et al., 1995). In this stage the individual can live quite normally.

 During the second stage, the mild dementia stage, there is a significant impairment of learning and memory but it can be found aphasic and visuoconstructional deficit too. There is a loss in plan capacity and difficulties in speaking (Locascio et al., 1995). The patients may suffer spatial disorientation and constructional apraxia (Moore et al., 1984). Non-cognitive disturbances are frequent in particular depression (Burns et al., 1990).

 In the moderate dementia stage a severe impairment of recent memory and in language capacity occurs (Beatty et al., 1988; Romero et al., 1995). The patients can’t read and write correctly, spatial disorientation becomes more severe, they can’t recognize familiar faces, normal actions as dressing or washing become impossible without help, changes of humour could fall in aggressive behaviours (Forstl et al., 1999).

 During the last severe dementia stage almost all the cognitive functions are lost, patients can’t speak correctly, they need help for movement and simple things like eating. Epilepsy may appear in some cases. Patients usually die because of infections, infarctions and septicaemia (Forstl et al., 1993).

Some of these features of AD were noticed in 1906 by Alois Alzheimer who was the first to define the clinico-pathological syndrome that bears his name but until 1960 Alzheimer’s observations weren’t followed.

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3 In the 1960s Michael Kidd and Robert Terry, employing electron microscopy, described the two classical lesions of neurons in AD: neuritic plaques and neurofibrillary tangles (fig.1). In the 1970s it was seen that cholinergic neurons degenerate and the acetylcholine synthesized and released is abnormal due to a decrease in the amounts and activities of choline acetyltransferase and acetylcholinesterase in the limbic and cerebral cortices. Cholinergic neurons aren’t the only kind of neuron degenerating as it has been observed in successive studies. Neurodegeneration has a macroscopic effect on the brain infact in AD patients’ hippocampus and cortex show shrinkage and cerebral ventricles are enlarged. After these discoveries researchers’ attention focused on the study of the two principal lesions of the disease, the plaques and the tangles. Studies about the genotype-to-phenotype relationship let researchers discriminate among familial forms of AD and non-familial sporadic ones.

Familial forms of AD present an incidence estimated from 5-10% to 50% and they are characterized by the presence of mutations and polymorphisms in some genes which codify for proteins implicated in the early or in the late onset of the illness i.e. APP, ApoE, Presenilin 1 and 2, ABCA7, Clusterin and many others whose inheriting rises the probability to develop AD (Karch et al., 2014; Selkoe et al., 2001).

As to the two most common lesions discovered in Alzheimer’s patients, the studies were and are focused on the characterization of these aggregates.

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Figure 1. Aggregation of Aβ peptide and Tau protein. The image shows the role of Aβ peptide and Tau protein in physiological (on the right) and pathological (on the left) conditions. During AD Aβ and Tau aggregate in toxic amyloid plaques and neurofibrillary tangles.

(Image courtesy of the National Institute on Aging/National Institutes of Health)

Neuritic plaques are extracellular microscopic foci of amyloid β-protein (Aβ) which aggregates outside the cell (Dickson, 1997). Neurites in proximity of these amyloid deposits become dystrophic, dilated and tortuous and are marked by ultrastructural abnormalities as enlarged lysosomes, numerous mitochondria and paired helical filaments (PHFs). Microgliosis and astrocytosis can be found in the regions of the brain in which neuritic plaques occur and seem to cause an inflammatory response which could enhance neuritic and neuronal injuries. Microglia around amyloid aggregates is activated and may respond to Aβ accumulation activating the complement cascade and cytokines which promote inflammation. (Itagaki et al., 1989; McGeer et al., 1995; Rogers et al., 1996; Selkoe et al., 2001). Amyloid microangiopathy is also present in brains affected by AD. Amyloid fibrils can be found in the abluminal basement membrane of the vessels sometimes with apparent extensions of fibrils into the surrounding perivascular neuropil. The presence of amyloid aggregates causes angiopathy and occasionally in AD vessels may rupture leading to cerebral haemorrhages (Verbeek et al., 2000). Finally experimental evidence suggests that the effects of Aβ aggregates, including the inflammatory response, may include excessive

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5 generation of free radicals and peroxidative injury to macromolecules (Harris et al., 1995); another effect is the excessive calcium entry in neurons which contribute to selective neuronal dysfunction and death (Mattson et al., 1992; Pike et al., 1993).

Molecular characterization of these plaques showed that the amyloid aggregates are constituted mostly by Aβ42, a peptide generated by the cleavage of APP. The other peptide found in the plaques is the Aβ40 obtained by cleavage of APP too but the latter is less hydrophobic than Aβ42 and seems to aggregate only after the Aβ42 deposition (Jarret et al., 1993). The cross-sectional diameter of neuritic plaques in microscopic brain sections varies from 10μm to plus than 120μm and the density of the amyloid fibrils shows great variation among plaques. Using antibodies against Aβ for immunohistochemical staining, it was discovered in the 1980s in limbic and cortical regions the presence of amorphous plaques referred to as “diffuse plaques” or “preamyloid deposits” (Joachim et al., 1989; Tagliavini et al., 1988; Yamaguchi et al., 1988). These deposits contain only Aβ42 and are considered as precursors for the formation of neuritic plaques (Iwatsubo et al., 1995).

The other lesion observed in AD is the presence of intracellular neurofibrillary tangles. Electron microscopy reveals that these fibers consist of pair of about 10nm filaments bound into helices (PHF) with a helical period of 160nm. Immunocytochemical and biochemical analyses suggested that the tangles were made of microtubule-associated protein Tau (Kosik et al., 1986; Nukina et al., 1986). The protein during AD migrates electrophoretically at higher molecular weight due to increased phosphorylation of Tau; moreover the tangles are insoluble (Selkoe, 2001). It’s not clear which one of the two lesions originates first because tangles of Tau can be found not only in AD but in other tauopathies too and neuritic plaques are present even in absence of tangles. The nature of these lesions and in particular of the two proteins, APP and Tau, implicated in the formation of these aggregates will be examined in next chapters.

APP and Tau are not the only proteins implicated in AD, there are different genes whose mutations promote early or late onset of the illness. Mutations in APP and presenilin are the principal factor of risk for early onset of AD and will be discussed below. The majority of AD risk genes affect Aβ clearance and production.

Late onset AD (LOAD) is characterized by the presence of polymorphisms in different genes implicated in lipid biology (APOE, CLU, and ABCA7), immune system function (CLU, CR1, ABCA7, CD33, and EPHA1) and cell membrane processes like endocytosis (PICALM, BIN1, CD33, and CD2AP) (Karch et al., 2014). Here I describe briefly some of the most important genes which are

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6 discovered to be important in increasing AD risk: ApoE, CLU, ABCA7, CR1, CD33, TREM2, BIN1, PICALM, CD2AP, SORL1. The identification of these genes is principally based on Genome Wide Association Study (GWAS) and further analyses by immunocytochemistry, biochemistry and transgenic animal models.

