Disentangle the link between nuclear Tau and gene expression regulation

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Master’s degree in Molecular Biotechnology

Disentangle the link between nuclear

Tau and gene expression regulation

Academic year 2018-2019 Candidate:

Giuseppe Madaro

Thesis supervisor:

Dr. Cristina Di Primio

Academic tutor:






The term Tauopathies indicates a group of neurodegenerative diseases linked by the formation of insoluble aggregates of the protein Tau and whose common symptoms include dementia, parkinsonism, akinesia and behavioural disorders.

The six Tau alternatively spliced isoforms are mainly expressed in neurons and glia, where they stabilize microtubules and promote axonal growth. At the early onset of the disease, Tau hyperphosphorylation and cleavages lead to its detachment from the cytoskeleton and accumulation in the cytoplasm. Either gain and loss of function mechanisms have been hypothesized to explain the distinctive pathological features.

Traditional studies and clinical trials mainly focused on Tau cytoplasmic role, but the hypothesis of other non-canonical functions opened further possibilities to search for new therapeutic targets. However, their impact on these pathologies has yet to be elucidated.

A small amount of Tau is physiologically localized into the nucleus where it directly interacts with nucleic acids, binds to intergenic regions of the genome, accumulates in stressful conditions, prevents DNA ruptures, promotes homologous recombination and chromatin architecture rearrangements. Our group recently discovered that Tau is involved in gene expression regulation. In particular, nuclear Tau modulates the expression of disease-related genes such as VGluT1 and NMDA receptors, ultimately impinging on synaptic transmission.

Recent studies identified the nuclear protein TRIM28 as a Tau interactor which mediates its translocation into the nucleus. Remarkably, TRIM28 is a universal transcriptional co-repressor involved in the formation of several nuclear complexes which affect gene expression directly or epigenetically by remodelling the chromatin architecture involving HDACs.

The aims of my thesis are: (i) to identify which Tau domain is involved in VGluT1 expression regulation; (ii) to investigate the underlying molecular mechanism.

First, I performed Tau protein dissection to test which of its domains retains the ability to increase VGluT1 expression in immortalized murine hippocampal HT22 cells.

The involvement of HDACs in VGluT1 expression regulation has been tested by administering the wide-spectrum HDACs inhibitor TSA. Moreover, I measured the global HDACs activity in the nuclear compartment of cells overexpressing Tau. To further investigate the involvement of nuclear Tau in TRIM28 complex rearrangements, I observed the relocation of its cofactor HDAC1 in the nuclear compartment through imaging and subcellular fractionation experiments. To study the interaction between Tau and TRIM28 cofactors involved in gene repression such as HDAC1, I performed overexpression and KD experiments and I checked for the expression of VGluT1 gene as functional readout. Finally, I investigated the molecular interaction between Tau and TRIM28 exploiting the Y2H technique.

Preliminary results indicate that even if TRIM28 is not directly affected, the interaction with its cofactors could be hampered by Tau over-expression. Tau could regulate HDAC1 function by displacing it from chromatin remodelling complexes rather than behaving as an enzymatic inhibitor. Nevertheless, HDAC1 knockdown alone is not enough to enhance VGluT1 expression, suggesting both a redundancy in HDACs activity or the involvement of other cofactors in a more complex mechanism.

In conclusion, Tau might modulate gene expression by altering the chromatin structure through a mechanism that will be deeply investigated in future experiments.





1.1 Dementia: a social issue ... 6

1.2 Tau gene ... 7

1.3 Tau protein ... 8

1.3.1 Tau in the axonal compartment ... 9

1.4 Non canonical functions... 10

1.4.1 Non-nuclear roles ... 11

1.4.2 Nuclear Tau ... 12

1.5 Pathological Tau alterations ... 13

1.5.1 Post-translational modifications ... 13

1.5.2 Truncation ... 16

1.5.3 Aggregation ... 18

1.6 Pathogenic mechanisms ... 19

1.6.1 Axonal transport impairment ... 19

1.6.2 Nuclear Tau alterations ... 20

1.6.3 Somatodendritic Tau accumulation ... 20

1.6.4 Mitochondrial dysfunctions in Tauopathies ... 21

1.6.5 Impaired Tau clearance and degradation ... 21

1.6.6 Unfolded protein response in Tauopathies ... 22

1.7 Tauopathies... 23

1.7.1 Genetic risk factors ... 23

1.7.2 Alzheimer’s disease ... 24

1.7.3 Tau and Synucleinopathies ... 24

1.7.4 Huntington disease ... 25

1.8 Interaction with TRIM28 ... 26

1.9 Aims of the thesis ... 28


2.1 Cell culture ... 30

2.1.1 HEK293T and HT22 ... 30

2.2 TSA treatment ... 30

2.3 Cloning ... 30

2.3.1 Tau-FLAG fragments ... 30



2.4 DNA Transfection ... 34

1.1.1 Transfection by Lipofectamine 2000 ... 34

1.1.2 Transfection by Polycation polyetilenim ... 35

2.5 Viral particles production ... 35

2.6 Transduction ... 36

2.7 Western blot analysis ... 36

2.8 Immunofluorescence ... 37

2.9 Image acquisition ... 37

2.10 HDACs activity assay ... 37

2.11 Yeast Methods ... 38

1.1.3 L40 yeast strain phenotype ... 38

1.1.4 Media and solutions for the Y2H ... 38 Media ... 38 Solutions ... 40

1.1.5 Yeast strain maintenance ... 40

1.1.6 LiAc transformation protocol ... 40

1.1.7 Characterisation of the chimeric constructs ... 41 Activity (auto-activation) ... 41 Expression ... 41

1.1.8 Yeast two hybrid screening ... 42

2.12 Statistical analysis ... 42

3 RESULTS ... 43

3.1 Full length Tau, but not single protein domains, regulates VGluT1 expression ... 43

3.2 TRIM28 expression and localization are not affected by Tau ... 44

3.3 Involvement of HDACs in VGluT1 expression regulation ... 45

3.4 Tau induces HDAC1 relocation in the nuclear compartment ... 46

3.5 HDAC1 alone is not sufficient to regulate VGluT1 expression ... 47

3.6 Tau does not directly interact with TRIM28 ... 48







1.1 Dementia: a social issue

Tau protein was first described forming insoluble aggregates in the brains of Alzheimer’s disease (AD) patients, along with Aβ (Alzheimer, 1907). The scientific community studying pathogenetic mechanism in AD was long divided by a harsh dispute on which of the two proteins was the primary driver of neurodegeneration: after decades of changing fortunes and the failure of drugs targeting Aβ, the so-called “Tau hypothesis” is now the path of choice for the development of new therapeutic strategies.

Tau aggregation is a common hallmark of a group of neurodegenerative disorders which fall under the denomination of Tauopathies. Tauopathies are among the main causes of dementia, a common term for neurodegenerative diseases altering cognitive function (Arendt et al. 2016). It is estimated that more than 50 million people in the world are affected by dementia and this number is expected to increase to more than 130 million people by 2050 (Fig. 1.1). The current cost of this group of diseases is about a trillion US dollars a year, and that’s forecast to double by 2030. (Alzheimer's Disease International, 2018). This scenario would have a massive psychological, social and economic impact.

Unfortunately, nowadays clinicians still do not dispose of any disease-modifying treatment as we are still far from having a comprehensive knowledge on the causes of dementia. Since the classical models have failed to explain the extraordinary complexity of Tau pathology, there is an urgent need of more thinking, more innovation and more dynamic entrepreneurship to find solutions and deepen our knowledge of the alternative roles of Tau in the cell.



1.2 Tau gene

MAPT (Microtubule Associated Protein tau) is a long gene (134 kb) located on chromosome

17q21.3. Its sequence is well conserved among mammals, exhibiting 97 to 100 % homology with primates. Phylogenetic analyses evidenced that MAPT shows high similarities with MAP4 gene, from which arose together with MAP2 because of an ancestral duplication event (Sündermann et al., 2016).

