1
DOTTORATO TOSCANO IN
NEUROSCIENZE
CICLO XXXI
COORDINATORE Prof. RENATO CORRADETTI
HTRA1 EXPRESSION AND FUNCTIONALITY IN HTRA1 MUTATION CARRIERS
Settore Scientifico Disciplinare MED/26
Dottorando Tutore
Dott. Fasano Alessandro Prof. Federico Antonio
Coordinatore Prof. Corradetti Renato
2
Summary
ABSTRACT 4 1. INTRODUCTION 6 1.1. HTRA1 PROTEIN 61.1.1. Gene and Protein structure 11
1.1.2. Protein Functions 14
1.2. CEREBRAL SMALL VESSEL DISEASE 21
1.2.1. CARASIL 22
Clinical features 22
Neuroradiological findings 23
Pathology 24
1.3. HTRA1 MUTATIONS 25
2. AIMS OF THE THESIS 31
3. MATERIAL AND METHODS 32
3.1. PATIENTS 32 3.2. HUMAN FIBROBLASTS 34 3.3 MICE MODELS 35 3.4. MICE TISSUE 37 3.5. PLASMID CLONING 38 3.6. TRANSFECTION 40 3.7. HEK293T CELLS 41 3.8. LTBP1 ASSAY 41 3.9. FITC-casein DEGRADATION 42 3.10. "RESCUE" OF HTRA1-R274Q ACTIVITY 42 3.11. IMMUNOFLUORESCENCE ANALYSIS 42
3
3.12. WESTERN BLOT ANALYSIS 44 3.13. REVERSE TRANSCRIPTASE-quantitative POLYMERASE CHAIN REACTION 45
3.14. STATISTICAL ANALYSIS 46
4. RESULTS 47
4.1. HTRA1 EXPRESSION 47
4.1.1. Human Fibroblasts 47
4.1.2. Mouse Embryonic Fibroblasts HTRA1-R274Q 51
4.2. HTRA1 ACTIVITY ON TGF-β PATHWAY 51
4.2.1. Human Fibroblasts 51
4.2.2. Mouse Embryonic Fibroblasts HTRA1-R274Q 54 4.3. DOMINANT NEGATIVE EFFECT 55 4.4. FIBRONECTIN ACCUMULATION IN BRAIN SMALL VESSELS 56 4.5. RECOVERY OF HTRA1 PROTEASE ACTIVITY 57
5. DISCUSSION 59
CONCLUSIONS 65
FUTURE PERSPECTIVES 66
4
ABSTRACT
High temperature requirement A1 (HTRA1) belongs to heat shock-induced serine proteases and is ubiquitously expressed in normal human adult tissues. HTRA1 plays a modulatory role in various cell processes, particularly regulates the transforming growth factor-β (TGF-ß) signalling. Biallelic mutations in HTRA1 lead to cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), a rare cerebral small vessel disease (CSVD). Nowadays, fifteen HTRA1 mutations have been identified.
Recent data reported that heterozygous HTRA1 mutations seem to be linked to
familial CSVD of unknown aetiology, which is characterized by a later age at onset. These data suggest that HTRA1 mutation could behave as autosomal recessive or
dominant mutation.
Our aim is to obtain further data about the pathogenic effect of various heterozygous HTRA1 mutations. We compared expression profiles of HTRA1 and intermediaries of TGF-β signalling proteins both in heterozygous carriers, with missense and stop codon HTRA1 mutations, and in heterozygous and homozygous mouse embryonic fibroblasts harbouring HTRA1-R274Q mutation. Moreover, we used heterozygous and homozygous murine models harbouring HTRA1-R274Q in order to evaluate in vivo the effects of mutant HTRA1.
Further, we performed supplementary studies to evaluate the possibility of a dominant negative effect on HTRA1-WT by HTRA1-mutants, and the possibility to rescue HTRA1-protease activity in homozygous HTRA1-R274Q carriers.
The cell lysates and culture medium of cultured cells were used to analyse the expression pattern of both HTRA1 and intermediaries of TGF-β signalling proteins by western blot and immunofluorescence analysis. RNAs extracted from cultured cells and from mice tissues were used to analyse HTRA1-RNA and CTGF-RNA expression level by RT-qPCR.
We found a ∼50% reduction in HTRA1 expression in human fibroblasts carrying heterozygous HTRA1-mutations compared to control. RT-qPCR analysis confirmed
5
these data for two of the analysed subjects, whilst showed no significant reduction in the remaining carriers compared to control. Analysis of the murine models showed that there is no alteration of HTRA1-RNA expression nor in heterozygous nor in homozygous HTRA1-R274Q mice. No significant alteration of Smad2/3 phosphorylation and CTGF expression, down- and up-stream intermediaries of TGF-β signalling pathway, respectively, were found, suggesting that dysfunction of TGF-β signalling in fibroblasts might not contribute to the pathogenesis of CARASIL and CSVD linked to heterozygous HTRA1 mutations reported in this study.
Heterozygous and homozygous HTRA1-R274Q murine cells displayed an increased fibronectin accumulation of 10-fold and 40-fold, respectively, than HTRA1-WT cells, suggesting that even the heterozygous HTRA1-mutations could be enough to cause deleterious phenotypic alterations in brain small vessels.
HTRA1-WT protease activity did not display any remarkable alteration in presence of HTRA1-mutants.These findings seem to rule out a dominant negative effect in
HTRA1 mutations we investigated. Finally, MEFs transfected with the rescue
protein give an outcome similar to that obtained with MEFs transfected with
WT, opening actual possibility to rescue the functionality of
HTRA1-mutants.
In conclusion, our results seem to suggest that CSVD, linked to heterozygous
HTRA1 mutations, may occur in the presence of ~50% expression of the protein.
Progressive tissue damage accumulation in small vessels leading to delayed and milder clinical expression with later onset with respect to classical CARASIL phenotype may be hypothesized.
Moreover, data collection on heterozygous HTRA1 mutants is still limited, so investigation on the HTRA1 expression and activity, in cells from a wider number of subjects harboring different heterozygous HTRA1 missense mutations, could be helpful to verify a possible correlation between specific aminoacidic variations and particular protein alterations.
6
1. INTRODUCTION
1.1. HTRA1 PROTEIN
High temperature requirement A1 (HTRA1) belongs to a group of heat shock-induced serine proteases that include oligomeric serine-proteases highly conserved from prokaryotes to humans (Kim et al., 2005; Singh et al., 2011). The serine proteases are subdivided into six clans based on sequence similarity, tertiary structures, and the sequential order of catalytic residues. HTRA1 belongs to the trypsin clan SA where the order of the catalytic triad is His-Asp-Ser. SA proteases have a two-domain structure with each domain forming a six-stranded β barrel. The active site cleft is located at the interface of the two perpendicularly arranged barrel domains. Many of the SA proteases are expressed as an inactive pro-form with an N-terminal inhibitory peptide and require proteolytic maturation for activation. Once the pro-peptide is removed, activation is irreversible and can only be abolished by trans-acting inhibitors. The active site is constructed by several loops located at the C-terminal side of both barrel domains. The participating loops of the N-terminal β barrel are termed LA, LB, LC, those of the C-terminal barrel L1, L2 , and L3 (Perona and Craik, 1995) (Figure 1). The over 180 members of this family (corresponding to 10% of trypsin-like proteases) are enzymes that catalyse the cleavage of peptide bonds and perform a variety of different tasks.
7
The main functions of HTRA family members are key aspects of protein quality control, some of them also exhibit a chaperone function that stabilizes specific proteins. In addition, HTRAs can trigger or modulate various signalling pathways by cleaving or sequestering regulatory proteins, deciding so cell fate (Clausen et al., 2011). All HTRA family members have in common an N-terminal segment with regulatory functions, a proteasic domain and at least a Post-synaptic density protein-95/Drosophila discs large tumor suppressor/Zonula occludens-1 protein (PDZ) domain. The latter is deputed to protein-protein interactions, it binds to target proteins on their firsts residues of the C-terminal (Clausen et al., 2002) while in some members of HTRAs family it is deputed to proteasic activity regulation.