Apolipoprotein E (ApoE) is the strongest risk factor for LOAD. It’s located on chromosome 19q13.2 and encodes a pleiotropic glycoprotein. There are 3 alleles ε2, ε3 and ε4 and the latter is associated with increased AD risk. Heterozygote individuals for ApoE4 increases AD risk 3 fold while homozygote ones increase risk of 12 fold (Coder et al., 1993; Guerreiro et al., 2012). About the 50% of AD patients carry ApoE4 allele. The protein is implicated in mobilization and redistribution of cholesterol, neuronal growth and regeneration, immune response and activation of lipolytic enzymes (Guerreiro et al., 2012; Kim et al., 2009). ApoE binds Aβ influencing its clearance and aggregation moreover ApoE4 has more affinity for the peptide resulting in accelerated fibril formation (Castellano et al., 2011; Kim et al., 2009; Sanan et al., 1994; Strittmatter et al., 1993). ApoE also affects Aβ metabolism indirectly by interacting with low-density lipoprotein receptor-related protein 1 (LRP1) implicated in endocytosis (Verghese et al., 2013). In APP transgenic mice, ApoE enhances Aβ aggregation in a way isoform-dependent (Holtzman et al., 2000). Neuropathological and neuroimaging studies demonstrate that ApoE4 carriers accumulates more Aβ42 plaques than ApoE4 negative individuals (Morris et al., 2010). Clusterin (CLU) is an apolipoprotein, stress-activated chaperone implicated in apoptosis, complement regulation, lipid transport, membrane protection, and cell-cell interactions (Jones et al., 2002). CLU gene is located on chromosome 18p21.1 and encodes 3 alternative transcripts. Different SNPs are identified which give protection against LOAD or enhance probability to develop AD. Elevated levels of Clusterin mRNA and protein can be found in AD brains and interacts with Aβ plaques (Demattos et al., 2002). The protein likely influences Aβ clearance, amyloid deposition, and neuritic toxicity.

ATP-binding cassette transporter A7 (ABCA7) is a member of ABC transporter superfamily which transports substrates across cell membranes. It’s located on chromosome 19p13.3 and generates two transcripts both expressed in brain (Kim et al., 2008). ABCA7 has a role in the efflux of lipids from cell to lipoprotein particles and it has been seen that it inhibits Aβ secretion; moreover increasing ABCA7 enhances phagocytosis of apoptotic cells, synthetic substrates and Aβ by microglia. Mice not expressing the transporter show more Aβ deposits (Kim et al., 2013; Jehle et al., 2006; Tanaka et al., 2011).

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7 The three genes described are linked to lipid biology such as transport and metabolism and influence Aβ clearance and aggregation. Other genes involved in regulation of immune system and endocytosis affect plaques deposit.

As to immune system regulation CR1, coded on chromosome 1q32, is a component of complement response (Krych Goldberg et al., 2002). Higher CR1 protein expression in associated with higher clearance rate of immune complexes but Aβ seems to activate the complement system exacerbating AD pathology (Gibson et al., 1994; Velazquez et al., 1997).

CD33 is a member of the sialic acid binding Ig-like lectin family of receptors expressed on myeloid cells and microglia. It’s located on chromosome 19q13.3. Sialic acid binds CD33 inhibiting monocyte activation moreover CD33 has a role in clathrin-independent receptor mediated endocytosis. The absence of the protein or the presence of SNPs which reduce its expression are correlated with a reduced accumulation of Aβ in the brain (Malik et al., 2013).

MS4A is a locus containing different genes associated with inflammatory response expressed in myeloid cells and monocytes (Howie et al., 2009). These genes are still under investigation but different SNPs has been found correlated with increase or reduction of LOAD risk (Hollingworth et al., 2011; Naj et al., 2011).

TREM2, located on chromosome 6q21.1, encodes for a receptor that stimulates phagocytosis and suppresses inflammation (Rohn, 2013). Missense mutations like R47H have been reported to increase LOAD risk probably affecting aggregates levels of Aβ and Tau or neuroinflammatory mechanisms (Bertram et al., 2013).

The last kind of genes linked to LOAD risk are involved in endocytosis, a critical aspect of APP processing.

Bridging integrator 1 (BIN1) is a protein involved in regulating endocytosis and trafficking, immune response, calcium homeostasis and apoptosis (Galderisi et al., 1999; Pant et al., 2009; Ren et al., 2013). Many SNPs indentified are linked to the increase of LOAD risk but because of the many functions of the protein it isn’t still clear the exact role of BIN1 in AD.

Phosphatidylinositol binding clathrin assembly protein (PICALM) encodes a protein expressed in neurons involved in clathrin assembly. It’s located on chromosome 11q14 and results in 23 alternative transcripts. PICALM recruits clathrin to cell membrane and plays a role in synaptic vesicle fusion to presynaptic membrane. PICALM colocalizes with APP in vitro and in vivo and the overexpression of the protein increases plaques deposition in transgenic mice (Xiao et al., 2012).

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8 CD2 associated protein (CD2AP) is a scaffolding protein, encoded on chromosome 6q12, that is involved in cytoskeletal reorganization and intracellular trafficking. The protein is required for synapse formation and is important for vescicular trafficking to the lysosomes. Some SNPs are related to increased LOAD risk but it’s not clear the way they can affect the disease (Cormont et al., 2003; Dustin et al., 1998).

Sortilin related receptor L (SORL1) is involved in vesicle trafficking from the cell surface to the Golgi endoplasmic reticulum. Some SNPs are associated with a reduction LOAD risk and SORL1-deficient mice have elevated Aβ levels. SORL1 directs APP for recycling and plays an important role in Aβ generation moreover the protein binds lipoproteins including ApoE-containing particles (Karch et al., 2014; Rogaeva et al., 2007).

This has been a brief description of the principal genes identified correlated with the increase of late onset AD risk, genes involved in particular with endocytosis, immune response and lipid biology above all related with clearance and aggregation of amyloid plaques.

Other polymorphisms in different genes have been discovered but their role in AD in still under observation. Some of these genes are CASS4, CELF1, DSG2, FERMT2, HLA DRB5 DBR1, INPP5D, MEF2C, NME8, PTK2B, SLC24H4 RIN3 and ZCWPW1.

The discovery of all these genes and aspects correlated with AD underlines the heterogeneity of the illness and the difficulty to find the causes of the degeneration and effective therapies.

As we can understand by the previous description of the illness, there are today many aspects of Alzheimer’s disease which need to be investigated and this is important not only to understand the etiology of the disease but also for the development of effective therapies to contrast the progress of AD.

