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1 Forshaw, T. E. (2017) The role of increased hbcatm in the endothelial cells of patients with Alzheimers disease. PhD, University of the West of England. Available from: We recommend you cite the published version. The publisher s URL is: Refereed: No (no note) Disclaimer UWE has obtained warranties from all depositors as to their title in the material deposited and as to their right to deposit such material. UWE makes no representation or warranties of commercial utility, title, or fitness for a particular purpose or any other warranty, express or implied in respect of any material deposited. UWE makes no representation that the use of the materials will not infringe any patent, copyright, trademark or other property or proprietary rights. UWE accepts no liability for any infringement of intellectual property rights in any material deposited but will remove such material from public view pending investigation in the event of an allegation of any such infringement. PLEASE SCROLL DOWN FOR TEXT.

2 Chapter 1 Introduction and main aims CONTENTS. 1.1 Branched chain aminotransferase hbcat enzyme kinetics The structure of the hbcat proteins Human branched-chain amino acid metabolism Redox regulation of hbcat and relation to other redox proteins hbcat distribution BCAT and the glutamate-glutamine cycle hbcat and the BCAAs in the brain Maple syrup urine disease Excitotoxicity pathway Alzheimer s disease Amyloid pathology Tau pathology Protein synthesis and autophagy Protein misfolding and redox pathology The blood-brain barrier Aims and objectives

3 1.1 Branched chain aminotransferase. Branched chain aminotransferase (BCAT) [E. C ] is a highly conserved enzyme essential for mammalian branched chain L-amino acid (BCAA) and branched chain keto acid (BCKA) metabolism (Bledsoe et al., 1997; Ichihara & Koyama, 1966; Schuldiner et al., 1996; Suryawan et al., 1998). The most described function of the BCAT enzymes is to catalyse the reversible transamination of the BCAAs (L-leucine, L-valine and L- isoleucine) and α-ketoglutarate (KG), to generate L-glutamate and the respective BCKAs (α-ketoisocaproate (KIC), α-ketoisovalerate (KIV), and α-keto-β-methylvalerate (KMV) (Scheme 1.1). Rat in vivo studies utilising 15 N-labelled L-leucine have determined that the preferred direction of transamination is towards L-glutamate production, although labelled L-isoleucine and L-valine were also detected (Kanamori et al., 1998; Sakai et al., 2004). Specifically, in rat neurons it has been observed that the BCAT enzymes are involved in the de novo synthesis of approximately 30% of brain glutamate (LaNoue et al., 2001). Scheme 1.1 Transamination catalysed by BCAT. Human BCAT (hbcat) exists as two isozymes, the cytosolic (hbcatc) and the mitochondrial (hbcatm), which show distinct cellular localisation (Hutson et al., 1988). Human BCATc and hbcatm have a molecular mass of 44.3 kda and 41.7 kda, respectively, and their primary amino acid sequence has 395 and 365 amino acids, respectively. These isozymes share 58% amino acid homology, with the greatest homology in the active sites (Davoodi et al., 1998). Both of the full length enzymes have been purified, first from rat tissue (Hall et al., 1993), and later the human protein from cloned genes overexpressed in E. coli (Davoodi et al., 1998). This has allowed for detailed kinetic and redox characterisation, as well as solving of the crystal structures (Conway et al., 2002; Conway et al., 2008; Goto et al., 2005; Yennawar et al., 2001). 2

4 As with all aminotransferases, hbcat uses a pyridoxal phosphate (PLP) co-factor which is bound to an active site L-lysine residue. The PLP-dependent enzyme family is divided into five different fold types by the specific orientation of PLP within the active site (Reviewed by Eliot & Kirsch, 2004). While most mammalian aminotransferases belong to fold type I, hbcat is the only mammalian type IV aminotransferase (Yennawar et al., 2001; Yennawar et al., 2002). The other fold type IV enzymes are bacterial D-amino acid aminotransferases (Martínez del Pozo et al., 1989); differing significantly from the type I and II in that the active site has a mirror image orientation (Yennawar et al., 2001). In fold type IV enzymes, the active site L-lysine residue is bound to PLP on the re face of the chiral centre, exposing the si face to solvent, while the opposite is true for type I aminotransferases (Sugio et al., 1995). This suggests that hbcat may have a different role in mammals from that of other aminotransferases. The genes for hbcatc and hbcatm are BCAT1 and BCAT2, respectively. Both genes can be transcribed by activating transcription factor 4 (ATF4) and hbcatm alone by C/EBP homologous protein (CHOP), in response to cellular stresses such as starvation, misfolded proteins, and oxidative stress (Harding et al., 2003; Han et al., 2013). Additionally, the BCAT1 gene may be promoted by c-myc, which is involved in cell cycle progression and apoptosis (Ben-Yosef et al., 1998) and the gene has been described as an oncogene (Zhou et al., 2013; Tönjes et al., 2013). There are five variants of BCAT1 mrna, however, four of these have only been observed in cdna arrays and only the longest variant is observed as hbcatc protein (Ota et al., 2004; Bechtel et al., 2007). Likewise, the longest transcript of the BCAT2 gene translates to the most widely observed hbcatm protein (Than et al., 2001). Unlike hbcatc, the BCAT2 gene has been obsevered to be transcribed by Kruppel-Like factor 15 (Klf15), a glucocorticoid receptor target (Kuo et al., 2013; Shimizu et al., 2011). There are two additional splice variants of BCAT2 (Than et al., 2001; Lin et al., 2001). The first, placental protein 18b (PP18b), is weakly expressed in many tissues, although its function is not clearly understood (Than et al., 2001). The variant protein has a molecular weight 3

5 approximately 8 kda lighter than hbcatm and lacks the N-terminal mitochondrial targeting sequence, resulting in cytoplasmic localisation (Than et al., 2001). The second transcript, termed P3, was identified as an inhibitor of thyroid hormone receptor (TR) transcriptional activity (Lin et al., 2001). This varient localises to both the mitochondria and nucleus of cells, acting both as a TR antagonist and as an inhibitor of TR binding to DNA. This suggests that hbcatm may control expression of other proteins, although P3 is currently the only isoform published as demonstrating this activity. 1.2 hbcat enzyme kinetics. Characterisation of hbcat has demonstrated that the enzymes both follow the common aminotransferase ping pong bi bi mechanism for multi-substrate enzymes, as described by Cleland (1963). For this it is a requirement that PLP is bound as a Schiff base to the Ɛ-amine of active site lysine (Lys202 for hbcatm and Lys222 for hbcatc) to form an enzyme-cofactor complex (E-PLP). The first step in catalysis is the modification of the complex by transfer of an amine group from a substrate L-amino acid to PLP, forming pyridoxamine phosphate (PMP) and releasing an α-keto acid as a first product (Figure 1.1). In the next step, a substrate α-keto acid abstracts the amino group from PMP in a reaction catalysed by the protein, restoring the molecule to the native PLP form and releasing an amino acid product (Figure 1.1). Through both steps the cofactor will disassociate and form external aldimine and ketimine intermediates with the substrate, before reassociation as an E-PLP or enzyme-pmp (E-PMP) complex (Yennawar et al., 2002). In addition to the earlier described substrates, the straight chain analogues of L- valine (norvaline) and L-leucine (norleucine) and the α-keto acid conjugate of methionine (α-keto-γ-methiobutyrate) are substrates for hbcat. However, neither of D-isoleucine, the aromatic essential L-amino acids L-alanine or L-aspartate were active substrates (Davoodi et al., 1998). Kinetic studies of the hbcat enzymes have determined that the first step of transamination is the slower rate determining step, while the second step has 4