In serine-proteases, hydrolysis of the peptidic bond occurs when the residues of the catalytic triad are aligned in such a way as to allow the electron transfer from Asp to Ser, via His residue. The –OH group of Ser acts as a nucleophilic centre, it attacks and breaks the peptidic bond on its carbonilic carbon. The distance
Figure 1 - Polypeptides can be cleaved either chemically or enzymatically. Enzymes that catalyse the hydrolytic cleavage of peptide bonds are called proteases. Proteases fall into four main mechanistic classes: serine, cysteine, aspartyl and metalloproteases. In the active sites of serine and cysteine proteases, the eponymous residue is usually paired with a proton-withdrawing group to promote nucleophilic attack on the peptide bond. Aspartyl proteases and metalloproteases activate a water molecule to serve as the nucleophile, rather than using a functional group of the enzyme itself. However, the overall process of peptide bond scission is essentially the same for all protease classes. Mechanism of action of the active site of: A) soluble serine proteases, B) cysteine proteases, C) aspartyl proteases, and D) metalloproteases. Erez et al. (2009)
8
between the residues of the catalytic triad is fundamental for the active site regulation, consequently loops’ orientation determines the protease activation or inactivation. Also the alignment of the residues that stabilize the negative charge on the intermediate reaction product is important, because it forms the oxyanion hole (Singh et al., 2011) (Figure 2).
DegP has been among the firsts serine-proteases to be identified in Escherichia
Coli: it code for a heat-shock protein deputed to homeostasis maintenance on the
periplasmic proteins carrying out chaperone and ATP-independent protease activities. The functional unit of DegP is a trimer, it has a funnel structure in which the proteasic domains are localized in the superior part while PDZ domains protrude to the outside. Two trimers can interact through their LA loops, so the specific composition of this region determine the degree of structuring of HTRA proteins (Clausen et al., 2002).
Figure 2 - A) Structure of HTRA1 trimer. Ribbon presentation of HTRA1 (inactive structure) in top and side views; monomers are shown in different colors. B) Structures of active and inactive HTRA1. The inhibitor bound to the active protease is not shown. The active-site residues are shown in stick mode, and functional loops are highlighted. Bottom, stereo presentation of the superimposed active and inactive HTRA1 conformations. C) Catalytic triads. Aligned catalytic triads of the active (yellow) and inactive (gray) HTRA1. The inactive conformation shows a distorted catalytic triad, with residue Ser328 being too distant from His220 (9.5Å) for proton transfer. Truebestein et al. (2011)
9
PDZ domains interact adjusting the access to the inner space, while every LA loop protrude into the active site of the opposite trimer, getting in contact with the active site’s loops L1 and L2. This triad blocks the entrance to the catalytic site, forcing the examer in an inactive state. The substrate binding and the eventual temperature increment induce strong conformational changes, with the consequent destabilization of the examer, the formation of bigger oligomers and the stabilization of the active site mediated by the interaction between PDZ1 and L3 (Singh et al., 2011).
The transition mechanism between the ordered-disordered states allow a reversible activation of the proteasic activity that guaranteed an immediate and flexible response to cellular stress (Wilken et al., 2004).
The proteolytic activity of DegP is irrelevant when the environment temperature is under 20°C, but it increases proportionally to the increment of the temperature. Therefore at low temperature it functions principally as chaperone, helping the protein folding; at high temperature the proteins are more damaged, so it acts prevalently as a protease (Singh et al. 2011; Clausen et al., 2002).
Until now, four members of the HTRA family have been identified in humans: HTRA1, HTRA2, HTRA3, and HTRA4. The human homologs can be divided into two group, mitochondrial HTRA2, which possesses a transmembrane anchor, and HTRA 1, 3, and 4, all contain predicted signal peptides as well as sections that are recognized as IGF binding and protease inhibitor domains. HTRA1 is the first identified members of the human HTRA protein family. It has a variety of targets most of which are extracellular matrix proteins such as type III collagen, fibronectin, and certain components of cartilage, and it is ubiquitously expressed in normal human adult tissues: appreciable levels are found in chondrocytes, endothelial cells, and fibroblasts (De Luca et al., 2003; Singh et al., 2011) (Figure 3).
10
Figure 3 – Schematic representation of the main HTRA family targets in the extracellular matrix and inside the cell (above), and in the mitochondria (below). Clausen et al. (2011)
11
1.1.1. HTRA1 Gene and Protein structure
HTRA1 gene is localized at chromosome 10q26.2. It consists of nine exons which
produce a transcript of 2138bp and encodes a 50 kDa polypeptide of 480 amino acid residues (Zurawa-Janicka et al., 2010; Singh et al., 2011) (Figure 4).
HTRA1 is a trimeric protein which resembles a flat disk. Each monomer is composed of:
- a signal peptide (SP) at the N-terminus, responsible for the addressing to the endoplasmic reticulum and to the secretory pathway;
- a domain with homology to Mac25 and insulin-like growth factor binding proteins (IGFBP);
- a Kazal type inhibitor motif: inhibitor of Serine proteases; - the evolutionarily conserved serine protease domain;
-a single PDZ domain at the C-terminal end, it is essential for the substrate binding and catalysis (Zurawa-Janicka et al., 2010) (Figure 5).
Figure 4 -Localization of the HTRA1 gene on the q branch of chromosome 10
12
The protease domain is made up of two β-barrel lobes (β1-β6 and β7-β12) in which the active site with the catalytic triad comprising His220, Asp250, and Ser328 is embedded. The structure of the active variant of HTRA1 (residues 158-480), comprising the serine protease and PDZ domains, has been solved with a covalently bound synthetic inhibitor peptide DPMFKLboroV to the active site serine residue via its boronic acid group. The binding of the inhibitor molecule led to the stabilization of the active site and proper positioning of the catalytic triad. The PDZ domain is dispensable for substrate binding and catalysis (Singh et al., 2011). HTRA1 activation implies strong conformational changes, allowing the formation of an activation pit, suitable for an efficient binding and for substrate’s
A
B
Figure 5 - A) Domain organization of HTRA1 gene. aa 1-22 Signal sequence, aa 22-100 Domain with homology to Mac25 and Insulin-like Growth Factor binding proteins (IGFBP), aa 101-155 Kazal type inhibitor motif, aa 204-364 Serine Protease domain, aa 365-467 PDZ domain. B) HTRA1 protein tridimensional structure in its funtional trimeric conformation. Truebestein et al. (2011)
13
catalysis, and an increased state of order of the protease domain (till 600 kDa) (Singh et al., 2011) (Figure 6).
Figure 6 - A) HTRA1 trimer with inhibitor. Ribbon presentation of the HTRA1 trimer in bottom view with bound inhibitor (blue). B) Electron density map of inhibitor. The active-site residues (green) and the inhibitor molecule (P1–P5) (blue) are shown in stick presentation. Residues in loop L3 forming hydrophobic interactions with the inhibitor molecule are shown in stick presentation (orange). C) Interactions of inhibitor with HTRA1. Schematic view of interactions between protein and inhibitor. (1) P4-Phe and P2-Leu form hydrophobic interactions with HTRA1; (2), (3) and (4) indicate hydrogen bonds to HTRA1 backbone; (5) indicates binding of the boronate to the active-site Ser328; (6) P1-Val points into S1 specificity pocket. Trubestein et al. (2011)
According to previous data on HTRA1 activation, Cabrera et al. (2017) proposed an allosteric mechanism where interactions between monomers alter protein conformation giving the active serine protease.
They performed in vitro simulations using the fully active and fully inactive HTRA1 variants. Importantly, they evidenced how the pattern and distribution of dynamic groups varies between the fully inactive and the fully active trimeric HTRA1.