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2. β-amyloid precursor peptide (APP)

2.1 Gene and protein

Purification and partial sequencing of the Aβ protein described above, led to the cloning of the gene encoding β-APP (Kang et al., 1987). Aβ is derived from its precursor protein by sequential proteolytic cleavage. APP gene is located on chromosome 21q21 and encodes a ubiquitously expressed transmembrane protein migrating between 110 and 140 KDa on electrophoretic gels (Selkoe et al., 1988). This heterogeneity is due to alternative splicing of the transcript, yielding 3 isoforms of 695, 751 and 770 residues, and due to a variety of post-translational modifications including O- and N-glycosylation, sulfation and phosphorylation (Selkoe et al., 1988; Yoshikai et al., 1990). APP751 and APP770 are expressed in non neuronal cells and also occur in neurons. APP695 is expressed mainly in neurons and at very low abundance in non-neuronal cells (Haass et al., 1991). The difference between the three isoform is that APP695 lacks of an exon which codes for a 56-amino acid motif homologous to the Kunitz-type of serine protease inhibitor (KPI) (Selkoe et al., 2001). APP is a transmembrane protein that is cotranslationally translocated into the endoplasmic reticulum and then post-translationally modified through the secretory pathway. Soon after biosynthesis it acquires N- and O-linked sugars. Proteolytic processing of APP occurs during and after the trafficking through the secretory pathway leading to the production of different fragments (Weidemann et al., 1989). The majority of APP is proteolyzed by and γ-secretase. α-secretase cleaves the protein 12 amino acids N-terminal to the single transmembrane domain releasing α-APPs into the lumen and extracellular space and restraining a 83-residue C-terminal in the membrane (αCTF). Alternatively APP can be cleaved sequentially by β- and then γ-secretase generating β-APPs and β-CTF. β-secretase, whose main neuronal representative is BACE1, cuts APP 16 residues N-terminal to the α-cleavage site. Processing of α- and β-CTF by γ-secretase leads to the formation respectively of p3 and Aβ peptides (Selkoe et al., 2001; Thinakaran et al, 2008; Vassar et al., 1999). γ-secretase activity is executed by a high molecular complex containing Presenilin, Nicastrin, Anterior pharynx defective, Presenilin enhancer and other proteins (Edbauer et al., 2003). Mutations in these genes, in particular in presenilin, lead to a cleavage of APP unbalanced towards the production of the more insoluble and pathogenic Aβ42. APP is internalized and Aβ is generated in the endocytic pathway and secreted into the extracellular space (Thinakaran et al., 2008).

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10 A number of possible functions have been described to APP holoproteins or their derivative. Soluble αAPPs acts as an autocrine factor and as neuroprotective and neuritotrophic factor (Mattson et al., 1993; Saitoh et al., 1989). It’s important for neuronal repair and synaptogenesis (Moya et al., 1994). The presence in APP770 and 751 of KPI motif lets them inhibit serine proteases such as trypsin and chymotripsin in vitro; moreover KPI-containing isoforms inhibit factor XIa in the clotting cascade (Smith et al., 1990). APP holoprotein in the membrane and secreted APP isoforms can confer cell-cell and cell-substrate adhesive properties in culture mainly due to their E1 and E2 domains and an integrine-like sequence (Ghiso et al., 1993; Soba et al., 2005). The APP intracellular domain (AICD) interacts with different proteins like Mint-a/X11a, Fe65 and C-Jun N-terminal kinase-interacting protein (King et al., 2004). Fe65 interaction stabilizes AICD but it’s also implicated in cell motility and growth cone dynamics (Sabo et al., 2001-2003). APP interacts with Mint/X11 family influencing synapse formation (Ashley et al., 2005). It can also undergo another cleavage downstream of the γ-side which causes the formation of Aβ49, the ε-cleavage (Weidemann et al., 2002). This processing releases an APP intracellular domain that translocates into the nucleus probably acting like a transcription factor (Gao et al., 2001). APP seems important for anterograde transport on axons mediated by Kinesin-1 infact APP interacts with the protein probably acting like a cargo receptor (Kamal et al., 2000). Knockout mice for APP are viable and fertile but they show a decreased locomotor and forelimb grip strength and they are also defective in long-term potentiation and GABA-mediated postsynaptic response (Seabrook et al., 1999; Zheng et al., 1995).

2.3 APP post-translational modifications

APP and Aβ are post-translationally modified by truncation, racemization, sulfation, isomerisation and metal induced oxidation but the most important modifications are glycosylation and phosphorylation.

APP N-glycosilation localizes the protein to the endoplasmic reticulum and Golgi; the protein is not subject to cleavage. In the Golgi APP is O-glycosilated and enters in the secretory pathway where it can be cleaved by α/β- secretase and γ-secretase (Tomita et al., 1998).

APP is a phosphoprotein carrying several phosphorylatable residues in its cytoplasmic and luminal regions. The main residue identified to be phosphorylated is Thr668 (APP695) in the cytoplasmic region of APP. Several kinases are implicated in this phosphorylation event including GSK-3, JNK3 (under stress) and CDK5. This modification is involved in regulating APP localization to the growth

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11 cones and neurites; moreover the protein is preferentially transported to the nerve terminals (Iijima et al., 2000; Muresan et al., 2005). Thr668 phosphorylated APP is also more represented in AD brains and it seems that Pin-1 isomerizes proline residues leading to alteration of APP processing and Aβ production (Lee et al., 2003; Pastorino et al., 2006). The modification causes a change in the interaction of APP with other proteins such as Fe65; Fe65 is liberated from the membrane and is translocated to the nucleus where it can absolve its function of stress protection (Nakaya et al., 2006-2008). Thr668 phosphorylation renders APP less vulnerable to cytoplasmic cleavage mediated by caspases-3 and -8 preventing the formation of cytotoxic peptides (Taru et al., 2004). This is the main phosphorylation identified in neurons even if other sites are phosphorylatable and are under investigation. Phosphorylation and other post-transcriptional modifications are specific for Aβ peptide affecting its capacity of aggregation. Before discussing Aβ modifications and their influence on the peptide biology it’s important to understand the effects of mutations on Aβ production and aggregation.

2.4 APP mutations

There are more than 30 mutations described in APP which accounts for approximately the 14% of early onset autosomal dominant cases of AD even though two recessive mutations, A673V and E693Δ, have also been reported to cause early onset AD (Guerreiro et al., 2012). The mutations are located either immediately before β-secretase cleavage site, shortly after α-secretase site or shortly C-terminal to the γ-secretase site (Selkoe et al., 2001). Some of these mutations have revealed important aspects of the molecular mechanisms of AD pathogenesis.

The Swedish mutations KM670/671NL increases plasma Aβ levels of 2-3 fold by altering β-secretase cleavage efficiency (Mullan et al, 1992).

Duplication of APP and its surrounding sequences leads to AD and cerebral amyloid angiopathy. Individuals affected by Down syndrome presenting trisomy 21, in most cases show AD; patients with Down syndrome with partial trisomy 21 which doesn’t include APP gene, don’t develop AD. Thus excess of Aβ is sufficient to cause AD (Guerreiro et al., 2012; Tokuda et al., 1997).

Several mutations occur at the C-terminal of Aβ domain. These mutations alter γ-secretase function shifting the cleavage site and increasing the Aβ42/Aβ40 ratio (Bergmans et al., 2010). Aβ42 is more prone to aggregate than Aβ40 enhancing the possibility to develop AD.

The Arctic mutation E693G and the Dutch mutation E693Q occur in the Aβ domain and increase its aggregation ability (Guerreiro et al.,2012; Nilsberth et al., 2001). The individuals carrying the Dutch

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12 mutation develop hereditary cerebral haemorrhage with amyloidosis with predominant vascular Aβ deposition and diffuse plaques in the parenchyma (Guerreiro et al., 2012).

As seen, variations in APP processing or in Aβ aggregation lead to AD with variable neurological and neurovascular phenotypes.