6 Figure 1.1 Ping-pong kinetics of the BCAT enzymes. The timeline illustrates the course of transamination from amino acid to α-keto acid. Starting from the left side, the amine group from an amino acid is transferred to enzyme-plp complex to form PMP and an α-keto acid. The α- keto acid is then released and an enzyme-pmp complex is formed. An α-keto acid may then react with the complex and abstract the amino group from PMP, resulting in release of amino acid and restoration of the enzyme-plp complex. Abbreviations: PLP co-factor. PMP amine modified co-factor. E-PLP/PMP complex of enzyme and co-factor. BCAA branched-chain amino acid. Glu L-glutamate. 5

7 a turnover rate approximately double that of the first (Table 1.1). Further characterisation by Yennawar et al. (2006) determined that the equilibrium constant (K eq) for each of the keto acid substrates is ten to fifteen-fold lower than that of the conjugate amino acid. It was found that in the PLP form, the order of substrate specificity (by lowest K eq) was L- isoleucine L-leucine > L-valine >> L-glutamate. This is mirrored by the conjugate α- keto acids whereby the specificity for the PMP form is KMV KIC > KIV >> KG (Yennawar et al., 2006). Of the two isozymes hbcatc has the faster turnover rate (K cat) and requires a lower concentration of substrate to achieve half maximal activity (K m) for each respective substrate (Table 1.1). There is a clear preference for L-isoleucine as a substrate for hbcatm, while hbcatc has a marginally higher affinity for L-leucine, (Yennawar et al., 2006), which may be indicative of subtle differences in active site conformation. Finally, the second order rate constant (K cat/k m) for each substrate analysed shows that for both steps of the catalytic cycle, transamination was considerably faster when catalysed by hbcatc as opposed to hbcatm (Davoodi et al., 1998). Knowledge of substrate affinity will contribute to the design of specific hbcat inhibitors, a key objective of this thesis, particularly when coupled with protein structural data by mimicking the substrate binding. 1.3 The structure of the hbcat proteins. The generation of several crystal structures of hbcatc (Goto et al., 2005) and hbcatm (Figure 1.2) (Yennawar et al., 2001) complexed with substrates or inhibitor compounds allows for further characterisation of the hbcat enzymes. Both enzymes were identified as homodimers with a small and large domain linked by an interdomain loop. On superimposition, a low variation between the crystal structures of the hbcatc and hbcatm isozymes (computationally quantified by measuring root mean square deviation) was observed, indicating that the subunit and overall structures were similar (Goto et al., 2005). 6

8 Table 1.1 hbcat isoform transamination. (Adapted from Davoodi et al., 1998). Abbreviations: Kcat substrate molecules turned over per enzyme molecule, per second. Km substrate concentration to reach halfmaximum enzyme activity. Kcat/Km specificity constant. Chapter 1 Introduction and main aims. Forshaw, T. E. (2016) 7

9 PLP K202 Figure 1.2 Structure of hbcatm protein. The full molecular surface of an hbcatm dimer is shown. Each of the two monomers is shown in a different colour. Residues from opposite monomers are required to form the substrate tunnel. The PLP co-factor is visible as a stick configuration through the substrate tunnel (circled) bound to K202. Image of entry 1EKP (Yennawar et al., 2003) acquired from the protein databank ( (Berman et al., 2000) visualised with UCSF Chimera (Pettersen et al., 2004). 8

10 For hbcatc, the small domain (residues 1-188) of each monomer is folded into an α/β open structure, while the large domain (residues ) is folded into a pseudo-barrel (Goto et al., 2005), with the domains linked by an interdomain loop (residues ). A large cavity is formed at the interface of the large and small domains of each monomer, which is completed by two loops from the small domain of the other monomer. Within this is located the active site where PLP is covalently bonded to the Ɛ-amine group of K222, and hydrogen bonded to Y227 to adjust orientation. Additional hydrogen bonding with E257 activates the co-factor by increasing the electron withdrawing effect of the PLP pyridine ring. Finally, the hbcatc active site residues T260 and A334 form a hydrogen bond with the substrate to secure it in place, while the bulky aromatic ring of Y193 appears to shield substrate within the protein from solvent (Goto et al., 2005). It has also been determined that hbcatc has a larger substrate pocket for L-amino acid side chains than hbcatm, which may affect substrate preference (Goto et al., 2005). The structure of hbcatm was found to be similar to that of hbcatc, particularly at the active site (Yennawar et al., 2001; Goto et al., 2005). The PLP co-factor is again bound as a Schiff base to K202, close to the interface of the two domains. As with all fold type IV aminotransferases, PLP is bound to the bottom of the active site and in hbcatm is secured into place by hydrogen bonding between Q237, Y207, and the PLP pyridine ring, as well as Y313, A99, V269, and V270 with the phosphate group oxygen atoms. Using the substrate L-isoleucine, Yennawar et al. (2002) were able to identify the substrate side chain pocket as nonpolar residues F30, Y141, Y207, F75, T240, A314, V155*, Y70*, and L153* (where * indicates an amino acid from the second monomer). Furthermore, with respect to hbcatm, closure of the substrate tunnel was observed by Y173, which was dependent on oxidation state (Yennawar et al., 2006). Analysis of the amino acid sequence revealed that within the substrate binding sites, hbcatm differs from hbcatc by one amino acid; G316 in hbcatm is V336 in hbcatc. This similarity may make the development of specific inhibitors for each isoforms more challenging. 9