The 6-kb transcript is primarily expressed in the central nervous system (CNS), mainly in neurons but also in astrocytes and oligodendrocytes. The gene encompasses 16 exons, of which exons 0 and 14 are transcribed but not translated and four undergo differential splicing. Alternative splicing of exons 2 and 3 results in three Tau isoforms which may be devoid (0N) or contain one (1N) or two (2N) insertion of 29 amino acid each at the N-terminal of the protein. Exon 10 falls in the highly conserved microtubule binding domain (MTBD) which may contain three or four imperfect microtubule binding repeats depending whether it is spliced or not, resulting in 3R or 4R isoforms respectively (Fig. 1.2). The alternative splicing of E10 is also associated to different Tauopathies, according to the Tau isoform found in aggregates. The combination of these splicing events produces the 6 tau isoforms expressed in the CNS, whose predicted molecular weight ranges from 37 to 46 kDa. When they are separated SDS-PAGEs the apparent molecular weight becomes 60-74 kDa; the reason of the electrophoretic shift is still unclear (Avila et al., 2016; Goedert & Jakes, 1990).

Exons 4A, 6 and 8 are exclusively transcribed in the retina and in the peripheral nervous system through a 9-kb mRNA and give rise to a group of 110-120 kDa isoforms known as “big Tau” (Bukar Maina et al., 2016).

The functional outcome of the differences among Tau isoforms is still poorly understood. As expected, 4R Tau binds microtubules with higher efficiency than 3R Tau, due to the additional MT binding domain. Less is known about the function of the N-terminal inserts, which may affect attachment and spacing between Tau and other cell components, Tau localization or aggregation kinetics (Y. Wang & Mandelkow, 2016).

Isoforms expression is finely regulated during development and among brain areas, with a characteristic shift from 0N3R to higher molecular weight isoforms during embryonal and early natal development (Kosik et al., 1989). Distinct isoforms have different propensity for post-translational modifications such as phosphorylation, which may impact on Tau interactions and distribution in subcellular compartments (Sato et al., 2018).



Fig. 1.2 Tau gene, protein isoforms, and structure. The tau gene has 16 exons. Alternate splicing of exon 2 (blue), 3 (green) and 10 (yellow) in the CNS generates the widely known six isoforms. Light blue: constitutively transcribed in the CNS; orange: rarely expressed in the brain but included in mRNA of most peripheral tissues; grey: untranslated regions

(adapted from Maina et al., 2016).

1.3 Tau protein

Overall basic, the highly soluble Tau protein can be considered as an intrinsically unfolded protein, namely it behaves as a kind of random coil, lacking a well-defined secondary structure. Notwithstanding, a complex network of long-range transient contacts has been described (Skrabana et al., 2006). Only small segments of the sequence present transient elements of secondary structure: six segments display propensity to form β-strands, three segments show poly-Pro helices and, finally two segments composed of ten amino acids within the N-terminal projection domain and the C- terminal domain present transient α-helix structure (Mukrasch et al., 2009; Y. Wang & Mandelkow, 2016).

Reactivity of some antibodies such as Alz50 with residues present at the N- and C-terminal regions, suggests a possible tertiary structure in Tau protein (Carmel et al., 1996). Notably, the C-terminal end of Tau folds over into the vicinity of the microtubule-binding repeat domain (Fig. 1.3); both ends of the molecule approach one another with a short-lived interaction resulting in an fleeting paperclip structure (Jeganathan et al., 2006). The paperclip conformation is stabilized by Tau interaction with microtubules (Daebel et al., 2012; Di Primio et al., 2017).

The acidic N-terminal domain, which protrudes from MT surface in MT-bound Tau, plays a variety of roles in Tau spatial localization at different scales by long-range interactions with other cellular components. The number of N domains, along with the polyadenylation site, is responsible for Tau sorting to specific subcellular compartments: the 0N and the 2N isoform are predominantly targeted to the soma and the axon, the 1N isoform to the nucleus (C. Liu & Götz, 2013). The projection domain is also responsible for Tau localization in the axon via interaction with annexin 2A and annexin 6A (Gauthier-Kemper et al., 2018) and it mediates dephosphorylated Tau insertion in the neuronal plasma membrane via amphipathic helices (Brandt et al., 1995). On a smaller scale, force spectroscopy studies showed that its highly negative charge mediates long-range repulsive interactions responsible for Tau spacing on MTs in vivo (Chen et al., 1992; Saunders et al., 2012).



A progressive reduction in the length of the N-terminal projection domain decreases long-range interactions (Saunders et al., 2012) and hinders tubulin binding above all (Fellous et al., 1994). The C-terminal assembly domain includes the microtubule binding domain, spanning exons E9- E12, and the C-terminal regions, which regulate the interactions with the MTs and the plasma membrane. The microtubule binding domain contains either 3 or 4 repeats, each with 18 highly conserved amino acids separated by 13- or 14-amino acid long flexible spacers. The conserved residues bind at the interface between α- and β- tubulin, creating a local hairpin (Lee et al., 1989). The hinge between the projection domain and the assembly domain is a Pro-rich region with seven PXXP motifs for binding to signalling protein with the SH3 domain such as Fyn kinase. This region has high diagnostic potential, since it harbours epitopes for many antibodies against AD-specific phosphorylation (Mandelkow & Mandelkow, 2012).

Fig. 1.3 Model of transient intramolecular interactions within soluble tau determined by FRET experiments. The polypeptide chain of a four-repeat Tau (htau40) is shown with differently coloured repeats (R1 in blue, R2 in green, R3 in yellow, and R4 in red) and the adjacent N- and C-terminal sequences in grey. The positions of FRET partners are indicated, with arrows highlighting interactions between them. (Adapted from Jeganathan et al., 2006)

1.3.1 Tau in the axonal compartment

Tau protein was originally identified as a MT binding protein which was able to induce MT polymerization. The protein is mostly located in axons, where it interacts with MTs (Fig. 1.4). The main role of Tau is the stabilization of the axonal microtubules contributing to decrease their intrinsic dynamic instability (Weingarten et al., 1975). Tau interaction with MTs and its ability to promote their assembly, was first demonstrated in vitro. In fact, Tau co-purifies with MT isolated from porcine brains. Ultracentrifugation sedimentation assays show that in the absence of Tau tubulin is monomeric, while in the presence of Tau tubulin shows MTs-forming capacity (Weingarten et al., 1975). This finding was then confirmed in cells. Rat fibroblasts were microinjected with Tau protein and this resulted in an increase in tubulin staining in an immunofluorescence (IF) analysis. Moreover, IF showed that Tau injection is able to delay of nocodazole-induced microtubule disassembly (Drubin et al., 1984).

A structural analysis exploiting three-dimensional electron cryo-microscopy showed that Tau binds to a pocket in β-tubulin which overlaps to taxol binding site to MTs. Indeed, in vitro pelleting assays show that, in the presence of taxol, less Tau is bound to MTs, demonstrating a direct binding competition (Kar et al., 2003). This finding has been confirmed in cells; by using a FRET-based biosensor, Di Primio and colleagues demonstrated that Tau binding to MTs is less stable in the presence of taxol (Di Primio et al., 2017). For these reasons, it has been suggested that Tau protein might mediate its effects on MT in a similar way to taxol (Kar et al., 2003). Arnal and



colleagues showed that taxol induces a structural modification in tubulin which may make it more prone to polymerization (Arnal & Wade, 1995).

Besides regulating MT assembly and stabilization, Tau might be involved in regulation of axonal transport. In fact, Tau is able to interfere with the dynamics of motor proteins in vitro and knock-down of Tau protein is able to increase transport velocity in iPSC-derived dopaminergic neurons. Moreover, in SH-SY5Y cells, Tau is also able to bind p150 subunit of dynactin and to stabilize its binding to MTs, thereby promoting dynein transport (Beevers et al., 2017; Dixit et al, 2008; Magnani et al., 2007).