2.5 PSEN1 and PSEN2

Other important proteins involved in early onset AD are PSEN1 and 2. PSEN1 is located on chromosome 14q24.3 and PSEN2 on 1q31-q42 (Levi et al., 1995; Sherrington et al., 1995). These genes encode for Presenilin 1 and 2, structurally similar integral membrane proteins that contain nine transmembrane domains with a hydrophilic intracellular loop region. Presenilin is an important component of γ-secretase (Wakabayashi et al., 2008). The protein localizes in the endoplasmic reticulum and Golgi apparatus acting in protein processing. Presenilin holoproteins undergo constitutive endoproteolysis within hydrophobic portion of the cytoplasmic loop between the sixth and seventh transmembrane domains; once formed, fragment associate into higher molecular mass complexes (Podlisny et al., 1997; Yu et al., 1998). About 185 dominant mutations have been identified for PSEN1 accounting for 80% early onset familial AD cases; 13 dominant mutations have been identified for PSEN2 (Korch et al., 2014). These mutations increase Aβ42 presence of about 2-3 fold and affect γ-secretase function by three mechanisms leading to AD (Lemere et al., 1996): an inhibitory effect on the initial endoproteolytic step which releases the APP intracellular domain; a premature release of intermediary substrates of APP generating longer Aβ peptides; an effect on the cleavage site cutting APP preferentially at position 49-50 or 51-50 (Chavèz-Gutierrèz et al., 2012). These three mechanisms explain the effect of Presenilin mutations on the alteration of Aβ42/Aβ40 ratio.

2.6 Aβ post-translational modifications

Aβ peptides undergo different post-translational modifications that modify its behaviour: truncation, racemization, isomerisation, pyroglutamination, and phosphorylation (Kumar et al., 2011).

Aβ can be truncated at the N-terminal at aminoacids 3, 11 and 25 and these forms are found in senile plaques and vascular amyloid deposits. Truncated Aβ25-35 enhances aggregation in vitro and it’s toxic for cells (Harting et al., 2010; Pike et al., 1995).

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13 Racemization of Aβ at Asp7, 23 and Ser26 influences aggregation, in particular racemization at Asp23 increases it, while Asp7 slows down fibrils formation (Mori et al., 1995; Tomiyama et al., 1994).

Isomerization and pyroglutamination also result in enhancement of Aβ aggregation (Schilling et al., 2008; Shimizu et al., 2000).

As to phosphorylation, Aβ has three sites that can be modified at Ser8, Ser26 and Tyr10. Aβ can be phosphorylated extracellularly by protein kinase A and the modification promotes the formation of toxic aggregate. Phosphorylated Aβ can be detected in the centre of the aggregates and is detected as early as two months in APP transgenic mice, accumulating with age. Experiments in transgenic Drosophila with pseudo-phosphorylated Aβ demonstrate that the peptide increases cell degeneration and aggregates more than the normal peptide (Kumar et al., 2011; Milton et al., 2001; Moloney et al., 2010).

2.7 Aggregation

The tendency of proteins to aggregate into amyloid assemblies is a general property of the backbone. These proteins have the intrinsic ability to self-organize into polymeric assemblies stabilized by intermolecular hydrogen bonds between the peptide bonds in parallel or antiparallel polypeptide stretches in the beta-strand conformation. Side chains are important to determine the environmental conditions where proteins can aggregate. Natural folding or intermolecular interaction are competitive processes and the latter is favoured by protein crowding, the propensity of the polypeptide to gain secondary structures and hydrophobicity. Even the presence of surfaces improves aggregation by concentrating amyloidogenic polypeptides (Chiti et al., 2003; Stefani; 2012). As showed in fig.2 amyloid aggregation is characterized by different steps whose nucleation is the limiting one. As to Aβ, physiologically the peptide is characterized by the presence of α-helical and unordered structures. Under pathological conditions monomeric Aβ is misfolded and acquires β-sheet conformation which leads to oligomer formation. This event is the nucleation phase followed by the elongation from the oligomer, then there is the fibrils and plaques formation (Ni et al., 2011). It has been demonstrated that prefibrillar aggregates are highly toxic for the cell, more than fibrils and plaques. However it has been seen that cytotoxicity is also associated to fibrils underlining that the scenario in AD is more complex than it seems (Lambert et al., 1998; Lorenzo et al., 1994; Stefani, 2012; Walsh et al., 2004; Tanker et al., 1990).

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Figure 2. Representation of the two phases of amyloid aggregation. Formation of amyloid aggregates is characterized by two phases: the nucleation phase which is a slow phase of aggregation of misfolded monomers in oligomers; elongation phase is a rapid phase leading to mature fibrils. The graph represents all the process of aggregation from the monomer to mature fibrils (green curve). The red curve represents the formation of fibrils when seeds of misfolded protein are added to the system reducing the lag time.

(Image courtesy of Kumar et al., (2011), “Phosphorylation of amyloid beta (Aβ) peptides - a trigger for formation of toxic aggregates in Alzheimer's disease” Aging; 3(8):803-812)

2.8 Aβ toxicity

A question remains to be answered: how the Aβ aggregates are toxic for cells? It’s not fully clear the mechanisms of Aβ cytotoxicity but there are several evidences. The cells in the brain of AD patients show high amounts of oxidatively modified proteins, lipids and DNA; this molecular damage is prominent in the environment of plaques and tangles. The oxidative stress is due to Aβ aggregation which generates hydrogen peroxide damaging macromolecules. Lipid peroxidation caused by Aβ impairs the function of important proteins such as ion-motive ATPases, glucose and glutamate transporters and GTP-binding proteins as a result of covalent modifications by the aldehyde 4-hydroxynonenal (Butterfiled et al., 2001; Mattson, 1997). Disturbing ion homeostasis and energy metabolism, oxidative stress can render neurons vulnerable to exotoxicity and

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15 apoptosis. Ca2+ homeostasis is no more maintained due to the oxidative damage of ion pumps; Aβ fibrils also creates channels in membranes deregulating ion homeostasis and Aβ oligomers can interact with membrane receptors promoting Ca2+ influx (Le et al., 2001; Mattson et al., 2003). Another mechanism of toxicity is the activation of apoptosis, indeed many proapoptotic proteins are associated with Aβ deposits and it seems that the peptide can activate proteins of the caspases cascade. Deregulation of Ca2+ influx is implicated also in the activation of caspases (Mattson, 2004). Coincident with the increased production of Aβ in AD is a decrease in the amount of αAPP produced, which may contribute to the death of neurons since αAPP is known to increase the cell resistance to oxidative and metabolic insults (Mattson et al., 1997). Because of the transport of APP along the axon, Aβ accumulates on synapses where the amyloid aggregates injure the correct functions of the cells (Kamenetz et al., 2003. Selkoe et al., 2001).

3. Tau

3.1 Gene and protein

The other lesion found in AD is constituted by neurofibrillary tangles (NFTs) consisting of aggregated, aberrantly phosphorylated forms of protein Tau. It’s evident that Tau-mediated neurodegeneration is the result of the combination of toxic gain-of-function acquired by aggregates and the detrimental effects that arise from the loss of physiological functions (Ballatore et al., 2007). Human Tau gene is located on chromosome 17q21 and contains 16 exons. Upstream of exon 1 there are consensus binding sites for transcription factors like AP2 and SP1 (Andreadis et al., 1992; Sadot et al., 1996). Tau mRNA undergoes alternative splicing yielding to different isoforms of the protein. The expression of different Tau isoforms with different number of exons is characteristic during brain development (Goedert et al., 1989). Isoforms lacking exon 10 are found during early developmental stages or in specific cell types like granule cells and dentate gyrus. Other Tau isoforms lack exon 2 and/or 3. Exons 2, 3 and 10 are alternative spliced leading to six Tau isoforms and are adult brain specific (fig.3).