11 1.4 Human branched-chain amino acid metabolism. The BCAAs are important molecules in nutrient sensing and have a role in regulating protein synthesis, cell survival, and proliferation. Their catabolism from BCAA to BCKA, and finally to Krebs cycle constituents acetyl-coa or succinyl-coa (Figure 1.3) is an important source of cellular energy and nitrogen (Harper et al., 1984; Hutson et al., 2005). As previously described, transamination by hbcat is the first step in catabolism of the BCAAs. The second step is irreversible oxidative decarboxylation of the BCKAs by the branched-chain α-keto acid dehydrogenase (BCKD) complex to acyl-coa derivatives using an NAD + cofactor, which is reduced to NADH during catalysis (Harper et al., 1984). This mitochondrial complex is part of the pyruvate dehydrogenase and α- ketoglutarate dehydrogenase family of complexes, which all form acyl-coa derivatives of their substrates (Islam et al., 2007; Koike et al., 1963; Perham, 1991). These complexes are formed of three components and have a similar mechanism of action, in particular, the E3 component is common to all three complexes (Perham, 1991). The BCKD complex was first purified and characterised in bovine kidney (Pettit et al., 1978) and is made up of a BCKA decarboxylase component (E1) [E. C ], a dihydrolipoyl transacylase component (E2), and a dihydrolipoamide dehydrogenase component (E3). Activity of the BCKD complex is controlled by phosphorylation of S293 of the E1 subunit with BCKD kinase, which inhibits oxidative decarboxylase activity (Shimomura et al., 1990; Harris et al., 1997). This is reversed by protein phosphatase 2Cm, which is activated by the BCAAs (Lu et al., 2009). Furthermore, hbcatm in the PLP state and the E1 component of BCKD have been shown to form a protein-protein interaction, which increases E1 component activity 12-fold (k cat) (Islam et al., 2007). This can only occur when hbcatm is reduced, indicating that a shuttling of substrate between hbcatm and BCKD is dependent on mitochondrial oxidation state. Interaction is disrupted by treatment with NADH, suggesting that NADH produced by a complete BCKD catalysis cycle causes disassociation of the complex (Figure 1.3). 10

12 Figure 1.3 Metabolism of the branched-chain amino acids. The BCAAs are first transaminated to the conjugate α-keto acid, before decarboxylation to an acyl-coa derivative. After further metabolism, these enter the Krebs cycle as either acetyl-coa or succinyl-coa. In this study, four enzymes involved in this process will be analysed by Western blot analysis; hbcatm, BCKD, AUH, and GDH. Abbreviations: BCKD Branched-chain α-keto acid dehydrogenase. AUH AU-rich binding homolog of enoyl- CoA hydratase. GDH Glutamate dehydrogenase. KG α-ketoglutarate. 11

13 In addition to BCKD, the metabolic protein glutamate dehydrogenase (GDH) 1 has also been shown to interact with hbcatm (Islam et al., 2010). While BCKD was shown to bind to the PLP state of hbcatm (Islam et al., 2007), GDH1 exclusively bound to the PMP state (Islam et al., 2010). It was proposed that a BCAA metabolon was formed (Figure 1.4). In the first step hbcatm in the PLP state is bound to BCKD. Upon transamination of a BCAA, hbcatm is modified to the PMP state and the resulting BCKA is shuttled to BCKD for decarboxylation. The complex would disassociate and hbcatm in the PMP form would bind instead to GDH1. L-glutamate is then oxidatively deaminated by GDH1, producing free ammonia, and the KG shuttled to hbcatm which would regenerate the L-glutamate. This would have the net effect of producing ammonia from the BCAA, without affecting the glutamate pool, and could be used to synthesise L- glutamine using the enzyme glutamine synthase (Islam et al., 2010; Hutson et al., 2011). Early research by Frame (1958) sought to determine the concentrations of individual amino acids in serum during a state of fasting or following consumption of a high source of protein. The BCAAs were unique in that the concentration increase was among the highest and remained so consistently for up to eight hours, while other amino acid concentrations were low or fluctuated. This was considered to be due to the unusually low activity of BCAT in the liver compared to other amino acid catabolic enzymes, as had been observed in rat liver preventing first pass metabolism (Harper et al., 1984). Further studies on rat biology determined that in skeletal muscle BCAA uptake and BCAT activity was high, and that BCKD expression was near absent (Harper et al., 1984; Hutson et al., 1981; Shinnick et al., 1976). With this considered, it was originally hypothesised that in humans, skeletal muscle would be one of the main sites of BCAA transamination and that BCKD activity would be very low, promoting the release of BCKAs which would then be metabolised by the liver (Harper, 1984). Through this pathway, the BCAAs would serve as donors of nitrogen for the transamination of KG, taken from the Krebs cycle, into L-glutamate. The L-glutamate produced is used as a nitrogen source to synthesise L-alanine and L-aspartate from the respective aminotransferases and restore KG into 12

14 Figure 1.4 hbcatm-bckd-gdh metabolon. BCATm, BCKD, and GDH1 form a metabolic shuttle and channel substrates from one protein to the other (Islam et al., 2007; Islam et al., 2010). Here, the PLP form of hbcatm is modified by the transfer of an amine group from a BCAA such as L-leucine and the resulting BCKA shuttled to BCKD for further oxidation. The generation of hbcatm-pmp then stimulates the disassociation of hbcatm and BCKD. In the next step, GDH1 binds to the PMP form of hbcatm, and catalyses the oxidation of L-glutamate to KG and ammonia. The KG is shuttled to hbcatm-pmp and where it is reduced back to L-glutamate. This results in the net product of L-glutamate, ammonia, and a BCKA-CoA (in this example, isovaleryl-coa). Abbreviations: GDH Glutamate dehydrogenase. BCKD Branched-chain α-keto acid dehydrogenase. KG α-ketoglutarate. KIC α-ketoisovalerate. 13