Linked to the axonal trafficking regulation function, Tau promotes axon elongation and maturation during development: in cultured rat neurons, it is necessary and sufficient for neurite formation, as demonstrated by knockdown and overexpression experiments (Caceres & Kosik, 1990). However, since MAPT-knockout mouse lines are viable and not all of them recapitulate this phenotype, a possible compensation by other microtubule-associated proteins has been suggested (Takei et al., 2000). As for neurite maturation, Tau is recruited to the axon terminal by NGF stimulation (Yu & Rasenick, 2006) and potentiates NGF and EGF-induced MAPK signalling by potentiating AP-1 transcription factor activation (Leugers & Lee, 2010).

Fig. 1.4 Model of full-length tau binding to microtubules and tubulin oligomers. Tau interacts with MTs through the MTBD four repeats binding in tandem along tubulin protofilaments (PFs). The region corresponds to the PGGG motif (grey), does not promote any strong interaction, resulting highly flexible. Tau binding at the interdimer interface, interacting with both α- and β-tubulin, promotes association between tubulin dimers (adapted from Kellogg et al., 2018).

1.4 Non canonical functions

The disruption of Tau-MT binding and even Tau protein aggregation, which surely are hallmarks of Tauopathy, are not enough to explain the widespread pathological alterations observed during the pathology progression. An increasing number of evidences suggest a much more complex role of the protein Tau in the cell, which may exert a plethora of “non-canonical” functions that we could have neglected (Sotiropoulos et al., 2017). Several lines of evidence indicate that Tau can



induce neurodegeneration through mechanisms that the scientific community have begun to consider in the last decade (Fig. 1.5).

Fig. 1.5 Schematic overview of the suggested roles of Tau in different subcellular compartments such as neuronal axon, nucleus, post- and pre-synaptic compartments (Sotiropoulos et al., 2017).

1.4.1 Non-nuclear roles

Under physiological conditions a small amount of Tau is capillary distributed through dendritic arborizations and spines, where interacts with the scaffold protein postsynaptic density protein 95 (PSD-95). This validated interaction could evidence a still not fully established role in the molecular organization of the synapsis (Ittner et al., 2010).

Recent studies on cultured neurons and hippocampal slices provided some evidences that hint a role in synaptic maturation and plasticity, demonstrating that Tau translocates from the dendritic shaft to spines following synaptic activation (Frandemiche et al., 2014). Evidences of learning and memory mechanisms impairments also derive from in vivo models. Tau knockout mice exhibit a selective deficit in long-term depression, although not in long-term potentiation (LTP), in the Cornu Ammonis 1 (CA1) region of the hippocampus (Ahmed et al., 2014). Some papers elucidated



the impact of Tau post translational modifications (PTMs) on its role on synaptic plasticity, reporting that hyperphosphorylation is responsible for synaptic dysfunction (Hoover et al., 2010; L. M. Ittner et al., 2008). At the base of these observations there could be Tau ability to regulate RNA granules translocation affecting the subset of activity-dependent transcripts targeted to the post-synaptic compartment (Vanderweyde et al., 2016).

Lastly, Tau seem to be involved in morphological maturation of new-born hippocampal granule cells, in providing sensitivity to neurogenesis modulators and in the correct assembly of the post synaptic density (Leugers & Lee, 2010; Pallas‐Bazarra et al., 2016).

Regarding the endoplasmic reticulum (ER) and Golgi, Tau colocalizes with distinctive marker of both these organelles, such as KDEL and Golgin 97 respectively, especially in AD brains (Tang et al., 2015). In this compartment, Tau accumulation promotes the interaction with ER membrane and its proteins involved in ER-associated degradation (ERAD), subsequently activating the unfolded protein response (UPR) (Abisambra et al., 2013).

Furthermore, Tau has long been known to colocalize with ribosomes both in neurons and astrocytes in AD brains (Papasozomenos, 1989). An increased association with ribosomes in AD patients has been confirmed by proteomics and coimmunoprecipitation screenings, providing a possible explanation to the reduced RNA translation. Indeed, the expression of many plasticity-related products is strongly affected in AD, which may drive synaptic depotentiation and cognitive decline (Meier et al., 2016).

Other studies focussed on cytoplasmic Tau interactors involved in key signal transduction cascades. The PRD provides several recognition sites for many partners, whose activity is pivotal in signal transduction cascades. Direct interactions between Tau and Src homology-3 (SH3)-containing proteins have been reported, including the Src family of protein kinases, such as Lck, Fgr, and Fyn. Other evidences have been reported about bridging integrator 1 (Bin1), peptidylprolyl cis/trans isomerases, NIMA-interacting 1, the p85α regulatory subunit of phosphatidylinositol 3-kinase (PI3K), phospholipase C (PLC) γ1, PLCγ2, and growth factor receptor bound protein 2 (Kim et al., 2005). In addition, proline-rich regions in proteins are the target of several other protein-interacting motifs, such as WW and Enabled/VASP homology 1 (EVH1) domains. Furthermore, second messengers such as phosphatidylinositol and phosphatidylinositol bisphosphate have been demonstrated as PRD binding partners (Surridge & Burns, 1994). All these observations point towards a possible modulation of the signalling functions of Tau.

1.4.2 Nuclear Tau

The first evidence that Tau can interact with nucleic acids was collected years before the discovery of its role as a microtubule binding protein, which subsequently absorbed research efforts for the following thirty years. The relationship with nucleic acids has returned to be a live issue since the discovery that Tau protein is also located in the nucleus of neuroblastoma cells (Loomis et al., 1990)

Subsequently, Tau has been localized into the nucleus of different neuronal cells subtypes in the brain of several species, human included, in neuronal and even in non-neuronal cell lines, when exogenously expressed (Rady et al., 1995). Even if traces of all the six isoforms has been reported, 1N4R isoform is the most abundant in adult mice neurons’ nuclei, which is less concentrated in the axonal compartment, in opposition to the other Tau isoforms. Both phosphorylated and non-phosphorylated Tau species coexist into the nucleus with a clear prevalence of the latter (C. Liu &



Götz, 2013). Some reports evidence that an increased phosphorylation status can alter intranuclear localization and the properties related to its nuclear role (Loomis et al., 1990; Sultan et al., 2011).

From in vitro studies, many features of Tau interaction with DNA have been highlighted. NMR spectroscopy revealed that the interaction between Tau and DNA is provided by the second half of the proline-rich domain and the second repeat of the MT binding domain (Qi et al., 2015a). As already discussed, Tau has 6 main isoforms and can undergo several PTMs, for these reasons, there is an increasing interest in clarifying nuclear Tau molecular profile. It has been discovered that in adult mice brains 1N Tau isoform is overrepresented in the soluble nuclear fraction, while 2N Tau isoform is the most abundant in the chromatin-bound fraction (C. Liu & Götz, 2013). The main evidence for the fundamental role of Tau in DNA protection from stressful condition comes from a study on a mouse model of hyperthermia: nuclear Tau pool increases in stressful conditions and protects genomic DNA in adult cortical and hippocampal neurons of wild-type mice. In contrast, in Tau-deficient mice, hyperthermia provokes more DNA breaks. (Sultan et al., 2011). Similarly, RNA integrity was negatively affected by heat shock in Tau deficient but not wild-type mice (Violet et al., 2014). Recent studies have demonstrated that Tau accumulates in neurons nuclei in tauopathies patients (Rousseaux et al., 2016). Notably, some works underly a role in DNA repair mechanisms, with Tau promoting homologous recombination (Rossi et al., 2013a).

Besides a possible DNA protective role, Tau shares some characteristics with minor groove architectural binding proteins, such as high mobility group proteins. They influence DNA conformation, causing it to unwind, and encourage the formation of multi-protein-DNA complexes which initiate or silence transcription (Bewley et al., 1998). Some reports evidence the bending of DNA structure because of the interaction with Tau (Sultan et al., 2011).