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Figure 3. MAPT gene and Tau splicing isoforms. The MAPT gene encodes for the Tau protein. It’s made of 16 exons and alternative splicing leads to the formation of six Tau isoforms discriminated by the presence of three or four repeats at the microtubule binding domain and for the presence or absence of the 29-aminoacids inserts at the N-terminal domain.

(Image courtesy of Jos Luna-Mu oz et al., (2013) “Phosphorylation of Tau Protein Associated as a Protective Mechanism in the Presence of Toxic, C-Terminally Truncated Tau in Alzheimer's Disease”, Understanding Alzheimer’s

Disease, ISBN: 978-953-51-1009-5)

Tau protein is a hydrophilic polypeptide which appears as a random coiled protein (Cleveland et al., 1977; Hirowaka et al., 1988). It can be divided in two large domains: the projection domain and the microtubule binding domain. In addition, the projection domain contains the amino-terminal region with high proportion of acidic residues and the proline-rich region; the microtubule-binding domain is subdivided into a basic true tubulin binding domain with three or four repeats and the acidic carboxy-terminal region. The six Tau isoforms differ from each other for the presence of three or four microtubule-binding repeats of 31 or 32 residues (isoform 3R or 4R) and for the presence or absence of one or two 29-amino acids inserts at the N-terminal portion of the protein (Avila et al., 2004; Binder et al., 1985). The projection domain plays several roles that are determination of space between axonal microtubules, interaction with other proteins and cation binding (Chen et al., 1992; Hirowaka et al., 1988). Motifs identified in this region include the KKKK

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17 sequence involved in heparin binding and the PPXXP/PXXP motifs in the proline-rich region for the binding of Tau with proteins containing SH3 domains (Avila et al., 2004; Arrasate et al., 1999). The repeats of the microtubule-binding domain can be divided in two parts, one composed of 18 residues containing the minimal region with microtubule binding capacity, while the second region of 13/14 residues is known as the inter repeat (Avila et al., 2004). The repeats bind microtubules stabilizing them but Tau 4R has more affinity to tubulin than Tau 3R (Lu et al., 2001).

Tau is mainly a neuronal protein although it can also be found in different types of glia cells and in different types of tumour cells. In neuronal cells Tau can associate with membranes, and the strength of this association can be modulated by phosphorylation (Brandt et al., 1995; Arrasate et al., 2000). Tau has also been found in nuclei, in particular in the nucleolus and in pericentromeric regions (Brady et al., 1995; Sjoberg et al., 2006). Tau distribution is influenced by phosphorylation, for example phosphorylation in the proline-rich region localizes Tau protein mainly in the somato-dendritic compartment, whereas, if the proline-rich region is dephosphorylated or if the phosphorylation occurs in the C-terminal domain, Tau localizes in the distal axonal region (Dotti et al., 1987; Mandell et al., 1996).

3.2 Physiological role

Tau is a microtubule-associated protein (MAP) and its primary function is to stabilize microtubules (MTs) (Weingarten et al., 1975). Tubulin binding repeats bind to specific pockets in β-tubulin at the inner surface of the MTs while the proline-rich regions, positively charged, are bound to the negatively charged MT-surface; the negatively charged N-terminal domain branches away from the MT-surface probably because of electrostatic repulsion. Moreover the β-tubulin pockets of adjacent protofilaments may be occupied by different repeats of the same MT-binding domain causing crosslinking of three or four dimers (Amos, 2004; Kar et al., 2003). Tau protein affects axonal transport due to its tight bind to MTs and probably detaches the cargoes from kinesin without influencing the speed of kinesin with cargoes (Trinczek et al., 1999).

The projection domain determines spacing between MTs in the axon and may increase the axonal diameter. It interacts with other cytoskeletal components like spectrin and actin filaments allowing Tau-stabilized MTs to interconnect with neurofilaments that restrict the flexibility of MTs lattices (Kolarova et al., 2012). Tau projection domain also interacts with mitochondria and neuronal plasma membrane (Brandt et al., 1995; Jung et al., 1993). The proline-rich domain binds SH3 domains of several proteins including Fyn, a tyrosine kinase from the Src-familiy involved in

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18 protein trafficking. Tau binds Fyn in dendritic spines too and this interaction regulates N-methyl-D-aspartic acid (NMDA) receptor signaling; Tau interacts also with glutamate receptors. In oligodendrocytes the association of Tau with Fyn regulates the outgrowth of cytoplasmic process (Cardona-Gomez et al., 2006; Klein et al., 2002; Ittner et al., 2010).

Another role of Tau protein in neurons is its ability to protect the cell from oxidative damage and heat shock DNA injuries. It has been demonstrated that Tau can bind RNA and DNA and that it can localize in the nucleus contributing in the organization of the nucleolus and protecting DNA (Sjoberg et al., 2006; Sultan et al., 2010).

3.3 Post-translational modifications

Tau protein undergoes several post-translational modifications which modify its function, these modifications are also involved in the pathogenesis of AD because of Tau gain or loss of function.

Phosphorylation

Phosphorylation is the main post-translational modification of Tau. There are 79 putative serine and threonine phosphorylation sites (ref. isoform 441). Tau protein is much more phosphorylated during embryonic development followed by its disappearance after post natal period. Tau phosphorylation controls MT dynamics during normal neurite growth and maturation. Tau is not hyperphosphorylated in adult individuals but during AD it becomes hyperphosphorylated; this form of Tau aggregates in paired helical filaments (PHFs). All six Tau isoforms can be detected in PHFs and the protein is phosphorylated in 40 residues identified mainly by antibodies, mass spectrometry and sequencing of Tau. Of all these phosphorylated sites only Ser262, Ser285, Ser305, Ser324, Ser352 and Ser356 are located in microtubule-binding domain (Avila et al., 2003; Pevalova et al., 2006).

Most of the kinases involved in Tau phosphorylation (GSK3, Cdk5) are proline directed protein kinases (PDPK); other kinases are non proline directed (PKA, CAMKII).

 GSK3

Glycogen synthase kinase-3 (GSK3) is expressed ubiquitously but it can be found at high levels in the brain where it localizes predominantly in neurons. GSK3 plays an important role for different functions of the cell and it regulates Tau phosphorylation under normal and pathological conditions (Pevalova et al., 2006). Two types of GSK3 phosphorylation have been proposed: primed phosphorylation which follows a previous phosphorylation of the substrate by another kinase, or unprimed phosphorylation in which the substrate isn’t phosphorylated (Cho et al., 2002).

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19 GSK3β phosphorylates Thr231, one of the most prominent sites in Tau protein for the regulation of its activity and the beginning of AD. This is an example of primed phosphorylation because it occurs after Ser235 phosphorylation. The modification of Thr231 causes a conformational change in Tau affecting its stability and capacity to bind MTs. pThr231 probably is an early event in the pathogenic processes of AD (Daly et al., 2000). Also the Ser396 is phosphorylated by GSK3 after Ser400 but it doesn’t appear to affect MT binding (Li et al., 2006).