15 the Krebs cycle, or to synthesise L-glutamine (Frame, 1958; Elia et al., 1989; reviewed by Rudderman, 1975). This is debated by Rennie et al. (2006) in a review of previous work by Millward et al. (1982) finding that athletes administered 13 C radiolabelled L-leucine would exhale significant quantities of 13 CO 2 when exercising. It was found that L-leucine was consumed at a linear rate, in correlation with an increase in VO 2 max, suggesting that it was oxidised as an energy source. This is further supported on a molecular level as human BCKD is significantly expressed in skeletal muscle tissues compared to rat tissue, although in humans the majority is in the deactivated phosphorylated state (Suryawan et al., 1998). It was proposed that BCKD is accumulated in skeletal muscle cells in an inactive form and activated in response to a demand for energy. Indeed, exercise was found to be an activator of BCKD in exercising human individuals, particularly during anaerobic respiration (Wagenmakers et al., 1989). Furthermore, while earlier rat studies found that neither BCAT isoform was expressed in the liver, Taniguch et al. (1996) found that hbcatm was active in human liver and was approximately 100 fold more active than BCKD, and more so in diseased liver. Regardless, when comparing total organ BCAT activity the skeletal muscle contains by far the greatest rate of transamination, totalling 65% of total body BCAT activity (Suyawan et al., 1998). The brain contains the second greatest site of transamination, accounting for 15% of whole body activity, while the liver is the third most with 8% of activity. The reason for this is not fully understood, although the authors again suggest an inter-organ shuttling of the BCKAs (Taniguch et al., 1996; Suyawan et al., 1998). The final section in BCAA catabolism is the multi-step conversion of the branched-chain acyl CoA to either acyl-coa or succinyl-coa which can be integrated into the Krebs cycle (Figure 1.4) (Reviewed by Engel & Wierenga, 1996). At this point the three BCKAs branch and have a variety of different oxidative catabolic pathways (Ikeda et al., 1983; Ikeda et al., 1985; Matsubara et al., 1989; Izai et al., 1992; Rozen et al., 1994). 14

16 Ultimately, L-leucine is metabolised to acetyl-coa, L-valine is metabolised to succinyl- CoA, and L-isoleucine is metabolised to either acetyl-coa or succinyl-coa. Of particular interest is the enzyme 3-methylglutaconyl-CoA hydratase (AUH) [E. C ] in the catabolism of L-leucine, as it is one of the few multiple role enzymes in a specialised metabolic pathway (Nakagawa & Moroni, 1997). As well as the metabolic enoyl-coa hydratase activity which is the penultimate step in acetyl-coa synthesis, the enzyme also has an AU-rich mrna binding domain, and as such is generally termed AU-rich binding homolog of enoyl-coa hydratase or AUH (Nakagawa & Moroni, 1997). Although AUH has not yet been observed to bind to any endogenous mrna (Mack et al., 2012), other proteins with this domain have been found to bind to the AU-rich elements in the 3 untranslated region of c-myc, c-fos, tumour necrosis factor α, and cyclooxygenase 2 mrna, leading to degradation of these mrna as a method of reducing protein expression (Barreau et al., 2005). This suggests that a protein involved in the catabolism of L-leucine may be involved in regulating other pathways, and it is an aim of this thesis to investigate this enzyme further. 1.5 Redox regulation of hbcat and relation to other redox proteins. A novel function of hbcat is that it is regulated by different redox environments (Davoodi et al., 1998; Conway et al., 2008). Both isozymes require a reducing environment for maximal transamination (Davoodi et al., 1998) and are inhibited in a dose-dependent manner by H 2 O 2 (Conway et al., 2002; Conway et al., 2008). In proteins, the reactive thiols of cysteine are particularly sensitive to oxidation (Barford et al., 2007). The thiol specific reagents DTNB and N-ethylmalemide (NEM) identified two and six reactive cysteine residues in hbcatm and hbcatc, respectively (Conway et al., 2002; Conway et al., 2008). Using these specific reagents, titration of one hbcatm thiol resulted in a 50% loss of activity, while titration of both thiols resulted in 85-95% inhibition (Conway et al., 2002). Similarly, titration of two thiols in hbcatc resulted in 33% inhibition, while titration of an additional two thiols increased inhibition to 80% (Conway et al., 2008). The 15

17 two solvent-accessible cysteine residues were subsequently identified using 125 I-β-(4- hydroxyphenyl)ethylmaleimide-nem labelling for hbcatm (Conway et al., 2002) and site-directed mutagenesis coupled with NEM labelling for hbcatc (Conway et al., 2008). They were identified as C315 and C318 in hbcatm, and C335 and C338 in hbcatc. The CXXC motif is a highly conserved structure existing in proteins of many cells and is commonly involved in redox regulation (Reviewed by Fomenko & Gladyshev, 2003). In hbcat, the two different residues of the CXXC motif appear to have different roles in regulating transamination. Studies using site-directed mutagenesis of the BCAT genes identified that the N-terminal residue mutations C315S or C335S decreased L-isoleucine turnover (k cat) by 70% compared to wild-type hbcatm or hbcatc, respectively. However, the C-terminal CXXC motif mutation (C318S or C338S) decreased turnover by no greater than 40% (Conway et al., 2004; Conway et al., 2008). Furthermore, it has been determined that the N-terminal cysteine of each isozyme is a redox sensor, as the thiol is readily oxidised to sulphenic acid under oxidising conditions (Conway et al., 2002, Conway et al., 2008). When this occurs the C-terminal cysteine rapidly reduces the N- terminal cysteine and a disulphide bridge forms between the two cysteines, decreasing transamination (Conway et al., 2002, Conway et al., 2008). It was found that air oxidation alone was able to inhibit BCAA transamination by hbcatc 40-45% (Davoodi et al., 1998) and completely inhibit hbcatm (Conway et al., 2008). However, this may be reversed by the addition of the reducing agent DTT; indicating that the CXXC motif acts as a nanoswitch, permitting transamination under physiological reducing conditions, while inhibiting transamination under oxidising conditions (Figure 1.5). It has been proposed that an hbcat nanoswitch has two roles. First, to control the activity of hbcat so that transamination is regulated during periods of oxidative stress, possibly to conserve L-leucine as an important anabolic signal (Conway et al., 2004). Second, to protect the redox sensing cysteine of the enzyme from oxidative stress, as the thiol may become further oxidised to sulphinic or sulphonic acid and these oxidative 16

18 Figure 1.5 Oxidation of the CXXC motif in hbcat. When hbcat is exposed to oxidising conditions, such as treatment with hydrogen peroxide, the N-terminal cysteine of the CXXC motif can be oxidised to sulphenic acid (Image adapted from Conway et al., Reprinted with permission). Abbreviations: A Brønsted Lowry acid. Cys Cysteine residues of hbcat. X Non-cysteine amino acid. 17