A study on HeLa cells has revealed a conserved pattern of Tau colocalization with pericentromeric heterochromatin contributing to the hypothesis of an epigenetic control of rRNA synthesis (Sjöberg et al., 2006). Moreover, the widespread reduction of heterochromatin markers, such as HP1 and histone methylations, has been linked with Tau overexpression, supporting the view that Tau may have a role as an epigenetic regulator of gene expression (Mano et al., 2017; Frost et al., 2014).

Both Tau knockout and overexpression impinge on gene expression, with some genes resulting downregulated and others overexpressed (Frost et al., 2014). Notably our group has discovered that Tau is able to modulate VGluT1 expression, a protein involved in vesicular loading of neurotransmitter in glutamatergic synapses. The nuclear amount of Tau correlates with VGluT1 expression level in a cell-type independent manner suggesting a direct control of the transcription machinery or a broader epigenetic control (Siano et al., 2019).

1.5 Pathological Tau alterations

1.5.1 Post-translational modifications

Tau undergoes a wide range of PTMs (Fig. 1.6) finely regulated by dozens of different binding partners, whose role in a more general perspective of Tau functions regulation are not completely understood. A toxic outcome has been associated to the unbalance of the competition existing between different PTMs.


14 Phosphorylation

Phosphorylation is the most well characterized PTM as it was firstly linked with Tau self-aggregation. Tau contains 85 putative phosphorylation sites (Alonso et al., 1994; Wang & Mandelkow, 2016), including 45 serine, 35 threonine and 5 tyrosine residues. The phosphorylation state of the protein is finely regulated and is crucial in tuning its functions and inducing pathological alterations since it impacts on development, spreading, subcellular location, partners interaction and of course PHFs formation and evolution (Camero et al., 2014). Moreover, NMR experiments carried out on a peptide derived from Tau protein have shown how phosphorylation stabilizes secondary structures on Tau (Sibille et al., 2012), therefore suggesting a mechanism promoting aggregation.

Elevated Tau phosphorylation detaches Tau from microtubules and induces missorting from axons into the somatodendritic compartment, compromising axonal microtubule integrity and synaptic activity (Hanger et al., 2009; Sengupta et al., 1998; Hoover et al., 2010). Further, phosphorylation of Tau can disrupt its intracellular route of degradation. For example, Tau phosphorylated on Ser262 or Ser356 is not recognised by the C-terminus of Hsp70-interacting protein - Hsp90 (CHIP-HSP90) complex and is thereby protected from degradation by the proteasome (Dickey et al., 2007). In contrast, phosphomimic Tau is selectively cleared by autophagy compared to endogenous Tau (Rodríguez-Martín et al., 2013). Finally, phosphorylation alters the association of Tau with its interacting partners, such as cytoplasmic membrane, DNA and Fyn, disturbing the functions of Tau in a range of signalling pathways (Hanger et al., 2009).

Phosphorylation state is controlled by the delicate equilibrium between kinase and phosphatase activity. Tau is physiologically phosphorylated with a stoichiometric ratio of 3:1, which may increase to 11:1 in pathological conditions (Hanger et al., 2009).

Tau kinases can be grouped in three classes:

● Proline directed serine/threonine-protein kinases. Among these, glycogen synthase kinase (GSK) 3α/β activity have the biggest impact on Tau phosphorylation: of the 40 predicted phosphorylation sites, 29 are related to AD (Hanger et al., 2009). GSK3 overexpression and high activity level correlate with more severe progression through Tauopathy, facilitates subsequent phosphorylation and increases neurofibrillary tangle burden (Hanger et al., 1992; Pei et al., 1997). The other representatives of the group such as cyclin-dependent kinase-5 (Cdk5), mitogen-activated protein kinases (MAPKs) and several other kinases including those activated by stress, are linked to neurofibrillary degeneration and colocalize with tangles in AD and other Tauopathies (Cruz et al., 2003; Noble et al., 2003).

● Non-proline-directed serine/threonine-protein kinases, such as Tau-tubulin kinase 1/2 (TTBK1/2), casein kinase 1 (CK1), which accounts for 46 overlapping phosphorylation sites (Hanger et al., 2007), dual-specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A), whose priming activity on Ser208 has been proposed to facilitate GSK3-mediated phosphorylation (Ryoo et al., 2007), microtubule affinity-regulating kinases (MARKs), Akt/protein kinase B, cAMP-dependent protein kinase A (PKA), protein kinase C, protein kinase N, 5′ adenosine monophosphate-activated protein kinase (AMPK), calcium/calmodulin-dependent protein kinase II (CaMKII), and thousand and one amino acid protein kinases (TAOKs) 1 and 2 (Guo et al., 2017);

● Protein kinases specific for tyrosine residues, such as Src, Abl, Lck and Syk, which drive aggregation in specific Tauopathies, and Fyn, which may regulate axonal transport (Guo et al., 2017).



Conversely, fewer phosphatases can oppose kinase action: protein phosphatase 2A (PP2A) and PP5 are both crucial for neuronal homeostasis (F. Liu et al., 2005; F. Liu et al., 2009). They account for 90% of phosphatase activity on Tau, which is severely downregulated in dementia. Interestingly, PP2A inhibits GSK3β by dephosphorylating Ser9 which in turn inhibits PP2A (X.-Q. Yao et al., 2011). GSK3β and PP2A are also inhibited by Akt/mTOR pathway suggesting a regulatory loop on Tau phosphorylation, whose unbalance can impairs Tau functions (Meske et al., 2008).


Tau acetylation is getting more attention since it has been observed to compete with phosphorylation on key residues, being either protective or a trigger for more severe phenotypes (Y. Wang & Mandelkow, 2016). This PTM is induced by cAMP-response element binding protein (CREB)-binding protein (CBP) pathway and is erased by sirtuin1 (SIRT1) and HDAC6 (Cook et al., 2014; Min et al., 2010). Tau itself possesses auto-acetylase catalytic activity: residues Cys291 and Cys322 located in R2 and R3 microtubule binding repeats act as donors and acceptors of acetyl groups (Cohen et al., 2016). CBP- mediated acetylation of lysine residues 259, 290, 321 and 353 is protective against aggregation. On the other hand, acetylation on lysine 259, 290, 321 and 353 is a common pattern in tauopathy affected brain (Cook et al., 2014). Different acetylation patterns can promote or hamper Tau turnover and degradation and finally, accumulation of toxic species (Cohen et al., 2011). Studies on mouse models expressing pseudo-acetylated Tau isoforms at Lys274 and Lys281 showed a decrease in long term cognitive abilities underlying an impaired synaptic molecular architecture and activity (Tracy et al., 2016). The same isoforms have shown in primary neurons culture to unsettle the cytoskeletal structure leading to somatodendritic accumulation of Tau, providing a possible explanation for the observed AMPA receptors misplacement (Tracy et al., 2016).

Other Tau modifications

Besides the previously outlined, others Tau modification have been observed specifically in Tauopathy affected brains only.

N-glycosylation, namely the conjugation of an oligosaccharide to a nitrogen atom, is a characteristic signature of diseased brains which promotes aggregates formation. It is likely to induce hyperphosphorylation and inhibit phosphatase activity through conformational changes in Tau backbone. The mechanism by which this PTM is fostered and regulated are still not completely clear but seem to be related to Tau localization in aberrant cellular compartments (F Liu et al., 2002).

Unlike N-glycosylation, glycation is not enzymatically regulated and is probably induced by alteration in alterations in energetic metabolism. As well as glycosylation, it promotes further modifications, mostly phosphorylation, and seems to impede Tau interaction with microtubules through conformational changes (Reyes et al., 2012).

Since conjugation with O-linked N-acetylglucosamine (O-GlcNAc) competes on serine and threonine residues for phosphorylation, it may be protective against Tau mediated-neurotoxicity. Indeed, O-GlcNAc suppresses Tau aggregation in cellulo and is significatively reduced AD brains owing to the reduced O-GlcNAc transferase expression (Watanabe et al., 2004).