Major rearrangements have place between the sites of the catalytic triad, two residues of the L2 loop, the LD loop and the L3 loop, leading the protein through five intermediate stages which end with the formation of the active site. They observed that Leu345 stands obstructing the entrance of the substrate to the
14
binding pocket. The dynamics of the L2 loop, and particularly Lys346, are key to initiate the conformational transition to remove Leu345 (Figure 7).
1.1.2. Protein Functions
HTRA1 is express during the embryonal development in those areas in which TGF-β proteins play important regulation processes. Specifically, high presence of HTRA1 has been revealed in cerebral tissue, suggesting a possible inhibitor role of this protein that could be direct or mediated by different targets related to the nervous system. The localization of HTRA1 in the developing nervous system could also indicate its possible function in neuronal migration, trophic support or plasticity (De Luca et al., 2004).
Figure 7 - Sequence of states in the activation process from computational simulation. The residues Ser328 (surface, red), His220 (surface, magenta), Asp250 (surface, orange), L2 loop residues Leu345 and Lys346 (surface, blue), the LD loop (purple) and the oxyanion hole forming loop (cyan) are highlighted. A) Fully inactive state that closely resembles the X-ray inactive structure. B) The L2 loop adopts an intermediate conformation. C) Leu345 and Lys346 have evolve to an active position from disorganized intermediate B. D) The catalytic triad and all the remaining elements are now aligned in an active configuration. Only the oxyanion hole-forming loop remains disordered and not functional. E) The structure is completely active and resembles the crystallographic active conformation. Loop L3 stabilizes to adopt a well-defined secondary structure. (Cabrera et al., 2017)
15
HTRA1 has also been found in the development of the placenta, resulting fundamental for the function of this structure (De Luca et al., 2004) such as in the development of heart and gonads, more precisely in the epithelial-mesenchymal transition (EMT), another process regulated by TGF-β family (Oka et al., 2004). Deregulation of the HTRA1 expression has been observed in numerous tumors and it is associated to chemotherapic resistance and to metastatic phenotype. Overexpression of HTRA1 can be used as a prognostic parameter for mesothelioma (Baldi et al., 2008); it can also be involved in lung cancer (Esposito et al., 2006) and in melanoma progression (Baldi et al., 2002). Negative regulation of the HTRA1 expression has been observed in ovarian and gastric tumor representing an additional factor in the chemoresistance mechanism (Chien et al., 2006).
Like other proteins related to specific tumors, HTRA1 has been reported as a microtubules associated protein (MAP) able to combine with microtubules network through its PDZ domain and regulate cellular motility functions (Chien et
al., 2006).
HTRA1 is a secreted protein essentially located in the extracellular space. Only a small percentage of the protein remains into the cells (Canfield et al., 2007), however several evidences show the presence of this protease in the cytoplasm. Clawson et al. (2008) have shown that processed forms of HTRA1 are found intracellularly and intranuclearly, and the active intranuclear form of HTRA1 shows a Mr ̴29000 (Clawson et al., 2008). Campioni et al. (2010) showed that
HTRA1, besides the fact that it is a protease known to cleave extracellular proteins such as fibronectin, collagen, or components of cartilage, acts as a protease also in the cytoplasm.
HTRA1 has been found in the synovial fluids of rheumatoid arthritis and osteoarthritis affected patients and among the astrocyte’s secreted proteins. Basing on this data, can be assumed that HTRA1 is able to operate on different substrates, whether they are growth factors or extracellular matrix (ECM) proteins, playing a key role both in physiologic and pathologic pathways (De Luca
et al., 2004). In fact, loss of activity of these proteins is correlated with severe
16
degradation of ECM compounds and to loss of chondrocytes) (Hu et al., 1998), cancer, familial ischemic cerebral small vessel disease, cerebral autosomal recessive arteriopathy with subcortical infarct and leukoencephalopathy (CARASIL), age-related macular degeneration, Parkinson’s disease and Alzheimer’s disease (Clausen et al., 2011).
Has been also found that HTRA1 has a role in the pathogenesis of tuberous sclerosis acting on an 1807-amino acid/220-kDa protein called TSC2 (Campioni et
al., 2010). Tuberous sclerosis complex (TSC) is a rare inheritable disorder
characterized by the development of hamartomas in the brain and in other vital organs such as kidneys, heart, eyes, lungs, and skin (Cheadle et al., 2000; Green et
al., 1994). Two tumor suppressor genes responsible for TSC have been identified: TSC1 located on chromosome 9q34 and TSC2 on chromosome 16p13, encoding
hamartin and tuberin, respectively. It has been elucidated that TSC1 and TSC2 physically interact to form an intracellular heterodimer (TSC1/TSC2 complex) participating in the control of cell growth and division. Mutations in TSC1 or TSC2 genes lead to uncontrolled cell cycle progression and tumor formation.
Campioni et al. in 2010, found that HTRA1 interacts with TSC2, but not with the other component TSC1. HTRA1 binds to TSC2 through the mac25 domain. Because this domain sometimes is cleaved off during HTRA1 activity, it is possible to suggest that this proteolytic event is important for HTRA1 function, as it could reduce the ability of HTRA1 to interact with other proteins (Figure 8).
17
TGF-β signalling is temporally and spatially regulated by a balance among maturation, sequestration and presentation (Dubois et al., 1995; ten Dijke et al., 2007). TGF-β is synthesized as a homodimeric proprotein (proTGF-β) and is subsequently cleaved into: 1) a N-terminal dimeric pro-peptide, latency-associated peptide (LAP) and 2) a C-terminal mature TGF-β by a pro-protein convertase, such as furin, in the trans-Golgi network. LAP forms a non-covalent complex with a dimer of mature TGF-β. This complex binds to a latent TGF-β-binding protein (LTBP), and the bound complex is then secreted and anchored to the extracellular matrix, resulting in sequestration of the mature TGF-β in the extracellular space. The sequestered mature TGF-β is activated by serine protease, matrix metalloproteinase or acidic microenvironments in the extracellular space (Annes
et al., 2003). The extracellular matrix, which stores TGF-β in a complex with LAP
and LTBP, also regulates the bioavailability of TGF-β (ten Dijke et al., 2007). The activation of mature TGF-β is the rate-limiting step for TGF-β signalling. The tight regulation of bioavailability of TGF-β in intracellular and extracellular spaces is important to regulate its signalling (Shiga et al., 2011).
Figure 8 - Schematic presentation of TSC pathway and the possible role of HTRA1. A) following the activation of the pathway, insulin receptor substrate, activated by insulin-like growth factor, recruits phosphoinositide 3-kinase converts phosphotidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5,-trisphosphate and activates the serine/threonine kinase Akt. Active Akt inhibits TSC2 activity through direct phosphorylation; thus, Akt-driven TSC1/TSC2 complex inactivation allows Rheb to accumulate in a GTP-bound state. Rheb-GTP then activates the protein kinase activity of MTOR include S6K and 4E-BP1. Once phosphorylated by mTOR, 4E-BP1 and S6K can initiate translation. B) Hypothetical role of HTRA1. Campioni et al. (2010)
18
Once this signalling is activated, it will initiate a cascade of phosphorylation and ubiquitination events. TGF-β family members exert their cellular effects by forming heterotetrameric complexes of type I and type II serine/threonine kinase receptors. In the complex, the type II receptor phosphorylates and activates the type I receptor, which thereafter phosphorylates downstream effectors of the Smad family (Feng et al., 2005; Massagué et al., 2005). The Smad family consists of eight members, which form three subfamilies; receptor-activated (R-)Smads (Smad2 and Smad3 are phosphorylated by TGF-β and activing receptors, and Smad1, Smad5, and Smad8 by BMP receptors), a single common-mediator (Co-)Smad (Smad4), and two inhibitory (I-(Co-)Smads (Smad6 and Smad7). After R-Smads have been phosphorylated in their C-terminals by type I receptors, they form oligomeric complexes with Smad4, which are translocated to the nucleus where they, in collaboration with other nuclear factors, regulate the expression of specific genes (ten Dijke, 2007). I-Smads are induced by Smad signalling and act in negative feedback control mechanisms (Itoh et al., 2000).