 Cdk5

Cyclin-dependent kinase 5 (Cdk5) has been characterized as a PDPK that contributes to phosphorylation of human Tau on Ser202, Ser205, Thr212, Thr217, Ser235, Ser396 and Ser404; these sites are phosphorylated in AD brains. Cdk5 is active in post-mitotic neurons, it’s implicated in cytoskeleton assembly and its organization during axonal growth. It interacts with p35 and it has been seen that conversion of p35 in p25 causes prolonged activation and miss-localization of Cdk5 and hyperphosphorylation of Tau (Maccioni et al., 2001; Tsai et al., 2004).

 PKA

Other kinases are non-proline directed kinases like Cyclic-AMP-dependent kinase (PKA) or Ca2+/Calmodulin-dependent protein kinase II (CaMKII).

PKA is a ubiquitous serine/threonine kinase activated by cAMP. PKA phosphorylates Tau at Ser214, Ser217, Ser396/404 and at Ser416. PKA also phosphorylates Tau at Ser262 (Drewes et al., 1997). The modification of these sites influences Tau binding on MTs, aggregation and cleavage.

 CaMKII

CaMKII regulates important neuronal functions including neurotransmitter synthesis and release, modulation of ion channel activity, synaptic plasticity and gene expression. The kinase phosphorylates Tau at Ser262, Ser356, Ser409 and Ser416 and these sites are phosphorylated in brains of AD patients. If Ser262 and Ser356 are phosphorylated, the ability of Tau to bind MTs and promote the assembly is reduced (Singh et al., 1996; Steiner et al., 1990).

 Phosphatases

Several studies have shown that three major protein phosphatases, PP2A, PP2B and PP5, can dephosphorylate Tau protein. PP2A is localized on MTs and binds Tau directly; PP2B is associated with developing of MTs and microfilaments. PP5 is associated with MTs and dephosphorylates Tau in neuronal cytoplasm. The inhibition or the absence of these phosphatases in the cells cause the hyperphosphorylation of Tau protein suggesting a possible role of these proteins in AD development (Pevalova et al., 2006).

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 Critical phosphorylation sites

As to critical sites involved in Tau aberrant activity implicated in AD and in other tauopathies, AT8 epitope (Ser199/Ser202/Thr205) has an important role. The hyperphosphorylation of these three residues is sufficient to cause MTs remodelling and instability, diminished mitochondrial transport, cell death and neurodegeneration (Alonso et al., 2010; Shahpasand, 2012). The same effect is due to the hyperphosphorylation of residues Thr212/Thr231/Ser262. Studies in vitro demonstrated that pSer262, pSer231 and pThr235 inhibit Tau binding to MTs by 35%, 25% and 10% respectively.

In vitro kinetic studies of the binding between hyperphosphorylated Tau and normal Tau suggest

that Ser199/Ser202/Thr205, Thr212, Thr231/Ser235, Ser262/Ser356, and Ser422 are among the critical phosphorylation sites that convert Tau to pathological molecule that sequesters normal microtubule-associated proteins from MTs. Moreover phosphorylation of Ser231, Ser396 and Ser422 promotes Tau aggregation. Pseudophosphorylation of Ser396 and Ser404 converts the protein in a more fibrillogenic one (Abraha et al., 2000; Alonso et al., 2004; Gong et al., 2004; Haase et al., 2004; Sengupta et al., 1998). Pseudophosphorylation of Ser422 has shown an increase in aggregation capacity but also a reduced cleavage at Asp421, one of the main cleavage sites of Tau protein (Guillozert-Bongaarts et al., 2006).

Glycosilation

Glycosylation is a co-/post-translational modification through which oligosaccharides covalently link to the side chains of polypeptides. There are two kinds of glycosylation: O-glycosylation in which sugars are linked to the hydroxyl group of serine or threonine; N-glycosylation in which sugars are linked to amide group of asparagines side chain. The aberrant glycosylation of Tau is an event that precedes hyperphosphorylation and is site-specific; the effect at each phosphorylation site is different (Gong et al., 2005). Deglycosylation of PHF tangles converts them in bundles of straight filaments and successive dephosphorylation results in increased release of Tau monomers (Wang et al., 1996).

Glycation

Glycation is the non-enzymatic linkage between a reducing sugar and the amino side chain of polypeptide. Glycation has been found in PHFs isolated from AD brains while it’s not present in normal Tau. It’s a late event and normally leads to subsequent oxidation, dehydration, condensation and formation of heterogeneous products called advanced glycation end products (Munch et al., 2002; Yan et al., 1995).

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21 Ubiquitination leads to the linkage of ubiquitin, a 76 aminoacids protein, to Tau protein. In particular polyubiquitination is important for protein degradation but in AD it has been found that PHF-Tau is ubiquitinated but not degraded and it deposits as NFTs. Ubiquitination is a late event in Tau modification (Pevalova et al., 2006).

Nitration

Protein tyrosine nitration is a post-translational modification implicated in physiological processes including signal transduction. Nitrated Tau is found in NFTs of AD but it’s not clear the implications of this modification. It has been demonstrated that tyrosine nitration of Tau inhibits MTs-binding activity (Pevalova et al., 2006).

Polyamination

Tissue tranglutaminase is upregulated in AD brain and localizes to NFTs with the Tau protein. Tissue transglutaminase can incorporate polyamines into Tau. Polyamination doesn’t affect its microtubule binding but makes Tau less susceptible to the degradation by calpain. Tissue tranglutaminase also catalyzes the linkage between glutamine residues and primary amines of lysine residues leading to insoluble and protease-resistant high molecular weight complexes (Appelt et al., 1997; Tucholski et al., 1999).

Acetylation

Lysine acetylation has been revealed to affect Tau properties. This modification neutralizes charges in the MT-binding domain interfering with Tau binding to MTs. Acetylation of Lys280 increases cytosolic Tau fraction and it’s correlated with Tau hyperphosphorylation. Acetylated Lys280 is present mostly in intracellular NFTs rather than in pre-tangles or extracellular aggregates and it precedes Tau truncation (Cohen et al., 2011; Irwin et al., 2012).

3.4 Tau cleavage

During AD Tau protein aggregates producing amyloid neurofibrillary tangles (NFTs) that are toxic for the cell. Above post-translational modifications, Tau protein which forms these aggregates is cleaved at different sites. In PHFs has been identified a 12KDa fragment, from His268 to Glu391, containing MT-binding domain and it’s referred to as the PHF-core. Truncated Tau at Glu391 is found in NFTs in AD brains and it’s associated to a late stage in NFTs development (Novak et al., 1993). A caspase-cleaved Tau species (ΔTau), cleaved at Asp421, is present in NFTs (Gamblin et al., 2003). Tau conformational studies suggest that cleavage of Tau at Asp421 is an early event in the pathogenesis of AD (Guillozet-Boogarts et al., 2005; Rissman et al., 2004). Caspase cleavage of Tau

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22 at Asp421 can be inhibited by phosphorylation at Ser422 (Guillozet-Bogaarts et al., 2006). Phosphorylation and truncation together influence Tau aggregation and this is shown in cells with or without GSK3 transfected with ΔTau in which the presence of the kinase causes the formation of aggregates (Cho et al., 2004). Tau protein can be processed by a variety of proteolytic enzymes which cut the protein at different sites.