Abnormal nitration on tyrosine residues 18, 29 and 394 destabilizes microtubules interaction and promotes aggregation, but when different sites are involved, aggregation is reduced (A. C. Wang et al., 2016).

Ubiquitination and Sumoylation play an antagonistic role on Tau turnover. Ubiquitination of Lys48 directed by CHIP or TRAF6 promotes proteasome-mediated degradation and could be hindered by nitration. Sumoylation, mainly on Lys340, opposes to degradation and correlates with hyperphosphorylation and aggregation (Luo et al., 2014).

Cys322 is modified by oxidation, a modification that regulates Tau self-assembly (Y. Wang & Mandelkow, 2016).

Finally, methylation has been observed on the same lysine and arginine residues involved in acetylation and ubiquitination, with a negative impact on the overall Tau solubility and ability to stabilize microtubules (T. Guo, Noble, & Hanger, 2017b).

Fig. 1.6 Sites of the most common Tau post translational modifications (from Morris et al., 2014).

1.5.2 Truncation

Cleavage of disease modifying proteins is a common hallmark of many human neurodegenerative disorders including AD, Pick’s disease (PiD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), some variants of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and Huntington’s disease (HD) as well (T. Guo et al., 2017b). The discovery of truncated Tau fragments in the protease resistant core of tangles was possible thank to antibodies which recognize Tau when its N or C terminus are cleaved generating neoepitopes. Truncated Tau fragments ranging between 9 and 12 kDa was found in AD but not in control brains (Vechterova et al., 2003). Their resistance to proteinase K suggests that fragmentation could enhance protein misfolding and prion-like aggregation. Once the insoluble core is formed, overcoming the slow aggregation kinetic of heavier isoforms, it may induce the secondary recruitment of more soluble full-length Tau species. The most commonly described Tau fragments, their physio-pathological features and the proteases involved in their formation are summed up in Tab. 1.1.

Tau fragment Amino acid residues



C-term cleaved Tau M1- 40–53 Present in synaptosomes from AD brain. C-terminus not identified.

Delta Tau


Associates with tangles in AD brain. Identified in the brains of aged wild-type and transgenic 3xTg-AD and hTau mice, which develop tangles, amyloid plaques and synaptic dysfunction. Induces Tau filament formation and inversely correlates with cognitive function. Induced by Aβ in neurons and leads to apoptosis. Tau is cleaved at D13 by caspase-6 and at D421 by caspase-3

NH2-Tau Q26-R230 Enriched in synaptosomal mitochondria in AD brain. Induced by apoptosis in SHSY-5Y neuroblastoma cells. Present in hippocampus in AD11 transgenic mice which have chronic NGF deprivation during adulthood and display AD-like molecular and behavioural phenotypes.

E45-R230 17 Detected in AD, ALS, and control brain. Generated in neurons by exposure to Aβ or by thapsigargin-mediated

inhibition of autophagy. Induces neurodegeneration when expressed in mice. Not toxic when expressed in N2a or CHO cells, or neurons. Generated by calpain-1 cleavage.

S129-(S71 in

0N3R Tau)

33 Isolated from tangles in AD brain. Decreased ability to bind to tubulin. C terminus not identified.

Q124-L441 43 Present in human brain. Increased acetylation and

detyrosination of tubulin when expressed in N1E-115 neuroblastoma cells.

A125-R230 17 Present in AD and control brain. Not toxic when expressed in N2a or CHO cells, or neurons. Generated by calpain-2 cleavage (see related fragment E45-R230 above).

I151-A391 29 Present in the neurofibrillary tangle core in AD brain. Expression of either 3R Tau151–391 (lacking 275–305) or 4R

Tau151–391 in transgenic rats induces tangle formation. Muscle

weakness develops only in 4R Tau151–391 rats.

Tau35 E187-L441 33–37 Present in AGD, PSP, and CBD, but not control brain. Includes four microtubule binding repeats. Expression of Tau35 mice in transgenic mice induces Tau pathology, cognitive and motor dysfunction.

Tau-CTF24 L243-L441 24 20–28 kDa C-terminal Tau species detected in AD, CBD, PSP, and FTLD-Tau, but not control brain. Includes four microtubule binding repeats. Present in Tg601 mice which exhibit increased Tau phosphorylation and synapse loss.

Tab. 1.1 Tau fragments that have been detected in human brain that is potentially associated with the development of tauopathy. The tau cleavage products are listed in order of their most N-terminal amino acid (single letter code). Aβ amyloid-β peptide, AD Alzheimer’s disease, AGD argyrophilic brain disease, ALS amyotrophic lateral sclerosis, CBD



corticobasal degeneration, CHO Chinese hamster ovary, FTLD frontotemporal lobar degeneration, PSP progressive supranuclear palsy (adapted from Guo et al., 2017b).

1.5.3 Aggregation

According to current hypothesis, Tau exerts its pathogenic mechanism through aggregation into insoluble oligomers. At the onset of tauopathies, mainly two types of Tau filaments have been described: paired helical (PHF) or straight filament (SF) which assemble into higher order structures, named neurofibrillary tangles (NFTs; Fig. 1.7). These Tau deposits engulf neuronal cytoplasm, being an obstacle for trafficking and other fundamental function, causing neuronal death (Ward et al., 2012). Indeed, Tau burden is tightly linked to the progression of Tauopathies and has long be used to describe disease stages (Braak et al., 1995).

Aggregation is promoted by two hexapeptides lying in the second and in the third microtubule binding repeat (von Bergen et al., 2000). They are respectively referred as PHF6* (residues 317-335: Val-Gln-Ile-Ile-Lys-Asn) and PHF6 (residues 306-311: Val-Gln-Ile-Val-Tyr-Lys). Importantly, because of its location, PHF6* hexapeptide is only expressed in 4R Tau isoforms, making them more prone to aggregation (Pérez et al. 1996). These two fragments self-assemble into parallel, in register β-sheet secondary structures (Meraz-Ríos et al., 2010). When put in solution, even without any chemical trigger, they spontaneously form homo and heterodimers. When a consistent nucleation centre is formed, which is the limiting kinetic step, the filament elongation proceeds as a function of time and concentration, following a prionic mechanism (Barghorn & Mandelkow, 2002).

Despite the propensity toward aggregation of its subdomains, full length Tau is resistant to self-assembly due to its overall positive charge of basic residues. Substitutions involving the hexapeptides could foster or hamper the process depending on whether secondary structures are stabilized or not becoming incompatible with sheet arrangement (Bulic et al., 2010). Not only the MTBD, but also insertions in NTD can modulate the kinetic od Tau aggregation, perhaps influencing the total protein charge: exon 2 retention promotes aggregation, exon 3 instead has an inhibitory effect (Zhong et al., 2012).

Regarding PTMs, phosphorylation and acetylation play a central role in Tau physiopathology, as they counterbalance or neutralize positive charges and mitigate repulsive interactions. Natural and artificial polyanions have a similar buffering effect and act as aggregation inducers. For example, heparin, fatty acids, tRNA, and polyglutamic acid have been reported to stabilize assembly competent Intermediates binding to Tau at several sites (Kuret et al., 2005; Wilson et al., 1997).

Although aggregation kinetic has been studied in detail, it is debated if tangles are the real responsible for the neurotoxic effect, as it was classically believed, since recent studies produced contradictory results (Cowan & Mudher, 2013). Several evidences collected by means of in vivo and in vitro models links tangle formation with cytotoxicity and apoptosis. Puzzling results has recently been obtained from a mouse model expressing P301L mutant Tau under control of and inducible promoter. When Tau expression were switched off in diseased individuals, both memory and neurotoxicity underwent a significant improvement, even if Tau burden remained unchanged (SantaCruz, 2005). This might suggest that larger aggregates could not be the main toxic species but an inert deposit of misfolded proteins, while the pathology trigger should be sought between soluble oligomers that might be generated during tangle deposition. Even if the exact nature of neurotoxic Tau isoform has not been revealed, more evidences support the last hypothesis



(Spires-Jones et al., 2011). Further, the great variety of PTMs, solubility and morphology of aggregates could account for the phenotypic variants of Tau pathology operating through different mechanisms (Sanders et al., 2014).