Although Smad pathways are of major importance in TGF-β signalling, there are examples of non-Smad pathways activated by TGF-β also. These pathways include Extracellular signal-regulated kinases (Erk), c-Jun N-terminal kinases (JNK), and p38 Mitogen-activated protein (MAP) kinases, phosphatidylinositol-3’-kinase, and Src kinase. (Moustakas et al., 2005). In addition, the ligand-induced receptor oligomerization brings together the E3-ligase TRAF6, recently found to have a fundamental role in TGF-β-induced apoptosis in prostate cancer cells. TRAF6 binds constitutively to a binding site in TβR-I. Ligand-dependent oligomerization leads to auto-ubiquitination of TRAF6. Active TRAF6 subsequently causes poly-ubiquitination of the MAP kinase kinase (MKK) kinase TAK1. Activated TAK1 then phosphorylates the p38 MAP kinase activators, MKK3 or MKK6. The Smad and TRAF6 pathways together lead to growth arrest, apoptosis and epithelial-mesenchymal transition (EMT) (Heldin, Landström and Moustakas, 2009) (Figure 9).
19
Figure 9 - Schematic presentation of TGF-β pathway highlighting the role of TGF- β receptor II (TβRII) in the phosphorylation of Phospho-Smad2/3 and p38/JNK. These phosphorylation events, together with subsequent interaction with other proteins (e.g. Smad4), end in growth arrest, epithelial to mesenchymal transition or apoptosis of the cell.
Secreted HTRA1 may binds to ECM around the cell through interactions with the PDZ domain, becoming fully active and allowing the interaction of HTRA1 linker region and serine protease domain with TGF-β proteins. Hence the binding and inhibitory activities of HTRA1 on TGF-β signalling depend on the integrity of
HTRA1 serine protease domain, while the PDZ domain seems to be not required for
these functions (Oka et al.; 2003). For the downregulation mechanism of TGF-ß signalling by HTRA1, it has been proposed that HTRA1 cleaves the pro-domain of proTGF-ß1 in the endoplasmic reticulum before furin process it in the trans-Golgi network (Shiga et al., 2011). The aberrant cleaved products of proTGF-ß1 are degraded by the endoplasmic reticulum-associated degradation system, leading to a reduced amount of mature TGF-ß1. In contrast, it has been reported that HTRA1 cleaves mature TGF-ß1 or TGF-ß1 receptors in extracellular space (Oka et al., 2004; Launay et al., 2008; Graham et al., 2013). However, all results concerning the downregulation of TGF-ß signalling by HTRA1 were obtained in overexpression conditions; thus, the downregulation mechanism under physiological conditions is
20
still unclear. In physiological conditions, HTRA1 interacts with pro-TGF-ß in the intracellular space, rather than with secreted mature TGF-β. The protease cut LAP in the endoplasmic reticulum, thus the products are degraded through Endoplasmic-Reticulum-Associated protein Degradation (ERAD), a control mechanism which allow to steer the unfolded proteins to the cytoplasm where they are digested by the proteasome (Shiga et al., 2011).
The regulation mechanism would be similar to that of Emiline1 that binds the pro-TGF-ß inhibiting processing by furin (Zacchigna et al., 2006) (Figure 10).
Figure 10 – Model of regulation of TGF-β synthesis by HTRA1. A) TGF-β1 is synthesized as a pre-pro-protein with signal peptide (grey), which undergoes proteolytic processing in the ER. B) The pro TGF-β1 is then clave by furin convertase (black arrowheads) in the trans-Golgi network (TGN). The cleaved products yield a small latent TGF-β complex, in which the latency associated peptide (LAP; orange) and the mature peptide (red) are connected by non-covalent binds. C) The LAP- TGF-β1 complex is secreted into the extracellular space and anchored in the extracellular matrix. Thrombospondin-1 (THBS1), matrix metalloproteinase (MMP) and serine protease can cleave LAP (black arrowheads) to release the mature TGF-β. D) HTRA1 cleaves pro- TGF-β1 in the ER. The cleaved products are degraded by ERAD, resulting in the reduction of mature TGF-β. Shiga et al. (2011)
21
1.2 CEREBRAL SMALL VESSEL DISEASES
Cerebral Small Vessel Diseases (CSVDs) are a major disease burden in most developed countries. The term CSVD refers to a group of pathological processes with various aetiologies that affects the cerebral small arteries, arterioles, venules, and capillaries, which altogether result in damage of the white and deep grey matter of the brain. At the moment is not possible to visualize clearly small vessels but it is possible to track the effects of their malfunctions in the brain through imaging techniques, especially with magnetic resonance imaging (MRI). The most common morphologic findings on brain MRI associated with CSVD include white matter hyperintensities (WMH, leukoaraiosis), small subcortical infarcts, and micro-bleeds.
In most cases, CSVD is sporadic, with age, hypertension, dyslipidemia, and diabetes mellitus representing the prevailing risk factors. The main clinical manifestations are motor slowing and loss of the cognitive functions’ control, caused principally by the presence of ischemic lesions, which break off pre-frontal and subcortical circuits.
Many hereditary or idiopathic CSVDs also have been identified. Among these, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL, OMIM 125310), the most common single-gene disorder of the cerebral small arteries with more than 500 families reported caused by NOTCH3 mutations, inherited cerebral amyloid angiopathies associated with mutations of COL4A1 and COL4A2, CARASIL, retinal vasculopathy with cerebral leukodystrophy (RVCL, OMIM 192315) and Fabry disease (FD, OMIM 301500) (Joutel et al., 1996; Gould et al., 2006; Revesz et al., 2009; Federico et al. 2012) (Table1).
22
Table 1 – Inherited small vessel diseases. Federico et al. (2012)
However, molecular screening of those genes in routine diagnosis identifies the causative mutation in <20% of patients referred for a familial CSVD, which strongly suggests that other genes may be involved (Verdura et al., 2015).
1.2.1. CARASIL
CARASIL is a rare form of inherited CSVDs. CARASIL is a very rare disease whose prevalence has not been defined yet. Only 14 HTRA1 mutated CARASIL families have been reported so far and the majority of affected patients have been reported in Japan. However, in the last years have been identified patients from different ethnicities, ranging from the Middle-East to Eastern Europe. This disease affects predominantly males, with a male:female ratio of 3:1, and 50% of patients have consanguineous parents (Fukutake, 2011).
Clinical features
In 1976 Maeda et al. reported familial unusual encephalopathy without hypertension in siblings whose parents were consanguineous. They showed early adult-onset of dementia, pseudobulbar palsy, and pyramidal and extrapyramidal
23
symptoms. Post-mortem studies revealed diffuse and focal demyelination with sparing of U-fibers, multiple small foci of perivascular softening in the cerebral small arteries and long arteries in the cerebral white matter. The other characteristic features were sever lumbago and alopecia during the teenage years (Maeda et al., 1976). In 1995, Fukutake proposed new disease criteria to define CARASIL summarized in the clinic triad leukoencephalopathy, alopecia and lumbago.
The main clinical manifestation of CARASIL are recurrent stroke or stepwise deterioration of motor ability. Cognitive deficits are seen in almost all patients who developed dementia by age 30-40 years. These deficits are distinct from those of cortical dementia (e.g. Alzheimer’s disease) and subcortical dementia (e.g. Huntington’s disease), and thus can be categorized as white matter dementia. CARASIL is characterized by several extraneurological symptoms that, combined with its earlier onset, make it differs from CADASIL. These systemic symptoms comprehend arteriopathy, acute lumbago or spondylosis deformans, which is present in approximately 80% of patients with CARASIL, and alopecia, found in 90% of patients. Alopecia is the most common initial symptom and occurs as early as adolescence. (Fukutake, 2011; Federico et al., 2012). Mood changes (apathy and irritability), pseudobulbar palsy, hyper-reflexia, Babinski sign, and urinary incontinence are frequently observed. An acute ischemic stroke event has been reported in 23,1% of patients, and no haemorrhagic stroke events have been reported. The patients do not have hypertension or diabetes mellitus, which are the major risk factors for sporadic CSVD (Nozaki et al., 2014).