 Caspases

Caspases can cleave in three consensus sites: Asp22-Asp25, Asp345-Asp348 and Asp418-Asp421, the last one is the preferred cleavage site. The consensus sequence at Asp421 is recognized by Caspase 7 and 3 and the mutation of this site in glutamate prevents the proteolysis (Canu et al., 1998; Fasulo et al., 2000). ΔTau fragment is toxic for cells inducing cell death (Chung et al., 2001). Caspase 6 cuts Tau at a N-terminal residue, Asp13, and it seems that this fragment is lost during NFTs evolution in AD (Horowitz et al., 2004).

 Calpains

The calcium-activated cysteine proteases, Calpain 1 and 2, have been shown to cleave Tau which retains its N-terminus (Johnson et al., 1989; Yang et al., 1995). In AD brains it has been found that there is an increase in Calpain activity and a depletion of Calpastatin, an endogenous Calpain inhibitor (Rao et al., 2008; Saito et al., 1993). The activation of Calpain generates a Tau fragment of 17KDa resulting from the cleavage at Leu43 and Val299; this fragment increases apoptosis in hippocampal cells revealing neurotoxic properties (Park et al., 2005).

 Thrombin

Thrombin is an extracellular serine protease which induces neurite retraction and modulates morphological changes in glial cells (Akiyama et al., 1992). In vitro the protease degrades Tau in a stepwise manner at sites Arg155, Arg209, Arg230, Lys257 and Lys340 (Wang et al., 2007). The initial cleavage at Asp155 produces a fragment of 37 kDa and then it is cleaved at Arg230 leading to a fragment of 25KDa (Olesen, 1994). Inhibition of Thrombin in brain lysates completely inhibits Tau degradation and Thrombin and Prothrombin can be found associated to NFTs in AD (Arai et al., 2006). Tau phosphorylation increases resistance to Thrombin cleavage for example GSK3 inhibits all but the initial cleavage at Arg155 while PKA induces a resistance at all the sites (Wang et al., 1996).

 Cathepsins

Lysosomal dysfunction in aged and AD brains causes the release into the cytoplasm of lysosomal enzymes including the protease Cathepsin D (Cataldo et al., 1997; Nakashini et al., 1997).

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23 Cathepsin D cuts Tau protein in vitro between residues 200 and 257 resulting in the generation of a 29kDa product (Kenessey et al., 1997). In human neuroblastoma cells destruction of lysosomes leads to formation of aggregates of Tau and it has been shown that Cathepsins B and L are involved in Tau cleavage (Hamano et al., 2008).

 PSA

Puromycin-sensitive aminopeptidase (PSA) has been identified as being protective against neurodegeneration (Karsten et al., 2006). PSA can digest Tau in vitro although insoluble Tau from AD brains in more resistant to cleavage (Sengupta et al., 2006).

3.5 Tau aggregation

All the modifications of Tau protein affect in different ways its properties, functions and aggregation. These changes in the protein activity are the causes of the development of pathological conditions like AD. As seen above some post-translational modifications and cleavage enhance the formation of amyloid aggregates whose formation is similar to that described for Aβ peptides. Previous studies have revealed a correlation between hyperphosphorylation, conformational change and cleavage of Tau. Hyperphosphorylation (and other post-translational modifications) of Tau determines a change in the conformation of the molecule which enhances its cleavage initially at Asp421; the cleavage and the phosphorylation pattern of the polypeptide induce the protein aggregation in early NFTs. With the development of AD other conformational changes provoke further truncation at the Glu391, linked to late stages of AD. After this truncation Tau is digested in smaller fragments until the PHF-core of 12KDa composed mainly of the repeated domains of the protein (Garcia-Sierra et al., 2008). Tau molecule has long stretches of positively and negatively charged regions which don’t allow intermolecular hydrophobic association. The β-structure is present in repeat regions, in particular in R2 and R3, which can assemble by their own in filaments (Von Bergen et al., 2000). Self-aggregation is inhibited by the presence of intact N- and C-terminal domains but when these ones are aberrantly phosphorylated and/or cleaved, the conformational structure changes exposing the sticky repeat regions which lead to the formation of PHFs (Alonso et al., 2001). Oxidation too seems to increase aggregation of monomeric Tau protein because it produces disulfide cross-linking between cysteines. Isoforms of Tau containing three repeats (3R) have only one cysteine residue in the MTs binding domain respect the isoforms 4R which have two cysteines. 3R Tau, because of the presence of a single cysteine, is more prone

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24 to aggregate rather than 4R isoforms which may form intramolecular disulfide bonds (Barghorn et al., 2002).

In AD Tau role is ineffective to keep the cytoskeleton well organized in axonal processes because the protein can’t bind MTs. This loss of physiological functions is due to conformational changes and misfoldings which lead to aberrant aggregation in fibrillary toxic structures inside neurons. Most of the pool of Tau in the disease is redistributed in the somatodendritic compartment and isolated processes of affected neurons (Kolarova et al., 2012). The destabilization of MTs affect trafficking along the axon in particular the plus-end-directed transport by kinesin (Ebneth et al., 1998). Inhibition of transport slows down exocytosis and the localization of organelles; the absence of these mechanisms causes a decrease in glucose and lipid metabolism, ATP synthesis and a loss of Ca2+ homeostasis which lead to a distal degeneration process (Futerman et al., 1996; Trojanovski et al., 1995). Phosphorylated Tau is also more affine to kinesin and this could explain why in AD NFTs are visualized initially at distal end of neurites (Chuchillo-Ibanez et al., 2008). It has been seen that aberrant Tau not only can create aggregates sequestering normal Tau but it can also remove from MTs the two other major neuronal MAPs, MAP1 and 2 (Alonso et al., 1997).

3.6 Secretion

A final aspect of Tau protein implicated in AD is its extracellular secretion and diffusion in adjacent cells and compartments. Tau was found first intracellularly and the protein detected in the extracellular space was considered as a release from dead cells (Medina et al., 2014). Recent works demonstrated that Tau protein is secreted by different cell types like neuroblastoma cells, stem cell derived neurons, Tau-expressing non-neuronal cells and primary neurons (Kim et al., 2010; Shi et al., 2012). Tau has been reported to be released in naked form, associated to exosomes or other membrane vesicles. Tau secretion was suggested to be a way for the cell to eliminate excess of Tau protein which can be detrimental (Medina et al., 2014). However it has been seen that hyperphosphorylation and cleavage of Tau enhances its secretion and this observation can have pathological relevance considering the fact that aberrant Tau is found in AD and aggregates (Plouffe et al., 2012). Tau can be toxic when applied extracellularly to cultured cells. Several mechanisms for the uptake have been proposed such as internalization of soluble uncoated Tau via receptor-mediated endocytosis, dynamin-driven endocytosis of soluble Tau aggregates or even proteoglycan-mediated macropynocytosis. It has also been suggested that extracellular Tau can stimulate M1/M3 muscarinic receptors leading to endocytosis of Tau. The

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25 phosphorylation of Tau inhibits the interaction with M1/M3 receptors and it has been proposed that such modifications can be involved in the transmission of Tau pathology (Medina et al., 2014). Release and subsequent uptake of Tau fibrils that contact directly native protein in recipient cells, mediate propagation of Tau misfolding among cells (Frost et al., 2009; Kfoury et al., 2012). Moreover intracerebral inoculation of synthetic preformed Tau fibrils induces NFTs formation that propagates from the injected site to connected brain regions in a time dependent manner (Iba et al., 2013).