19 states are not readily reversible within the cell (Conway et al., 2004). The structural impact of the intramolecular CXXC disulphide bond was determined by Yennawar et al. (2006) by studying the crystal structures of enzymes in varying states of oxidation or catalysis. It was found that in hbcatm the thiol-thiolate interactions between C315 and C318 are responsible for orientating the residues of a β-turn (residues ). When correctly aligned for catalysis, the β-turn creates a positive dipole moment in the active site, this attracts the negatively charged phosphate group of PLP, as well as the carboxylate group of amino acids, with the effect of improving substrate orientation. Disruption of this positively charged region is proposed to decrease the catalytic capacity of the enzyme to transfer an amine group from an amino acid to PLP; demonstrated by the reduced equilibrium constant (K eq) value observed in the oxidised enzyme (Yennawar et al., 2006). Furthermore, the residues of the β-turn also form hydrogen bonds with residues of the binding site and substrate channel, helping form both an ionic and hydrophobic substrate pocket in these regions. However, oxidation of the enzyme results in a change of the hydrogen bonding between the β-turn and the substrate pockets, leading to an inhibitory conformational change. Understanding how hbcat is regulated and the respective changes in conformation will be important for designing a specific inhibitor, which is a principal aim of this thesis. In addition to oxidation, transamination by hbcatc is also inhibited by S- glutathionylation, suggesting a role for S-glutathionylation in hbcat regulation (Conway et al., 2008). Conversely, chaperone refolding activities of the protein have been described and appear to be enhanced by S-glutathionylation (El Hindy et al., 2014). The oxidation state of the CXXC motif is essential for protein-protein interactions between hbcat and other proteins. It was found that under reducing conditions, hbcatc will bind with several neuronal proteins including β-tubulin, septin 4, and kalirin RhoGEF (Conway et al., 2008). Under oxidising conditions, hbcat was not observed to bind to these proteins, possibly indicating that the proteins will not interact during periods of cellular stress. More recently, protein disulphide isomerase (PDI) (El Hindy et al., 2014) 18

20 has also been observed to bind to hbcatm under reducing conditions. Each of the aforementioned proteins, are known to be involved in synapse plasticity or protein repair and have been implicated in the pathology of neurological diseases such as Alzheimer s disease (AD) (Puig et al., 2005; Kinoshita et al., 1998; Mandela & Ma, 2012). In addition, by calculating the redox potentials of both hbcatc and hbcatm, Coles et al. (2012) showed that the isozymes were most active when the redox conditions within a cell were typical of a quiescent state as opposed to the slightly oxidising conditions of a proliferative state. This may be important in controlling the metabolic state of the cell and the production of neurotransmitters much as L-leucine, L-glutamate and γ- aminobutyric acid (GABA). Because of these interactions it is a further aim of this thesis to investigate how expression of hbcat may impact the metabolome and redox status of the cell. 1.6 hbcat distribution. The BCAT proteins have been mapped in a variety of different tissues using several different methods. They were first mapped and purified by Ichihara & Koyama (1966) in the liver, heart, skeletal muscle and kidney of rats by measuring activity of tissue lysates, although this was for total transamination and did not separate the isoforms. This was followed by tissue activity assays which identified and confirmed expression of BCATm in nearly all tissues, although expression of BCATm in the liver, brain, ovary, and placental tissues appeared limited and transamination was instead predominantly by another enzyme (Ichihara, 1975). An L-leucine specific enzyme was identified in rat liver (now identified as leucine transaminase) and BCATc identified in the brain, placenta and ovaries (Ichihara, 1975). These observations were later confirmed by further rat tissue activity assays (Schinnich et al., 1976; Hutson et al., 1988), and mrna expression mapping of BCATm by Torres et al. (1998). Additional immunoblotting by Hall et al. (1993) in rat tissues also again showed that BCATm appeared to be ubiquitously 19

21 expressed, while BCATc was restricted to a small number of specialised tissues and that tissues tended to express only one of the isozymes. In humans, immunoblotting has demonstrated that BCAT expression is similar to that observed in rat (Suryawan et al., 1998; Sweatt et al., 2004). Specifically, RT-PCR by Suryawan et al. (1998) and later western blot analysis by Sweatt et al. (2004) confirmed high hbcatm expression in the kidney and pancreas, moderate expression in the brain, muscle, and stomach, and lower expression in adipose and liver tissue. This closely maps with tissue activity, although liver activity appears to be unusually high for the low expression (Suryawan et al., 1998; Sweatt et al., 2004). Expression of hbcatc however was shown to be limited to the brain (Sweatt et al., 2004). Mapping of the BCATc in rat brain was first conducted by García-Espinosa et al. (2007), again by immunoblot, where expression was found throughout the brain and localised specifically to neurons. The greatest intensity was observed in the hypothalamus, basal ganglia, midbrain and brainstem, although there was also significant expression in the cerebral cortex; all of which contain primarily glutaminergic or GABAergic neurons. Furthermore, within glutamatergic pyramidal neurons expression was prominent in the axons and proximal dendrites, while in GABAergic neurons expression appeared to be localised to the cell body. All mapping of cholinergic and particularly dopaminergic neurons showed low expression of BCATc (García-Espinosa et al., 2007). Subsequent investigation of the BCATm isoform in rat brain found it solely expressed in the astrocytes throughout the brain, but not in any other cell type (Cole et al., 2012). Additionally, expression of BCKD in the rat brain was localised to neuronal cells, with a marked absence in the astroglia (Cole et al., 2012) and is considered further evidence for the expansion of a brain glutamate-glutamine cycle to incorporate the BCAAs (Cole et al., 2012; Hertz, 1979; Yudkoff et al., 1996). A specific mapping of the hbcat isozymes in the human brain has recently been published by Hull et al. (2012) and, as in rat tissue, demonstrated that hbcatc is 20

22 distributed throughout neuronal cells of the human brain. In particular, there was again a high density of positive staining in many of the glutamatergic and GABAergic neurons of the hippocampus, temporal cortex, basal ganglia, and midbrain. Less frequent but more intense positive staining was observed in the large neurons of the basal ganglia, pyramidal neurons, hypothalamus, and cholinergic neurons in the nucleus basilis of Meynert. This supports the concept that hbcatc may be important in the synthesis of the neurotransmitters L-glutamate and GABA, as well as new data that suggests hbcatc may be involved in hormone secretion (Hull et al., 2012). Contrary to studies of BCATm location in rats (Cole et al., 2012), hbcatm is not located in the astrocytes in human brain but rather is located in mitochondria of brain endothelial cells which make up the cerebral microvasculature and as such an alternative mechanism for the glutamate-glutamine cycle in humans has been proposed (Hull et al., 2012; 2015). 1.7 BCAT and the glutamate-glutamine cycle. It is well characterised that during glutamatergic neurotransmission there is a significant uptake of L-glutamate by astrocytic cells from the synaptic cleft (Reviewed by Danbolt et al., 1994). To enable recycling, the relatively inert amino acid L-glutamine is synthesised from L-glutamate by glutamine synthase within astrocytes. This L-glutamine is then transported back to neuronal cells, where it is hydrolysed by glutaminase to replenish L- glutamate (Reviewed by Hertz, 1979). This is however an oversimplification of the process as there is significant glutathione synthesis and anapleurotic oxidation of L- glutamate which would remove it from the glutamate-glutamine cycle, and several additions to the cycle have been proposed (Daikhin & Yudkoff, 2000; Yudkoff et al., 1989; Erecińska & Silver, 1990; Hassel & Bråthe, 2000). There is also a need for ammonia, released in neuronal cells by glutaminase to be returned to the astrocytes for L-glutamine synthesis. 21