Neuroradiological findings
All patients show white matter changes and around 50% of them presents also small foci with softening. The most characteristic brain MRI findings in CARASIL patients are white matter high-signal intensity (HSI) lesions and multiple lacunar infarctions in the basal ganglia and thalamus. HSI on T2-weighted or fluid-attenuation inversion recovery images is symmetrically distributed and located more often in the periventricular and in the subcortical regions than in the
24
superficial white matter (U-fibers) part. These lesions initially appears in the subcortical white matter by age 20, and then they extend into the basal ganglia, thalamus, brainstem, and cerebellum. This suggest that the white matter changes precede the onset of neurologic symptoms.
Involvement of the anterior temporal lobes and external capsules is a common feature with CADASIL. MRI changes progression appear to be different in CARASIL and CADASIL patients. In CARASIL, it seems to spread homogenously starting from subcortical white matter while in CADASIL it shows a punctiform or a nodular initial stage that later produce confluent lesion. Hypo-perfusion in the frontal lobe and other parts of the cerebrum has been verified through single-photon emission computed tomography (Fukutake, 2011; Nozaki et al., 2014).
Pathology
CARASIL, from a histopathological point of view, is characterized by intense arteriosclerosis prevalently in the small penetrating arteries, without granular osmiophilic materials or amyloid deposition (Nozaki et al., 2014).
In the cerebral small arteries, smooth muscle cells are extensively lost, even in arteries without sclerotic changes. Arteriosclerotic changes are limited in cerebral small arteries and not in intracranial large arteries and extra-cranial arteries. These changes lead to an increased artery dimension rather than to the development of stenosis. Tunica media of the cerebral small arteries presents hyalinosis and is immunopositive for fibrinogen. These changes might disturb auto-regulatory mechanisms for cerebral blood flow, resulting in ischemic changes in the deep white matter. The internal elastic membrane is split into multiple layers and fragmented. Some intima is thickened with fibrosis and involves myointimal cells. Arterial adventitia is thin and its immunoreactivity for type I, type III, and type VI collagen (Fukutake, 2011; Nozaki et al., 2014).
25
1.3. HTRA1 MUTATIONS
In 2009, Hara et al. with a linkage analysis on five families with CARASIL identified a linkage of the disease to the 2.4 Mb region on chromosome 10q, which contains the HTRA1 gene (Hara et al., 2009).
To date, 15 mutations in HTRA1 gene have been identified (Table 2). They include 12 missense mutations, 2 nonsense mutations and 1 deletion mutation (Figure 11). All the missense mutations were located in or around the protease domain of
HTRA1, causing the reduction in the protease activity.
Table 2 - List of the identified HTRA1 mutations in both Asian and Caucasian patients
Mutation type Nucleotide Protein Ethnicity Publication
Far-Eastern patients
homoz, ms c.754G>A p.A252T Japanese Hara et al. (2009) homoz, ms c.889G>A p.V297M Japanese Hara et al. (2009) homoz, ns c.904C>T p.R302X Japanese Hara et al. (2009) homoz, ns c.1108C>T p.R370X Japanese Hara et al. (2009) homoz, ms c.821G>A p.R274Q Japanese Nishimoto et al. (2011) homoz, ms c.854C>T p.P285L Chinese Chen et al.(2013) homoz, ms c.1091T>C p.L364P Chinese Wang et al. (2012) homoz, ms c.161_162insAG p.G56Afs*160 Chinese Cai et al. (2015) compound
heteroz
c.958G>A c.1021G>A
p.D320N
p.G341R Chinese Fei Xie et al. (2018)
Caucasian patients
homoz, ms c.517G>A p.A173T Pakistani Menezes Cordeiro et
al.(2015)
homoz, ms c.496C>T p.R166C Portuguese Khaleeli et al.(2015) homoz, ms c.883G>A p.G295R Spanish Mendioroz et al.(2010) homoz, ms c.1108C>T p.R370X Turkish Bayraki et al.(2014) compound
heteroz
c.126delG c.961G>A
p.E42Dfs*173
p.A321T Romanian Bianchi et al. (2014)
26
Figure 11 - Distribution of HTRA1 mutations. HTRA1 gene consists of 9 exons (squares): those encoding the insulin-like growth factor-binding protein domain (red; 22-100 aa); the Kazal-type serine protease inhibitor domain (blue; 101-155 aa); the trypsin-like serine protease domain (orange, 204-364 aa); and the PDZ domain (green; 382-473 aa). All individuals are homozygotes for missense or nonsense mutations, except for the patient with p.[Glu42fs];[Ala321Thr]. Nozaki et al. (2014)
Nozaki et al. (2014) found that the disease-associated mutant HTRA1, p.A252T, p.R274Q, p.V297M and p.R302X, decrease their protease activity of 21-50% than wild type HTRA1 protein (Nozaki et al., 2014).
The nonsense and deletion mutations cause the premature termination codons, particularly the p.R730X leads to mRNA degradation by nonsense mediated decay, a surveillance pathway that reduce errors in gene expression by eliminating mRNA transcripts that contain premature stop codons.
It has been reported that CARASIL-associated mutant HTRA1s exhibit decreased protease activity and fail to decrease TGF-β family signalling. Hara et al. (2009) showed that the mutant variant of HTRA1 with missense amino acids (p.A252T and p.V297M) lose the ability to repress signalling by the TGF-ß family members BMP-4 and BMP-2 as well as subsequent phosphorylation of SMAD proteins (downstream effectors of the TGF-ß family signalling pathway). Moreover, the same authors demonstrated that in fibroblasts from subject with CARASIL carrying p.Arg370X mutation, TGF-ß signalling was increased three times that in fibroblasts from control subject. Moreover, the ED-A fibronectin and versican, which are induced by increased TGF-β signalling, accumulate in the hypertrophic intima of cerebral small arteries in patients with CARASIL. TGF-β is also increased in the cerebral small arteries of patients with CARASIL (Hara et al., 2009). These findings indicate that increased TGF-β signalling plays a pivotal role in the pathogenesis of cerebral small-vessel disease in CARASIL, however, the precise molecular mechanism by which HTRA1 decreased TGF-ß signalling remains unknown.
27
Through a in vivo and in vitro experiments performed on zebrafish embryos, Kim et
al. (2012) demonstrated the involvement of the HTRA1 protease in modulating
crucial cellular processes and in life-threatening diseases. Examining the expression levels of several FGF and BMP family genes, which are known to be involved in dorso-ventral patterning of zebrafish gastrula (zFGF), they found increased levels of zFGF family in knockdown of zebrafish HTRA1 (zHTRA1), although no alteration in the expression of BMP family genes was noted. In particular, the authors showed an expression of zFGF8 3-fold higher than that of other zFGFs. Moreover in the zHTRA1 embryos they showed increased levels of activated ERK (phosphorylated ERK), protein downstream effector of FGF signalling. Based on these findings, Kim et al. (2012) proposed that the levels of
zFGF expression might be negatively regulated by zHTRA1 at a
post-transcriptional level. Finally, they suggested that in dorso-ventral patterning in zebrafish embryos, HTRA1 acts as a key protease in the regulation of the FGF signalling pathway via cleavage of FGF8 in the extracellular region (Figure 12).