3.7 Tau-Aβ interaction

A role for Aβ peptide in Tau aberrant activity has been hypothesized. Aβ peptide has been seen to activate caspases which process Tau at different sites leading to toxicity and aggregation. Other evidences show that Aβ and Tau protein can directly interact each other in vitro and in vivo. Aβ interaction with Tau enhances the phosphorylation of the latter by GSK3 but the phosphorylation of specific residues of serine and threonine blocks the binding. Probably intracellular Aβ binds Tau inducing pathological phosphorylation and acts as a nucleation site for insoluble accumulation (Guo et al., 2005).

All these aspects described above underline the importance of Tau protein in physiological and pathological conditions. Several studies aim to investigate the implication of this molecule in AD and how its post-translational modifications, secretion and aggregation are effectively involved in the development of the disease. There are many aspects that need to be examined in depth about all the aspects described above and different results are emerging about the role of Tau in AD and many other tauopathies.

4. FRET and FRAP

4.1 FRET

Fluorescence resonance energy transfer (FRET), is a mechanism of energy transfer between two light-sensitive molecules. An excited fluorophore can deactivate by non-radiative transfer of energy from the excited dipole of the donor fluorophore to the dipole of the acceptor one. The acceptor fluorophore in turn can return to its ground state by different mechanisms including

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26 photon emission, non-radiative dissipation or again energy transfer to another acceptor molecule. No light photons are transferred to the acceptor and no acceptor fluorescence is required for resonance energy transfer to occur. In order for FRET to happen, the donor emission spectrum must overlap the acceptor excitation spectrum. FRET efficiency is influenced by the distance (R) of the molecules involved. The efficiency decreases at the 6th power of R and increases with R0, the

distance at which the probability of the energy transfer between the donor and the acceptor is 50% (Förster distance): E= . The orientation of the dipoles also influences FRET efficiency infact no FRET occurs when the dipoles are perpendicular while it’s maximal for parallel dipoles. The choice of the fluorophores is fundamental for an analysis based on FRET technique. The most commonly used donor-acceptor pairs in biology are synthetic organic dyes and fluorophores followed in recent years by the introduction of synthetic nanoparticles, non-natural autofluorescent aminoacids, genetically encoded protein tags targeted by synthetic dyes and bioluminescent donors. Synthetic organic dyes are small molecules with favourable photochemical and spectral properties respect fluorophores. The advantage of fluorophores is that they can be genetically controlled outweighing the fact that are large molecules (Kalab et al., 2010). FRET is a useful technique to study protein interactions and conformational changes in the same molecule, it has also been used in different studies of Alzheimer’s disease. Takahashi et al, exploited FRET strategy to construct a sensor detecting Aβ oligomers using CFP and YFP respectively as donor and acceptor fluorophores (Takahashi et al., 2012). Kinoshita et al, used FRET to visualize the interaction between APP and β-secretase from the surface of the cells to the endosomal compartment (Kinoshita et al., 2003). Other studies employs FRET to study localization of Tau protein on MTs (Nouar et al., 2013). These examples are indicative of some aspects which FRET can reveal: the first example exploits a conformational change in the molecule, the last ones the interaction between two molecules.

4.2 FRAP

Developed in 1970s, FRAP (Fluorescence Recovery After Photobleaching) is based on bleaching definitely fluorescent molecules with a short intense laser pulse in a region of interest; subsequently the mobility of unbleached molecules from neighbouring areas to the bleached one is recorded by time-lapse microscopy. This technique is used to study the mobility of molecules. Two quantitative parameters can be detected by FRAP: the mobile fraction (Mob), which is the

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27 the minimum time required for the recovery of 50% of the fluorescence in the region of irradiation (ROI). t1/2 is inversely proportional to the diffusion coefficient of the fluorescent molecule which is

in turn influenced by the dimension of the molecule and binding interactions. FRAP analyses allow to plot a recovery curve reflecting diffusion and binding dynamics. These recovery curves can be fitted by two different equations: a FRAP curve fitted by a single exponential equation, representing a case in which diffusion and binding cannot be separated; a FRAP curve fitted by the sum of two exponentials where there is an initial short diffusive phase followed by a longer binding phase (Nouar et al., 2013). FRAP technique is often used to obtain data about the mobility of proteins and if there are interactions or less that can have an influence on the diffusion of molecules. Some studies reveal Tau mobility in cells in different conditions and treatments; the results show that Tau in physiological conditions has a FRAP recovery curve which can fit with a sum of two exponentials suggesting the presence of a diffusive phase and a binding one. The recovery curve can change in times of recovery or in the kind of fitting if conditions in the cell change for example stabilizing or destabilizing MTs (Breuzard et al., 2013; Nouar et al., 2013). In conclusion FRET and FRAP are very powerful techniques in order to understand mechanisms of molecule interactions and mobility.

5. Aims

The aim of this thesis is the characterization through molecular and microscopy approaches of the Tau FRET-based sensor. The sensor is a chimeric protein obtained by fusion of CFP at the N-term of Tau and YFP at the C-term. The peculiar structure of the sensor will allow to answer many unsolved questions regarding: the mobility of the unprocessed Tau, the subcellular localization of the unprocessed Tau and of the N-term and C-term fragments in AD conditions, the conformational changes of Tau after post-translational modification, the aggregation dynamic, the circuitry of secretion and uptake of truncated species or unprocessed protein, the interaction with proteins involved in Tau physiological and pathological processes, the role of Tau in cancer cells, the effects of drugs on the protein.

The future perspective of this work is to apply the sensor in animal models, such as mice, to study step by step Tau pathological modifications during AD progression.

The sensor could clarify the role of unprocessed Tau and of its fragments in the interaction with Aβ, the localization of Tau in the brain and in other regions of the organism during the disease, the

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28 effects of stress conditions typical of AD on the protein, it can be used as a marker to characterize by imaging the progression of AD or to screen therapeutic agents.

These are examples of Tau sensor potentialities for the study of Tau in different conditions, in particular in AD.

The first aim of the thesis is to check that Tau sensor shares the same main physiological characteristics of Tau protein in HeLa cells.

The second aim is to examine the effect of drugs on the sensor to study the response of Tau sensor in induced pathological conditions.

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MATERIALS AND METHODS

1. Plasmid construction

The plasmid pECFP-YFP derived from the pECFP-C1 (Clontech), already present in the lab, has been used for the cloning of the Tau sensor construct. The pECFP-YFP plasmid contains the YFP coding sequence cloned in frame with the ECFP. The sequence of TAU has been inserted in frame between the ECFP and the YFP into the BspeI site as shown in fig 1. The Tau isoform employed is Tau-D, a 4R isoform which lacks of the N-terminal domains and has 383aa. CFP and YFP genes are inserted respectively upstream and downstream the TAU gene and the sequence is separated from the fluorophores by a linker sequence coding the peptide RSIAT. The promoter for the expression of the construct is the CMV, a ubiquitous promoter which causes a high expression of the protein in several cell types.

a