23 Studies using primary astrocytes and neurons, and the specific BCATc inhibitor gabapentin, showed that L-leucine is a significant contributor to brain L-glutamate (Yudkoff et al., 1996; Hutson et al., 1998). The glutamate-glutamine cycle was therefore revised to include the BCAAs (Figure 1.6) as a BCAT-glutamine-glutamate cycle (Yudkoff et al., 1996; Hutson et al., 1998). It was suggested that in the astrocytes, KG from the Krebs cycle and L-leucine are transaminated by BCATm, producing L-glutamate and KIC. The L-glutamate is condensed to L-glutamine, before both KIC and L-glutamine are shuttled to neuronal cells. Within the neuron, L-glutamine is amidohydrolased to L- glutamate, producing ammonia which is then utilised by GDH to synthesise L-glutamate. L-glutamate can either be used for neurotransmission, or transaminated with KIC to enter the Krebs cycle and produce L-leucine to be shuttled back to the astrocyte, completing the cycle. As the pathway is catapleurotic in the astrocytes the cycle can be replenished by increased glucose uptake (Yudkoff et al., 1996; Hutson et al., 1998; Cole et al., 2012). On the other hand, mapping of the hbcatm isoform in humans to endothelial cells rather than astrocytes has led to a further model revision for humans (Hull et al., 2012; 2015). As before, L-glutamate is uptaken by astrocytes and shuttled back to neuronal cells as L-glutamine. However, in this model hbcatc in neurons is catapleurotic and generates L-glutamate and KIC from KG and L-leucine (Figure 1.7). L-glutamate is used for neurotransmission and KIC is shuttled back to the endothelial cells for decarboxylation. Human neurons have been shown to have pyruvate decarboxylase activity and this may help replenish the Krebs cycle (Hassel & Bråthe, 2000). Finally, L-glutamate could be shuttled between the astrocytes and endothelial cells to replenish or support removal from the glutamate-glutamine pool according to need. The net effect of the endothelial cells of the blood-brain barrier (BBB) is the transport of L-glutamate out of the brain, however L-leucine easily enters the brain through the large neutral amino acid transporter (LNAAT) (Oldendorf & Szabo, 1976; Reviewed by Hawkins, 2009). L-leucine may be transaminated to L-glutamate and condensed to L-glutamine in the endothelial 22

24 Figure 1.6 The rat model of the glutamate-glutamine cycle. L-glutamate is uptaken from synapses after neurotransmission and transported back to neurons as relatively inert L-glutamine. In astrocytes, L-glutamate may be synthesised from KG provided by the Krebs cycle. Likewise, in neurons, KG may enter the Krebs cycle and be oxidised as an energy source. Abbreviations: Cit Citric acid. Gln L- glutamine. Glu L-glutamate. GS Glutamine synthase. KG α-ketoglutarate. KIC α-ketoisovalerate. Mal Malate. OAA Oxaloacetate. PAG Phosphate-activated glutaminase. Succ Succinate. Chapter 1 Introduction and main aims. Forshaw, T. E. (2016) 23

25 Figure 1.7 The human glutamate-glutamine cycle. The glutamate-glutamine cycle maintains physiological concentrations of L-glutamate in the brain. An important part of this cycle is the synthesis and catabolism of L-glutamate by the BCAT enzymes. An investigation by Hull et al. (2012) found hbcatm located in the endothelial cells of the brain microvasculature, rather than astrocytic cells as observed in rat models (Coles et al., 2012). To account for this, an updated human glutamate-glutamine cycle was proposed (Hull et al., 2015). It has been hypothesised that changes in hbcat activity may affect glutathione synthesis and mtor activity which could have pathological implications (Hull et al., 2015). Abbreviations: Gln L-glutamine. Glu L-glutamate. GS Glutamine synthase. GSH Glutathione. KIC α-ketoisocaproate. KG α- ketoglutarate. mtor Mammalian target of rapamycin. PAG Phosphate-activated glutaminase. Chapter 1 Introduction and main aims. Forshaw, T. E. (2016) 24

26 cells with hbcatm, before being shuttled to the astrocytes for entry into the glutamateglutamine cycle. Alternatively, L-leucine may be directly transported to neurons to support transamination to L-glutamate by hbcatc (Hull et al., 2015). 1.8 hbcat and the BCAAs in the brain. Due to the unique role of the BCAAs and BCAT in metabolism, protein synthesis, and neurotransmission they have been studied extensively in a range of different models. In particular, the neurological disease maple syrup urine disease (MSUD) is characterised by a deficiency in catabolism of the BCAAs (Dancis et al., 1960). Additionally, the role of BCAT has been investigated in apoptosis (Kholodilov et al., 2000), cancer (Tonjes et al., 2013), and AD (Hull et al., 2015). There are indications that the BCAAs may be involved in regulation of nutrient sensing (Lynch, 2001), diabetes mellitus type 2 (Reviewed by Adams, 2011), and brain trauma (Ott et al., 1988; Evangeliou et al., 2009; De Bandt & Cynober, 2006). In particular, a role for BCAT in AD has far reaching implications. Indeed, L-glutamate toxicity, autophagy, impaired BBB integrity, and oxidative stress are all widely described in the disease and are introduced as linked to hbcat in this thesis (Hynd et al., 2004; Barnett & Brewer, 2011; Zipser et al., 2006; Perry et al., 2002). With this considered, in order to investigate the role of hbcat in neurological disease it would clearly be desirable to design a selective inhibitor of hbcatm. It would therefore be possible to investigate the extent to which hbcat plays a role in the homeostasis of the BCAAs and BCKAs, and particularly to neuronal glutamate and resulting GABA concentrations Maple syrup urine disease. There are several metabolic syndromes which are completely characterised by deficiencies in catabolism of the BCAAs, however MSUD is by far the most common and well-studied. First described as a case study by Menkes et al. (1954) the disease was 25