Figure 12 – FGF8 positively regulates FGF8 expression through an FGF signaling feedback loop. A) Working model of the FGF8-mediated positive feedback loop of the FGF signaling cascade. FGF8 binds to FGFR in the extracellular region and thereby activates the mitogen-activated protein kinase (MAPK) cascade. Notably, the FGF8 gene is transcriptionally activated via FGF signaling. The newly synthesized FGF8 protein is secreted into the extracellular region. B) Working model of HTRA1 as a novel antagonist in the FGF8-mediated positive feedback loop of FGF signaling. HTRA1 cleaves FGF8 in the extracellular region, which may be required to maintain FGF8 and FGF signaling at a constant level. Kim et al. (2011)
In a recent study, Beaufort et al. (2014) showed that a loss of activity by HTRA1 results in reduced TGF-β signalling. They performed studies on a knockout mouse model that generates a truncated transcript lacking exons 2-9 demonstrating, in brains of young and aged HTRA1 knockout animals, a significant decrease of
28
SMAD2/3 phosphorylation, an intracellular indicator of TGF-β activity, and a ∼50% reduction of the mRNA levels of connective tissue growth factor (CTGF). To analyse the TGF-β pathway, the same authors quantified TGF-β1 in immortalized wild-type and HTRA1-deficient mouse embryonic fibroblast (MEF) culture medium and unexpectedly observed a ∼40% reduction of TGF- β1.
To confirm these findings in human cells, Beaufort et al. (2014) used skin fibroblasts from a CARASIL patient of Pakistani origin. In culture medium of patient fibroblasts, both total and bioactive TGF-β levels were decreased by ∼80% in comparison with cells from a control individual. These findings, on the contrary respect to previous data reported by Hara et al. (2009), suggesting a facilitating role of HTRA1 in TGF-β signalling.
From data reported, it is difficult to assess the exact role played by HTRA1 on TGF-β signalling regulation and at the moment the CARASIL patogenic mechanism remains still unclear.
CARASIL was initially reported in siblings from a consanguineous family with unaffected parents. Interestingly, in three CARASIL families, cerebral MRI performed in either one or both parents was reported to show a leukoencephalopathy, raising the question of the causality of the heterozygous variants carried by these parents (Bianchi et al., 2014). Recently, Verdura et al. (2015), reported a surprising finding showing a pathogenic role of heterozygous mutations in HTRA1, suggesting that 5% of familial SVD of unknown aetiology are associated with heterozygous HTRA1 mutations. Verdura et al. (2015) used whole exome sequencing to identify candidate genes in an autosomal dominant CSVD family in which known small vessel disease genes had been excluded. The authors screened all candidate genes in 201 unrelated probands with a familial small vessel disease of unknown aetiology. A heterozygous HTRA1 variant (p.R166L), predict to be deleterious by in silico tools, was identified in all affected members of the index family. Ten probands of 201 additional unrelated and affected probands harboured a heterozygous HTRA1 mutation predicted to be damaging. Seven of these variants were located within or close to the HTRA1 protease domain, three were in the N-terminal domain of unknown function and one in the C-terminal PDZ domain. They also demonstrated a loss of function effect in HTRA1 mutants. Nozaki et al. (2016) screened 113 unrelated index patients with severe
29
leukoaraiosis who were younger than 70 years, they found 4 heterozygous missense mutations in HTRA1 gene in 6 cases; these mutations were located in the serine protease domain. Three mutations, p.G283E, p.R302Q, and p.T319I, were novel and p.P285L was reported as homozygous in one patient with CARASIL and heterozygous in the 65-year-old father of a proband with severe leukoaraiosis (Nozaki et al., 2016) (Figure 13).
More recently, Di Donato et al. (2017) analysed 142 NOTCH3-negative patients and they detected five different HTRA1 heterozygous mutations in nine subjects from five unrelated families. They observed that the pathogenicity of these HTRA1 variants is supported by several observations, e.g. located in highly conserved positions, absent in control subjects, absent or rare in databases, predicted to be deleterious by four out of five in silico tools. These data strongly suggest that in some cases HTRA1 mutations behave as autosomal recessive mutations, with heterozygous carriers being clinically unaffected. In other cases, HTRA1 mutations behave as dominant mutations, with heterozygous carriers being clinically affected.
Clinical features of autosomal dominant CSVD linked to HTRA1 heterozygous mutations differ from those of CARASIL by a later age at onset of stroke and cognitive decline, a milder phenotype, a long-lasting history of illness and the absence of the typical CARASIL extra-neurological symptoms (Verdura et al., 2015; Di Donato et al., 2017). However, Nozaki et al. (2016) reported presence of clinical
Figure 13 - Missense mutations in patients with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) or severe leukoaraiosis. The green box shows the protease domain of the
HTRA1 gene. Red highlighted mutations were studied by Nozaki et al. (2016). Mutations shown in black and p.P285L
30
hallmarks of CARASIL, such as spondylosis deformans, in all cases. Alopecia was detected in three patients in the frontal area, as observed in CARASIL.
Moreover, Di Donato et al. (2017) reported no significant clinical differences with respect to CADASIL except for the age at onset, usually earlier in CADASIL. Compared to common sporadic CSVD, the main differentiating feature are usually younger age at onset with a presenile pattern, the familial recurrence, and the substantial lack of correlation of clinical and MRI phenotype with vascular risk factors which may be occasionally present (Di Donato et al., 2017).
Differences in biochemical characteristics of heterozygous HTRA1 mutant were reported in two different works. Verdura et al. (2015) described that most of the analysed heterozygous HTRA1 mutants showed a loss-of-function, only two mutants revealed residual activity. Nozaki et al. (2016) demonstrated that mutant HTRA1s associated with CSVDs had dominant-negative effects on wild type HTRA1 protease activity, otherwise non-manifesting heterozygotes subjects did not show differences in the alteration of the proteolytic activities (Figure 14). These data indicated that a different localization of the mutation on distinct domains of HTRA1 could affect differently the protease activity.
Figure 14 – A) Protease assay for HTRA1 proteins. Fluorescein isothiocyanate (FITC)–labeled casein was incubated
with human HTRA1 proteins. The black line indicates wild-type HTRA1; dashed yellow line: S328A, which abolished the protease activity; blue lines: HTRA1 mutants associated with CARASIL; red lines: HTRA1 mutants found in patients with cerebral small vessel disease (CSVD) in the heterozygote state. B) Protease assay for HTRA1 protein complexes. FITC-labeled casein was incubated with human HTRA1 protein complexes. The dashed yellow line indicates the complex consisting of wild-type and S328A HTRA1; blue lines: the complex consisting of wild-type and each mutant HTRA1 associated with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy; red lines: the complex consisting of wild-type and each mutant HTRA1 found in patients with CSVD in the heterozygous state (Nozaki et al., 2016).
31
2. AIMS
The pathogenic mechanism linking HTRA1 mutations and TGF-ß dysregulation in CARASIL disease is still not fully understood. Recent data showed a role of HTRA1 heterozygous mutations in CSVD, however which variants truly contribute CSVD pathogenesis and how HTRA1 mutants cause CSVD in heterozygous individuals is still unclear. Therefore, many questions about the HTRA1 mutation behavior as autosomal recessive or dominant mutation remain unsolved.
On these bases, the aim of this work was to obtain further data about the effect of heterozygous HTRA1 mutations. Therefore, we planned to study the expression profile of HTRA1 and proteins related to TGF-β signaling pathway both in human fibroblasts from subjects carrying heterozygous HTRA1 variations and in mouse embryonic fibroblasts (MEF) harboring HTRA -R274Q mutation.
Moreover, using a polycistronic vector to transfect HEK cells, we investigated the potential dominant negative effect of an array of HTRA1 mutants.
Further, in aged heterozygous and homozygous mice models carrying HTRA1-R274Q mutation we analyzed brain small vessels to evaluate the presence of accumulation of fibronectin protein, which is the extracellular matrix substrate of HTRA1.