Fig. 1.7 Tau hyperphosphorylation leads to the formation of paired helical and straight filaments resulting in NTFs.

1.6 Pathogenic mechanisms

Understanding the pathological mechanisms that lead to Tau mediate neurotoxicity remains a hard challenge, notwithstanding the research efforts made until now. We could observe and describe an intricate net of alteration regarding Tau mutations, mis-splicing, aggregation, PTMs and truncations. Each of these alterations burden on synaptic functionality, protein compartmentalization and metabolic homeostasis and could imply a positive feedback towards disease progression and spreading.

1.6.1 Axonal transport impairment

Besides regulating tubulin dynamics, Tau dramatically impacts on organelles dynamics and motor protein activity (Utton, 2005). Microtubule provide a truck for motor proteins to move towards the axon terminus (kinesin) or the soma (dynein). The transport mechanisms are characterized by a multilevel complexity, requiring an intact cytoskeleton architecture, functional motor proteins, the efficient interaction with cargo and a ready source of energy provided by mitochondria in the form of ATP. The impairment of one or a combination of these elements result in pathological phenotypes shared with Tauopathies, including aberrant organelles accumulation, engulfment of neurites and their swelling. Indeed, axonal transport deficit is a very early sign described in several tauopathy models (De Vos et al., 2008). Additionally, the presence of tangles has been linked to anterograde and retrograde transport impairment in both axons and dendrites. As a microtubule binding protein, the detachment of Tau from the cytoskeleton prompted by hyperphosphorylation and other modifications is the first cause of its destabilization (Zhang et al., 2012). Tau mutations can alter tubulin binding affinity and promote Tau phosphorylation, but can also have an important effect on its subcellular localization (Michel Goedert & Jakes, 2005).

A different mechanism concerns the alteration of motor proteins activity provoked by Tau accumulation, which precedes Tau aggregation and clinical symptoms manifestation (Chiasseu et al., 2016). Both dynein and kinesin show a reduced velocity and frequency of binding to the microtubule track when Tau is overexpressed or dislocated (Dixit et al., 2008; Ebneth et al., 1998). Moreover, Tauopathy models evidence a reduced availability of motor proteins for cargoes (Morel



et al., 2012). The increased activation of GSK3β contributes to transport deficits by aberrant phosphorylation of kinesin’s light chain, resulting in premature release of kinesin from its cargoes (Morfini et al., 2002). Finally, Tau displaces the adapter molecule (JNK)-interacting protein 1 from microtubules. The accumulation of proteins and organelles in the soma provokes the impaired assembly of synaptic machinery altering its functionality. In particular, alterations of the mitochondria turnover reduces the energy available for the fast neuronal metabolism and induce oxidative stress (L. M. Ittner et al., 2008).

1.6.2 Nuclear Tau alterations

The abnormalities reported in Tauopathy models and diseased patients support the current theories about the functions exerted by Tau in the nucleus. In lymphocytes and fibroblast of FTDP-17 and PSP patients, Tau mutations have been linked with peculiar alterations of gene transcription, chromosome alterations and an increased susceptibility to oxidative stress (Rossi et al., 2014, 2013b, 2008). Phosphorylation reduces Tau ability to translocate into the nucleus (Lefebvre et al., 2003) and impairs its interaction with DNA (Y. Lu et al., 2013; Qi et al., 2015b), thus a possible loss of function may result from its mislocalization. Indeed, an increase of oxidative stress-related DNA and chromosomal damages has been observed a consequence of nuclear Tau depletion (Frost et al., 2014; Mondragón-Rodríguez et al., 2012; Violet et al., 2014). However, the role of phosphorylation seems to be much more articulated and toxic gain of function of a small amount of phosphorylated nuclear Tau cannot be excluded. Some reports suggested that phosphorylation lies upstream of oxidative stress damage. Nuclear accumulation of phosphorylated Tau can be induced by Aβ exposure or viral infection (Mondragón-Rodríguez et al., 2012) and can even lead to nuclear aggregate formation, as it has been observed in neurons of HD, FTLD, and AD patients (Fernández-Nogales et al., 2014, 2017). Last, phosphorylated Tau could interact with RNA stress granules thanks to TIA1, alter their dynamics and make the cell more prone to stress (Brunello, Yan, & Huttunen, 2016). In conclusion, the general chromatin relation induced by these changes cause aberrant gene expression and protein synthesis, promoting a deleterious cell cycle re-entry of the neurons.

1.6.3 Somatodendritic Tau accumulation

Several studies have linked the abnormal translocation of Tau in the somatodendritic compartment with pathological gain of function. It is commonly accepted that Tau modifications such as phosphorylation and acetylation are key factor which promote Tau redistribution, as they heavily impact on its interaction with microtubules. Notwithstanding, since newly synthesised Tau is missorted to the somatodendritic compartment prior to its phosphorylation by MAPK, the spatial and temporal occurrence of these events is still not clear (Zempel et al., 2013). Tau translocation in the somatodendritic compartment correlates with Fyn kinase accumulation due the direct interaction between the two proteins. There, Fyn phosphorylates the NR2B subunit of the N-methyl-d-aspartate receptors (NMDARs) stabilizing its interaction with PSD-95 and promoting synaptic hyperexcitability (Ittner et al., 2010).

Another protein whose localization in the dendritic compartment follows Tau displacement is tubulin tyrosine ligase-like 6 (TTLL6), which catalyses polyglutamylation of microtubules. This modification enhances spastin-mediated microtubules cleavage resulting in alteration of cytoskeleton structure, cellular trafficking impairment and abnormal accumulation of organelles (Zempel et al., 2013).



In mouse models characterized by a massive amyloid plaque deposition and the subsequent Tau aggregation, such as APPswe-transgenic mice and rTg4510 mice, both AMPA and NMDA receptors

activity abnormalities has been reported leading to synaptic dysfunction and cognitive deficits (Hoover et al., 2010).

1.6.4 Mitochondrial dysfunctions in Tauopathies

Alterations involving mitochondria activity and their turnover could be important mediators of tauopathy phenotype (J. Yao et al., 2009). Abnormalities in their localization have been evidenced in tauopathy brains and in mouse models expressing mutated Tau isoforms, associated with increased oxidative stress and significant mitochondrial depletion in the axonal compartment (Kopeikina et al., 2011; Rodríguez-Martín et al., 2016). Besides localization, Tau can also impair mitochondrial biogenesis, as suggested by the observation that Tau phosphorylation modulates their cytological and molecular phenotype. For instance, the interaction between phospho-Tau and dynamin-related protein 1 (Drp1), a protein involved in mitochondrial fission, result in mitochondrial deficiency and fragmentation in GSK3β overexpressing mice (Rockenstein et al., 2014). Conversely, when P301L Tau is overexpressed, Drp1 reduction allows a partial phenotypic recovery (Kandimalla & Reddy, 2016).

Not only phosphorylation, but also Tau truncation interferes with mitochondrial homeostasis. The N-terminal Tau fragment (Tau26–230), detected in cellular and animal models as well as in AD brain,

correlates with synaptic dysfunction and is enriched in AD mitochondria extracts. Its toxicity is related to the impairment of mitochondrial turnover through the unbalanced mitophagy (Corsetti et al., 2015). Another Tau fragment cleaved at Asp421, has been reported to increase molecular markers associated mitophagy both in transgenic mice and AD brains (M. J. Pérez et al., 2018). Finally, Tau protein overexpression results in its accumulation in the outer mitochondrial membrane causing the increase of membrane potential and again impairing mitophagy (Hu et al., 2016). Taken together, these findings suggest a possible impact of Tau on mitochondria integrity, providing additional explanations for synaptic dysfunction.