27 characterised by cerebral dysfunction and urine which smells of maple syrup. Further investigation with 14 C-labelled BCAAs was able to identify that the disease was a result of a deficiency in oxidative decarboxylation of the BCAAs (Dancis et al., 1960). Genetic studies have identified approximately 100 different genetic mutations in the BCKD complex which cause MSUD, including a form treated with thiamine, and a severe type affecting the E3 subunit which is also part of the pyruvate dehydrogenase and α- ketoglutarate dehydrogenase complex (Chuang et al., 2006; Nellis & Danner, 2001; Scriver et al., 1971; Robinson et al., 1977). The decrease in BCKA decarboxylation results in an increase in BCAA concentrations in plasma and cerebrospinal fluid (CSF), correlating with severity of disease, and which has several downstream effects (Wajner et al., 2000). For example, concentrations of other large neutral amino acids (L-phenylalanine, L-tyrosine, L-tryptophan, and L- methionine) in the brain are decreased during symptomatic periods of MSUD, which is considered to be due to excessive competition for the LNAAT through the BBB by the BCAAs (Wajner et al., 2000). This may result in a deficiency of monoamine, evidenced by the reduced concentrations of dopamine and serotonin observed in MSUD patients (Prensky & Moser, 1966). Furthermore, decreases in brain L-glutamate and GABA concentrations observed in MSUD (Sansaricq et al., 1989; Prensky & Moser, 1966) would be consistent with impairment of the leucine-glutamine-glutamate cycle (Figure 1.7). In a previous study, modelling of MSUD by infusion of KIC into the brains of rats in vivo was concurrent with a marked increase in L-leucine concentration and decrease in L-glutamate (Zielke et al., 1997). It was therefore proposed by Yudkoff (1997) that increased KIC is readily taken up by neurons and forces transamination in the direction of L-leucine production, using L-glutamate as a nitrogen source and depleting concentrations of the neurotransmitter. Finally, studies with rat models have also identified the BCKAs as key disruptors of brain energy utilization (Sgaravatti et al., 2003). Using tissue sections, it was determined that treatment with the BCKAs increased anaerobic respiration by increasing uptake and utilization of acetate and glucose 26

28 respectively, as well as decreased CO 2 output and increased lactate output (Sgaravatti et al., 2003) Excitotoxicity pathway. Early experiments had determined that L-glutamate was one of the few free amino acids in the brain and it was assumed that this was primarily in a metabolic role (Weil-Malherbe, 1950). It was considered that administration of L-glutamate as a medicine could help with mood disorders, however its administration was linked to seizures in dogs and a different role for L-glutamate was considered (Hayashi, 1954). Later, pioneering experiments by Lucas and Newhouse (1957) found excess concentrations of the excitatory neurotransmitter L-glutamate would cause necrotic lesions in retinal cells and a theory of glutamate toxicity, termed excitotoxicity, was described. During excitotoxicity, excessive L-glutamate agonist binding initiates a toxic increase in cytoplasmic calcium concentration, which via several mechanisms leads to cell damage, oxidative stress, and potentially the apoptotic or rapid necrotic death of the cell (Ankarcrona et al., 1995). The principal pathway for this is well-documented and involves the ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N- methyl-d-aspartate (NMDA), and kainate (KA) receptors, as well as the metabotropic glutamate receptors (Doble, 1999; Garthwaite & Garthwaite, 1991). In the first instance, binding at the AMPA and KA receptors initiates membrane depolarisation and opening of voltage dependent calcium channels (VDCC). Through this there is an influx of calcium by transmembrane Ca 2+ /Na + exchange, further stimulating depolarisation (Brorson et al., 1994). Binding of the metabotropic receptor also stimulates the endoplasmic reticulum to release stored calcium into the cytoplasm, while Ca 2+ can flow into the cell through ion channels opened by agonist binding at the NMDA receptor (NMDAR) (Ferraguti et al., Under healthy conditions, Mg 2+ ions block the NMDAR ion channels preventing the influx of Ca 2+. During neurotransmission 27

29 membrane depolarisation (from an action potential) repels this ion block to enhance the transmission cascade via NMDA channel opening. Once an excitotoxic threshold has been crossed, influx of Ca 2+ will inhibit the plasma membrane from returning to the resting potential; keeping the VDCCs open so that even if the agonist binding stops, the flow of calcium into the cell will continue (Doble, 1999). Additionally, depolarisation by excitotoxicity may completely repel Mg 2+ binding to the NMDAR, leading to a larger and pathological influx of Ca 2+ from the NMDAR (Schramm et al., 1989). In an attempt to reduce excessive Ca 2+ concentrations the mitochondria will take up Ca 2+, however, excessive uptake results in a loss of activity, particularly of adenosine triphosphate (ATP) production, which inhibits membrane repolarisation (Doble, 1999). There may be an immediate necrotic cell death, or delayed apoptosis due to increased activation of Ca 2+ dependent enzymes (Ankarcrona et al., 1995). For example, sustained activation of nitric oxide synthase (NOS) causes significant oxidative stress by the generation of excess NO. Nitric oxide is further oxidised to peroxynitrite, which may denature proteins and directly cause DNA damage (Perez-De La Cruz et al., 2005). Many proteases, caspases, phospholipases and endonucleases are also activated by Ca 2+ influx, leading to substrate degradation and apoptotic death (Figure 1.8) (Ankarcrona et al., 1995). Interestingly, in humans, after excitotoxic insult, levels of L-glutamate continue to remain at a slightly elevated concentration for a number of weeks before subsiding (Baker et al., 1993; Aliprandi et al., 2005). This contrasts dramatically against the minutes that glutamate is elevated in rat models of neuronal insult (Benveniste et al., 1984). Two theories have been postulated to explain this. First, that the concentration of L-glutamate remains at a reduced but still excitotoxic concentration for an extended period and will continue to cause cell death (Doble, 1999), and a second that the concentration of glutamate remains high as a result of increased synapatic activity to repair neurons and inhibit apoptosis (Ikonomidou & Turski, 2002). Neurotransmission is well-characterised 28

30 Figure 1.8 The glutamate-mediated excitotoxic pathway. Agonist binding of glutamate at the NMDA and AMPA receptors stimulates an increase in the cytoplasmic Ca 2+ concentration. This then results in the activation of Ca 2+ dependent enzymes such as apoptotic proteases, lipases and nucleases which disrupt function within the cell, and NO synthase which leads to an increase in harmful free radicals, mitochondrial collapse and cellular death (Image reprinted with permission; Doble, 1999). 29