Finally, in this study we investigated on the possibility to perform a “rescue strategy” in cells harboringHTRA1-R274Q mutation. We tried to restore HTRA1
protease activity in MEF harboring HTRA-R274Q mutation, known to impede the HTRA1 trimer formation.
The cell lysate and culture medium of cultured human fibroblasts, MEF, HEK293T, and homogenized mice tissues were used to analyze proteins, mRNA expression and brain pathology, by using Western blot analysis, Reverse Transcriptase– quantitative PCR analysis (RT-qPCR), and Immunofluorescence analysis.
32
3. MATERIAL AND METHODS
3.1. PATIENTS
We analysed four subjects harbouring heterozygous HTRA1 mutations, one HTRA1 compound heterozygous CARASIL patient and one healthy age-matchedcontrol subject. Demographic, clinic and molecular parameters of the subjects analysed are shown in Table 3.
1- CARASIL patient (FA). She is a 29-year-old Romanian female with unrelated parents, carrying two heterozygous HTRA1 mutation: the c.961G>A in exon 4, inherited from the father, and a G deletion (c.126delG), inherited from the mother, in exon 1. The missense mutation in exon 4 results in substitution of highly conserved alanine residue with threonine (p.A321T). The deletion in exon 1 causes a frameshift, altering the aminoacidic sequence from position 42 (p.E42Dfs*173), and introduces a stop codon at position 214.
Clinical feature of the patient is characterized by neurologic signs such as ataxic gait, gaze-evoked nystagmus, dysmetria, hypoactive deep tendon reflexes at legs, and no alopecia. She complained of chronic lumbar and cervical pain since the age of 14 years. She had two ischemic strokes with left hemiparesis and dysarthria at ages 24 and 29, respectively. Diffuse leukoencephalopathy including subcortical infarcts and evidence of microbleeds have been revealed by brain MRI, while spine MRI showed degenerative disc disease. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) has been ruled out by automatic sequencing of exons 2-24 of the NOTCH3 gene and no evidence of granular osmiophilic material in skin and muscle biopsy have been revealed.
33
2- Parents of the CARASIL patient (FM, FG). They are not consanguineous and are neurologically normal: the father shows the heterozygous missense HTRA1 mutation c.961G>A (p.A321T) in exon 4 and the mother shows the heterozygous HTRA1 mutation G deletion (c.126delG, p.E42Dfs*173) in exon 1. The 62 year-old father had negative history for neurologic and cardiovascular diseases and hypertension; the mother, aged 61 years, reported mild hypertension under treatment since the age of 54 years without other vascular risk factors. After genetic diagnosis of the daughter, the parents underwent brain MRI. The father showed mild supratentorial leukoencephalopathy and the mother diffuse infratentorial and supratentorial leukoencephalopathy.
3- SVD patients (FV, CC). FV is a 69 years-old male not related to the previous family, carrying heterozygous HTRA1 mutation c.883G>A in exon 4 that results in substitution of glycine residue with arginine (p.G295R). The patient presented a progressive reduction of cognitive performances with limitation of his daily activities, mood depression, and progressive motor impairment with speech difficulties (anarthria), history of lumbar spondylosis was reported. Diffuse leukoencephalopathy was evident at MRI. Leukoencephalopathy, lumbago and dementia were also found in three siblings and in his son. CC is a 60 year-old male who has had progressive gait disturbance for 1 year, speech disorder for some months and widespread leukoencephalopathy, white matter hyperintensity, brainstem lesions, microbleeds and status cribosum at brain CT scan. He was tested with neuropsychological tests that uncovered a relevant cognitive impairment. The patient harboured a novel heterozygous missense variant c.451C>A (p.Q151K) in exon 1, resulting in an aminoacid change from glycine to lysine. He had no vascular risk factor, nor juvenile alopecia, spondylosis or low back pain. In his family history, his mother and maternal uncle did present the same clinical pattern. Neurological examination showed ataxic gait with the need of support, dysphagia, and dysarthria.
34
All patients and healthy controls provided written informed consent before participation in the study.
3.2. HUMAN FIBROBLASTS
Primary cultures of skin fibroblasts from CARASIL patient, her parents, SVD patients and control subject were incubated for 15 minutes with trypsin at 37°C, then the fibroblasts were grown in 75 cm2 plastic flasks with 1 ml of Dulbecco’s
modified Eagle medium (DMEM) supplemented with 10% Fetal Calf Serum (FCS), 1% L-glutamine and 1% streptomycin-penicillin (100 IU/m and 100 µg/ml respectively). Flasks were maintained overnight at 37°C in a humidified atmosphere containing 5% CO2. Finally 5 mL of fresh DMEM were added to the
flasks and the cell were left to grow in standard conditions.
Analysis of sub-confluent fibroblasts cultures were carried out after 5-15 replications. At this time cells were rinsed with culture medium without FCS, then they were treated with 2 cc of trypsin. Detached cells were harvested in complete medium.
Patients Age
of Onset
Sex Leukoencephalopathy Cognitive Deficit
Gait
Disturbances Other HTRA1 Mutations
FA 23 F Yes - Yes Emiparesis Disarthria c.126delG c.961G>A
p.A321T p.E42Dfs*17 3
FM 61 F Yes - - - c.126delG p.E42Dfs*17
3
FG 62 M Yes - - - c.961G>A p.A321T
FV 65 M Yes Yes - Anarthria c.883G>A p.G295R
CC 60 M Yes Yes Yes Dysarthria, Dysphagia c.451C>A p.Q151K Table 3 – Clinical, genetic, and demographic data of HTRA1 mutation carriers.
35
Preparation Fibroblasts-Derived Samples
- Cells Lysate Fibroblasts harvested in culture media were separated by
centrifugation at 1,000 x g for 5 min. Fibroblasts were washed once in cold PBS, and lysed on ice with Mammalian Protein Extraction Reagent (M-PER) and Complete Mini Anti-protease Cocktail tablets (ThermoScientific) according to the manufacturer's instructions. Lysates were centrifuged for 20 min at 16,500 x g to remove small cell debris. The supernatant were harvested, aliquoted and stored at -80°C until analysis.
- Cells Culture medium were concentrated 10-fold by Amicon Ultra-15 PL
10K centrifugal filters for 4 min at 2,000 x g. Concentrated media were aliquoted and stored at -80°C until analysis.
Protein concentration in cell lysate and concentrated media were performed by Lowry-Folin colorimetric method (Lowry et al., 1951).
- Cell lysates RNA. After washing with PBS, human fibroblasts were lysed in
RLT buffer (QIAGEN) containing ß-mercaptoethanol (10µl/ml) in order to inactivate RNAses in the lysate. Total RNA was processed immediately using RNeasy Mini Kit (74104, QIAGEN), following the manufacturer's instruction. RNA was treated with DNAseI for 15 minutes at room temperature (RT) (79254, Qiagen), concentration and purity of total RNA samples were quantified using the spectrophotometer NanoDrop-1000 (Thermo Scientific).
3.3. MICE MODELS
Heterozygous mice for the HTRA1 mutation R274Q (HTRA1-R274Q) were generated with CRISPR/Cas9 genome editing by Dr. B. Wefer (Wefer et al., 2017) from Pr. Wurst’s workgroup at the German Center for Neurodegenerative Diseases (DZNE) in Munich. CRISPR is an abbreviation of Clustered Regularly Interspaced Short Palindromic Repeats. CRISPR/Cas9 technology derive from the adaptive immunity of Streptococcus Pyogenes where it is needed to arrest viral proliferation through genome cleavage. This activity was adapted to target a
36
specific sequence using a custom single guide RNA (sgRNA) which is able to direct the CRISPR-associated (Cas) nuclease to the target DNA site by base-pairing, resulting in Cas9-generated double strand breaks.
Mice were kept under standard conditions with food and water ad libitum. Animal experiments were performed in compliance with the German Animal Welfare Law and the Government of Upper Bavaria.