1.6.5 Impaired Tau clearance and degradation

In physiological conditions misfolded proteins are specifically targeted to degradation through ubiquitin- proteasome system (UPS). Alternatively, autophago-lysosomal system operates on long-lived proteins and cell organelles in bulk. Both the mechanisms are involved in Tau molecules turnover. Deciphering the ubiquitination sites of Tau provided solid evidences of its UPS-mediated degradation (M. J. Lee et al., 2013). Moreover, some members of the UPS pathway have been identified among Tau interactors, such heat shock protein 27 and CHIP (M. J. Lee et al., 2013). It has been observed that phosphorylated Tau may inhibit proteasomal activity by binding to its 20S subunit, but if UPS impairment is caused by or follows Tau aggregation is still a matter of debate (Keck et al., 2003). Since impairment of the UPS during Tauopathies progression has been reported in several animal models and in patients’ brains, it has been proposed as a possible therapeutic target. Indeed, increasing the 26S proteasome activity by stimulating the cAMP-PKA pathway proved to minimize the toxic effect of Tau oligomers and improve UPS activity (Lokireddy et al., 2015). The involvement of heat shock proteins in Tau degradation, instead, is less clear, but many evidences agree on the importance of CHIP’s role (T. Guo et al., 2017b).



Unlike physiological conditions, unfolded Tau has been observed to be mainly degraded via the autophago-lysosomal pathway. Therefore, its malfunctioning could be accounted among the possible pathogenic mechanisms exerted by Tau (Rodríguez-Martín et al., 2013). Indeed, immature autophagic structures and intermediates, such as autophagosomes and late autophagic vacuoles, has been observed in dystrophic neurites in AD brains, and in animal and cell models of AD, suggesting impaired degradation of autophagic vacuoles by lysosomes (Tan et al., 2014). On the other hand, some experiments put abnormal autophagy upstream to Tau accumulation, as stimulating mTOR, an inhibitor of autophagy, increases total and phosphorylated Tau in P301S Tau mice (Caccamo et al., 2013). Moreover, animal model lacking important elements of the autophagosomal mechanism evidence an increased accumulation of Tau aggregates (Khurana et al., 2010). Conversely, stimulating this pathway result in a general improvement in neuronal health conditions (Berger et al., 2006).

Finally, also Tau truncation have a strong impact on both the forms of protein degradation, resulting in the accumulation of insoluble products (Bondulich et al., 2016).

In general, pathological Tau forms could impair their UPS and autophagy or overload their clearance rate. The fine understanding of the mechanism ruling its preferential rate of degradation could provide new therapeutic targets.

1.6.6 Unfolded protein response in Tauopathies

The unfolded protein response (UPR) is activated at the level of the endoplasmic reticulum (ER) following external insults, ER stress and abnormal protein synthesis and accumulation. The UPS consists of three main routes, leading to different outcomes. The inositol-requiring transmembrane kinase/endonuclease 1 (IRE1) branch, result in the synthesis of a cluster of chaperons which help protein folding in the ER. The involvement of activating transcription factor 6 (ATF6) requires the cleavage of its cytoplasmic domain and its translocation inside into the nucleus. Here it promotes the transcription of genes whose function are related to correct protein folding, autophagy, redox balance, amino acid metabolism and apoptosis. The (PKR)-like endoplasmic reticulum kinase (PERK) pathway inhibits new protein synthesis through the phosphorylation of eukaryotic initiation factor 2α (eIF2α) (Scheper & Hoozemans, 2015).

UPS activation has been reported in several models of Tauopathy and in AD brains, both as a direct effect of Tau mutations, which enhance the interaction with UPR mediators, or indirectly caused by the impairment of organelles trafficking (abnormal protein degradation) or of mitochondrial functions (increased oxidative stress and widespread protein damages) (van der Harg et al., 2014). Tau phosphorylation and UPS have been proposed to interplay in a vicious cycle in which UPS activation promotes further phosphorylation and vice versa. Indeed, the activation GSK3β (which is one of the main Tau kinases), UPR markers and phospho-Tau often colocalize (Nijholt et al., 2012). GSK3β is indirectly activated by different UPR mediators, such as IRE1 and PERK and its interaction with Tau is favoured by the ER-associated chaperone BiP (Z.-C. Liu et al., 2012). Conversely, the activation of GSK3β and the induction Tau phosphorylation through the administration of okadaic acid, result in the upregulation of UPS markers in primary neurons (Ho et al., 2012). The impact of abnormal UPR activation could promote neuronal damage by inhibiting the synthesis of fundamental structural and functional proteins and inducing apoptosis.



1.7 Tauopathies

Tau has been recognized as the etiological cause of a large and heterogeneous group of dementia and movement disorders designed as Tauopathies, whose common hallmark is the accumulation of intracellular Tau inclusions in neurons and glia (Fig. 1.9). Mutations of MAPT gene genetic causes of fronto-temporal – like dementia. Many other pathologies report Tau lesions in association to deposits of other disease-associated proteins, demonstrating bot a causative role or a secondary induction of pathological features. Multiple pathogenetic mechanisms have been proposed, depending on the contribution of incident factors.

1.7.1 Genetic risk factors

Since the identification of P301L mutation as a cause of FTDP-17, many others MAPT gene mutations have been linked to familial and sporadic forms of Tauopathies (Michel Goedert & Jakes, 2005). Some of them fall in the domains involved in Tau primary functions related to MTs binding and stabilization (Dickson et al., 2011), others increase Tau propensity to pathological PTMs or impair expression regulation and 3R/4R isoforms balance (Alonso et al., 2004). If these mutations relate to the impairment of Tau non canonical functions remains an open question. Different mutations of the same gene results into slightly different clinical symptoms classifiable in different form of dementia, each with its characteristic physio-pathogenetic tracts (Fig. 1.8), again suggesting that the Tau world is much more faceted than what gravitates around cytoskeleton solely (Rossi et al., 2013b) .

It is clear that MAPT mutations are detrimental to neurons and likely to impact on the conformation of Tau, with resultant effects on its PTMs, interactions with other proteins and a variety of intracellular processes (Iqbal et al., 2010).

Fig. 1.8 The variability of molecular hallmarks of Tau-related neurodegenerative diseases in space and time. The deposited proteins adopt an amyloid conformation and show prion-like self-propagation and spreading in experimental settings, consistent with the progressive appearance of the lesions in the human diseases. a, Amyloid-β deposits (senile plaques) in AD neocortex. b, Neurofibrillary tangles in AD neocortex. c, α-Synuclein inclusion (Lewy body) in PD neocortical neurons. On the right, characteristic progression of specific proteinaceous lesions in neurodegenerative



diseases over time: Aβ deposits (e) and Tau inclusions (f) in AD, α-synuclein inclusions in PD (g), (adapted from Jucker &

Walker, 2013).

1.7.2 Alzheimer’s disease

In this pathology Tau has been described as an hallmark in association with amyloid deposits (Heiko Braak & Braak, 1995). Braak stages describe the progression and the severity of the pathology associated to the predictable pattern of Tau lesions, whose symptoms depend on the brain area affected by neural loss. The most susceptible brain region for the deposition of neurofibrillary tangle and amyloid plaque is the transentorhinal/peripheral cortex (Braak stage I). From here, the pathology follows a consistent path spreading through anatomically interconnected brain areas resulting in clinically relevant cognitive and behavioural alterations: the CA1 region of the hippocampus (Braak stage II), limbic structures (Braak stage III), amygdala, thalamus and claustrum (Braak Stage IV), isocortical areas (Braak stage V), and finally, primary sensory, motor and visual regions (Braak stage VI) (H. Braak & Braak, 1991; 1995).