Mice were bred to obtain:
- Embryos with genotype wt/wt, wt/mut and mut/mut;
- 6 months old animals with genotype wt/wt, wt/mut and mut/mut.
Mouse embryonic fibroblasts (MEF) were isolated from 12.5-day post coitus embryos. No obvious morphological defect was detected and embryos had similar weights. Upon removal of the head and internal organs, embryonic tissue was chopped and seeded in petri dishes with Dulbecco´s modified Eagle´s medium (DMEM) containing GlutaMAX, 10% (vol/vol) FBS, 100U/ml penicillin (Invitrogen) and 100 µg/ml streptomycin (Invitrogen). After 24h, fibroblasts started to proliferate and were transferred to 25 cm2 flasks. Once they reached confluence
they were transferred to 75 cm2 flasks and genotyping of cell lines was performed.
Genotype was assessed upon RNA extraction performed using RNeasy minikit (BIO-RAD). cDNA was synthetized using Omniscript Reverse-Transcription kit (Qiagen) according to manufacturer’s instruction using primers upstream and downstream exon 4 of HTRA1. Genotype was determined using a new restriction site for PstI formed with the insertion of HTRA1-R274Q mutation.
Cell lines were subdivided in wt/wt (WT), wt/mut (HET) and mut/mut (MUT). Cell lines were kept under a humidified atmosphere with 5% CO2 at 37°C. Culture lines
were subsequently immortalized by serial re-plating. Before, during and after the immortalization process, no obvious morphological or growth differences were observed among cell lines.
37
Preparation of MEF-Derived Samples
- Cell Culture Media. Cells were grown to high-density cultures, and then
maintained in culture for 24 or 72 h in FBS-free DMEM. Medium was collected and centrifuged 10 min at 400 x g, then it was concentrated 20-fold using Amicon centrifugal units (10 kDa cutoff; Millipore). Total protein concentration was determined using Bicinchoninic Acid (BCA) assay (Pierce);
- Cell lysates Proteins. After washing with PBS, MEF cells were lysed in
RIPA buffer (50 mM Tris-HCl pH 7,6, 150 mM NaCl, 1% Nonidet P-40, 0,1% SDS, 0,5% Na-deoxycholate) containing a mixture of protease and phosphatase inhibitors (Roche). Lysates were centrifuged for 20 min at 16,500 x g to remove small cell debris, aliquoted and stored at -20 °C. Total protein concentration was determined using the BCA assay (Pierce);
- Cell lysates RNA. After washing with PBS, MEF were lysed in RLT buffer
(QIAGEN) containing ß-mercaptoethanol (10µl/ml) in order to inactivate RNAses in the lysate. Total RNA was processed immediately using RNeasy Mini Kit (74104, QIAGEN), following the manufacturer's instruction. RNA was treated with DNAse I for 15 minutes at RT (79254, Qiagen). The concentration and purity of total RNA samples were quantified using the spectrophotometer NanoDrop-1000 (Thermo Scientific).
3.4. MICE TISSUES
Brains from perfused mice models were homogenized in a TissueLyser LT (Qiagen) and lysed with 50 mM Tris pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% SDS, 50 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4 supplemented with a mixture of
protease inhibitors (Roche). Proteins and RNA contents were extract as explained in “Preparation of fibroblasts-derived samples” paragraph.
Brains tissue were maintained at 4°C and, after inclusion in paraffin they were sliced in sections of 7µm thickness, placed on slides and stored at -20°C until use.
38
3.5. PLASMID CLONING
Cloning protocol was designed using NheI and NotI restriction sites, respectively on multiple cloning site (MCS) A and B, to insert in a polycistronic vector (pIRES) an array of HTRA1 mutants. pIRES is a mammalian expression vector that allows high level expression of two genes of interest from the same bicistronic mRNA transcript. The vector contains the encephalomyocarditis virus (ECMV) internal ribosome entry site (IRES) flanked by two multiple cloning sites (MCS A and B). Expression of the bicistronic transcript is driven by the constitutively active cytomegalovirus immediate early promoter (PCMV IE), located upstream of MCS A. An intervening sequence (IVS) known to enhance the stability of mRNA is located between PCMV IE and MCS A, and is efficiently spliced out following transcription. SV40 polyadenylation signals downstream of MCS B direct proper processing of the 3’ end of the mRNA. The vector contains an ampicillin resistance gene (Ampr), and a ColE1 origin of replication for selection and propagation in E. Coli. HTRA1-WT and a set of HTRA1-variants were inserted in MCS A and MCS B, respectively. Obtained pIRES vector (pIRESWT/ R166L, pIRESWT/ A173P, pIRESWT/H185R, pIRESWT/S284R) was then transfected in HEK293T cells and culture media were analysed by Western Blot, to detect HTRA1, and by FITC-casein degradation, to analyse the activity of the different constructs. pIRESWT/-, and pIRES-/- vectors were used as negative controls.
Inserts were obtained with Quick change lightning site directed mutagenesis kit (Agilent) in order to express HTRA1 variants as follow (Table 4):
p.R166L p.A173P p.H185R p.R284S Relevance late onset SVD late onset SVD late onset SVD late onset SVD Multimeric status Monomer Monomer Trimer Trimer
Table 4 – List of HTRA1 variants inserted in MCS B of different pIRES vectors. These variants were found in subjects with SVD.
39
PCR reactions were performed to amplify HTRA1-variant inserts. Primers were designed to hybridize DNA region surrounding HTRA1 using Agilent primer design program. (http://www.genomics.agilent.com/primerDesignProgram.jsp).
PCR reactions (50 µl) containing 10 µl of 5X Reaction Buffer (Sigma Aldrich), 100 ng DNA (in 1 µl H2O), 1 µl of Forward and 1 µl of Reverse primers diluted 1:10 in
H2O, 1 µl Accu Polymerase for GC-rich regions (Sigma Aldrich), completed to 50 µl
with deionized H2O (ddH2O). PCR were performed using the following cycling
conditions: an initial 5 min incubation at 95°C, followed by 35 cycles of 95°C for 30’’, 55°C for 30’’, 1 min 40’’ at 72°C and 10 min at 72°C. PCR-clean was performed using High Pure PCR Product Purification kit (Sigma-Aldrich) according to manufacturer’s instruction. After PCR-clean, the DNA digestion of insert and vector was performed. Reaction mix was prepared with 1µg of DNA (insert or vector), 1µl NheI (for MCS A) or NotI (for MCS B) restriction enzymes, 2 µl BSA diluted 1:10 in H2O, 2 µl Cutsmart 10x (BioLabs), and completed to 20 µl with ddH2O. Reaction
mix was incubated for 2h at 37°C.
In vector DNA digestion, a dephosphorylation passage is required. Vector dephosphorylation mix was prepared with 20 µl of digested vector, 2.5 µl 10x phosphatase buffer (BioLabs), 1 µl phosphatase (BioLabs), completed to 25 µl with ddH2O. Reaction mix was incubated for 1h and 30 min at 37°C.
Insert and/or vector purification were performed with an electrophoretic run in 1% agarose gel (50 ml Tris-acetate-EDTA Buffer, 0.5 g agarose, 6.5 µl SYBR SAFE (Thermo Fisher)), then bands were cut out and DNA was extracted from the gel using GenElute Gel Extraction kit (Sigma-Aldrich) according to manufacturer’s instructions.
Ligation of the insert and vector was performed using a 3:1 insert/vector ratio. Reaction mix was prepared with 1.5 µl ligation buffer 10X (BioLabs), 1 µl ligase (BioLabs), completed to 15 µl with ddH2O. This ligation mix was incubated for 1h
at RT.
2 µl ligation mix were added to competent E. Coli DH5α for the bacterial transformation. The bacterial culture was incubated for 30 min on ice, then heat-shocked for 90’’ at 42°C and cooled on ice. 200 µl of lithium borate (LB) medium