The main tangle components found in AD consist of paired helical and straight filaments. All six Tau isoforms can be reported in AD aggregates, in a 1:1 ratio between 3R and 4R variants (M. Goedert et al., 1995). Due to the their heavy hyperphosphorylated status, the addition to other PTMs and the abnormal interaction with SDS, all of them undergo an electrophoretic shift towards higher molecular weights, from 48 to 67 kDa to 68-72 kDa (Steiner et al., 1990).

Besides Tau deposition, the formation of amyloid plaque in the extracellular space and around blood vessels is an AD hallmark on which post-mortem diagnosis are based (Wong et al., 1985). Despite many efforts have been spent in this field, we are still far to understand which between amyloid and Tau is the causative agent of AD. Indeed, amyloid plaque does not correlate with cognitive decline and is often present in healthy aged brains (Jucker & Walker, 2013). Conversely, a strong correlation can be found between Tau tangles and neural loss (Lloret et al., 2011). However, the neurotoxic effect can be observed in a greater number of neurons than those engulfed by PHF, suggesting that the latter could not be the cause but a defence mechanism that neurons might adopt to cope with the progressive accumulation of toxic soluble of Tau aggregates (Gómez-Isla et al., 1997).

Genetic analysis of familiar AD forms allowed to link several mutations affecting MAPT gene but also loci that regulate both APP synthesis, cleavage and clearance evidencing a role for both the two proteins in AD aetiology (Deming et al., 2016). Indeed, a consistent piece of literature reported a direct relationship between Aβ-mediated toxicity and Tau pathology. One of the most relevant hypothesis suggests that an APP gain of function could trigger Tau hyperphosphorylation as it has been observed that in triple mutant mice for APP, Presenilin1 and Tau the injection of anti AB antibodies prevent amyloid plaque formation but also Tau hyperphosphorylation (Oddo et al., 2004). However, our understanding of the mechanism that link Aβ with Tau deposition remains poor.

1.7.3 Tau and Synucleinopathies

Parkinson’s disease (PD) is the most common synucleinopathy of the brain, whose preeminent physio-pathologycal feature is the progressive degeneration of dopaminergic neurons in the basal ganglia, with the substantia nigra pars compacta being the most affected area. PD is clinically diagnosed by motor symptoms, mainly tremor, bradykinesia, rigidity, and postural instability, and



non-motor symptoms including autonomic dysfunction and neuropsychiatric problems (Spillantini et al., 1997).

PD and all the other synucleinopathies including Parkinson’s disease dementia, dementia with Lewy bodies, and multiple system atrophy share the same histological hallmark which is the deposition Lewy bodies in subcortical brain regions. Even if synuclein is the main component of PD aggregates Tau has been repeatedly reported to colocalize and even promote synuclein aggregation (Spillantini et al., 1997).

Some mutations of the MAPT locus have been linked with familiar forms and rare cases of Parkinsonism (Nalls et al., 2014). For instance, the H1 Tau haplotype consisting of eight single nucleotide polymorphisms and a microsatellite marker, is an important risk factor for a “Tau-only” form of PD as it result in Tau overexpression (Vilarino-Guell et al., 2011). On the other hand, H2 haplotype exerts a protective role (Vilarino-Guell et al., 2011).

Since Tau pathology and α-synuclein aggregates colocalize in different diseases (Irwin et al., 2013), the effects of their interaction are getting more and more attention. Notably Lewy bodies are usually found in 50% of AD patients’ brains (Moussaud et al., 2014) and, vice versa, pronounced Tau pathology can be found in familial Parkinson’s disease dementia (Schneider et al., 2006). Additionally, these two proteins colocalize in the axonal compartment both physiologically and during aggregate formation (Esposito et al., 2007). Importantly, synuclein promotes Tau phosphorylation and fibrils formation through a mechanism involving GSK3β and MAPKs and in return Tau expression exacerbates detrimental α-synuclein molecular changes and accelerate PD progression (Jensen et al., 1999; Kawakami et al., 2011; Oaks, Frankfurt, Finkelstein, & Sidhu, 2013). These findings strongly suggest an important interplay between Tau and α-synuclein, which may share some pathogenic mechanisms in the formation of neuropathological lesions in Tauopathies and synucleinopathies.

1.7.4 Huntington disease

Tau has also been proposed as a possible effector of Huntington disease (HD), an autosomal dominant neurodegenerative disorder mainly affecting the striatum but with a strong cognitive component. HD is caused by the expansion of the polyglutamine tract of the huntingtin protein (Labbadia & Morimoto, 2013). The exact function of this protein is not known, but it plays an important role in nerve cells where it may be involved in signalling, axonal transport of vesicles and mitochondria and transcription regulation. Huntingtin forms filamentous aggregates which sequester the splicing factor SRSF6, involved in exon 10 spicing of MAPT transcript. This cause a neat shift of Tau pool composition toward 4R Tau isoforms, also enriched in HD Tau aggregates (Fernández-Nogales et al., 2016). Interesting results have been collected from the model R6/2, which overexpresses huntingtin exon 1 with an expanded polyglutamine repeat. Besides motor dysfunction, impaired learning and memory and intraneuronal huntingtin inclusions (Davies et al., 1997), increased Tau phosphorylation is evident in the brains of R6/2 mice in parallel with reduced amounts of protein phosphatases (Blum et al., 2015). Similarly, increased Tau phosphorylation is detectable in HD, along with elevated GSK3 activity (L’Episcopo et al., 2016).



Fig. 1.9 Tauopathies. Diagram illustrating the wide range of neuropathological conditions in which tau pathology is a significant feature. The central panel illustrates disorders in which tau pathology is the primary feature. The overlapping panels summarise conditions in which tau inclusions are accompanied by deposits of other disease-associated proteins.

1Chronic traumatic encephalopathy includes traumatic brain injury and dementia pugilistica; 2ARTAG, aging-related tau

astrogliopathy includes globular glial tauopathy; 3PART, primary age-related tauopathy includes tangle-predominant

dementia and clinically asymptomatic cases; FTLD, frontotemporal lobar degeneration (from Guo et al., 2017b).

1.8 Interaction with TRIM28

How Tau could enter the nucleus has been an open question for long time, since it lacks a known nuclear localization signal (NLS). It is currently hypnotised the presence of a cryptic NLS or alternatively the interaction with a cofactor which mediates its nuclear import. A recent work demonstrated that Tau interacts with the nuclear protein TRIM28 which is able to translocate it to the nucleus. This was shown tracking the Tau- TRIM28 complex exploiting bimolecular fluorescence complementation and was confirmed observing that TRIM28 overexpression was able to boost Tau shuttling to the nucleus. TRIM28 exerts the same effect on α-synuclein causing an overall exacerbation of the pathological phenotype. These observations suggest a convergence of the pathogenic nuclear mechanisms of PD and tauopathies (Rousseaux et al., 2016). Rousseaux and colleagues observed that nuclear Tau translocation is impeded by loss of function mutations of TRIM28 E3-ligase domain, which SUMOylate Tau onto Lys340 (Luo et al., 2014; Rousseaux et al., 2016). SUMOylation competes with Tau ubiquitination and increases its half-life and abundance. Hence the hypothesis of a functional more than a structural role of TRIM28 in Tau nuclear shuttling.

Tripartite motif-containing 28 (TRIM28), also known as transcriptional intermediary factor 1b (TIF1b) and KAP1 (KRAB-associated protein-1), is a nuclear protein mainly involved in transcriptional regulation and chromatin remodelling. This protein performs many core biological functions, including degradation of p53 tumour suppressor, regulation of retrotransposons activation, regulation of gene expression through heterochromatization, mediation of DNA damage response, maintenance of stem cell pluripotency and induction of autophagy in cancer cells (Fig. 1.9A; Czerwińska et al., 2017). Given its relevance in stem cells differentiation, TRIM28 is crucial in the maintenance of neuronal identity: a reduction in its levels is responsible for severe alterations ranging from lethality in early developmental phases (Cammas et al., 2000), to early differentiation (Quenneville et al., 2011), to alterations in postmitotic neurons (Jakobsson et al., 2008).




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