31 as contributing to synapse strength, and indeed the failure of NMDAR antagonists to improve recovery from stroke appears to support this possibility (Ikonomidou & Turski, 2002). Furthermore, NMDAR antagonists have been shown to cause neuronal apoptosis when administered to rats immediately following neuronal insult which could indicate that a L-glutamate neurotransmission recovery mechanism was blocked by the NMDAR antagonist (Ikonomidou et al., 2000). As BCAT is involved in the synthesis of brain L- glutamate is it possible that hbcat may be upregulated to increase the concentration of L-glutamate for neurotransmission. In support of this Kolodilov et al. (2000) have observed that in rats BCATc is upregulated in non-apoptotic surviving neurons after neuronal insult and postulated that the reason for the upregulation is to act as an antiapoptotic agent. Conversely to acute excitotoxicity, slow excitotoxicity, first hypothesised by Novelli et al. (1988) describes a condition where the concentration of L-glutamate is below that which would cause excitotoxicity under healthy conditions, but that further metabolic changes changes within the cell could reduce it below the excitotoxic threshold. The primary mechanism for this is described as insufficient production of ATP, which results in the diminished ability of Na + /K + channels to exchange ions and maintain resting potential. When this occurs the membrane could become slightly depolarised and unable to maintain the Mg 2+ block on the NMDAR as previously described, leaving the cell much more susceptible to depolarisation (Doble, 1999). This slow excitotoxic mechanism has been proposed for several neurodegenerative diseases. For example, the AD brain has decreased neuronal respiration, which may reduce the ability to repolarise (Heiss et al., 1991). This provides a role for BCAT as it can act as a metabolic enzyme by producing α-keto acids for oxidative decarboxylation to enter the Krebs cycle for ATP production (Hutson et al., 2005). 30

32 1.9 Alzheimer s disease. AD is the most common form of dementia, accounting for 45-75% of dementia cases depending on ethnicity, and currently affects over 35 million people worldwide (Fratiglioni et al., 1999; Wimo et al., 2013). In the United Kingdom, AD is estimated to cost 1500 per person, per month and as such carries a significant financial burden (Dodel et al., 2015). Current projections suggest that cases of AD will increase by 2-4 fold by 2050, with over 40% of those requiring high levels of care, which will have a significant economic impact (Mura et al., 2009; Brookmeyer et al., 2007). There are currently two types of approved treatment in the UK targeting AD; the acetylcholinesterase inhibitors donepezil, rivastigmine and galantamine, and the NMDAR channel blocker memantine (NICE guidelines, 2011). The efficacy of these drugs is debated (Ikonomidou & Turski, 2002), but it is generally accepted that they have a modest effect in reducing clinical symptoms of the disease for up to a year (Spalletta et al., 2014; Di Santo et al., 2013; Winblad et al., 2004). Initially, AD presents clinically as memory impairment, vasoconstrictive deficits, and aphasia (Förstl & Kutz, 1999). The disease is continually degenerative and over time the initial symptoms will increase in severity, while the individual may also suffer emotional instability, agitation, ataxia, dysgraphia, and anosognosia; although it should be noted that not every case of AD is typical (Förstl & Kutz, 1999; Dubois et al., 2010). Death is typically due to comorbid factors such as pneumonia or septicaemia (Förstl & Kutz, 1999). Diagnosis of AD is difficult due to atypical pathology and overlap with other dementias and mild cognitive impairment. It should be noted that the familial early onset cases make up 1-5% of AD cases (Rogaeva, 2002), and that the lexicon dictates that discussion of AD generally refers to the more common late onset sporadic form (Dubois et al., 2010). In the UK, initial diagnosis of probable AD is given after a series of psychological tests, and confirmation is generally only given after post-mortem analysis of histological markers (NICE guidelines, 2011). Ante mortem imaging techniques (MRI 31

33 and PET) and CSF assays are considered good indicators but are currently only used for research purposes and not for routine diagnosis (McKhann et al., 2011). Histologically, AD is most commonly characterised by the presence of extracellular amyloid β-peptide (Aβ) plaques, intracellular hyperphosphorylated tau protein tangles termed neurofibrillary tangles (NFT), and lowered brain volume (Parihar & Hemnani, 2004; Braak & Braak, 1991) Amyloid pathology. The most common association with AD are the Aβ plaques, most likely due to their central role in the early onset familial form of AD (Tanzi & Bertram, 2005; Rogaeva, 2002). Although there is debate that the soluble form of Aβ is more closely correlated with the disease, the plaques remain the major histological marker (Hannson et al., 2006; McLean et al., 1999; Sultana & Butterfield, 2010). The pathway of amyloid production has been studied extensively and is reviewed here. In healthy individuals, transmembranal amyloid precursor protein (APP) is cleaved first by an α-secretase before being released by a γ-secretase to produce the 1-40 Aβ peptides (Aβ40), which have a role in synapse regulation (Wang et al., 2012). However, in AD the APP is cleaved first instead by a β-secretase, producing Aβ peptides 1-42 protein (Aβ42) which form aggregated plaques on release and are considered toxic (Hardy, 1997; Parihar & Hemnani, 2004). In support of the amyloid theory of AD the majority of individuals affected by familial AD have mutations in either APP or in the 1-40 cleavage supporting presenilin genes (Hardy, 1997). Although the mechanism of Aβ42 toxicity is not completely understood there is evidence for potentiation of excitotoxicity and increased oxidative stress (Reviewed by Carrillo- Mora et al., 2014). With respect to excitotoxicity, Aβ42 has been demonstrated to reduce astrocyte reuptake of L-glutamate, as well as positively modulating activity of the NMDAR leading to an increase in neuronal Ca 2+ concentration (Harkany et al., 2000; Texidó et 32

34 al., 2011; Harada & Sugimoto, 1999). While oxidative stress may be a downstream effect of excitotoxicity (Doble, 1999), there is also evidence of a pathway of superoxide generation by Aβ42 interaction with endogenous metal cations (Hureau & Faller, 2009), as well as superoxide production from over activation of oxidase enzymes (Abramov et al., 2004). The Aβ plaques, however, are not consistently correlated to the neuropathological stage (Braak & Braak, 1991). Removal of Aβ plaques does not appear to have a consistent significant effect on disease progression, although improvements to trial methodologies are required (Lambracht-Washington & Rosenberg, 2013). Interestingly, the BCAA analogue valproic acid has been found to decrease formation of Aβ plaques in AD both in vivo and in vitro, potentially due to inhibition of glycogen synthase kinase 3 β (GSK3β) and tau kinases (Qing et al., 2008; Uchida et al., 2005) Tau pathology. The tau proteins belong to a family of microtubule-associated proteins (MAP) vital for cellular trafficking of proteins and organelles (typically exosomes and vesicles), as well as morphology in neurons (Spires-Jones et al., 2009). The tau MAP gene (MAPT) is highly conserved and alternatively spliced into many variants, suggesting an important multifunctional role (Goedert et al., 1988; Spires-Jones et al., 2009). In particular, it has been demonstrated that tau is required for rapid dendrite extension in developing neurons, and that overexpression may inhibit microtubule trafficking (Esmaeli-Azad et al., 1994; Stamer et al., 2002). A number of post-translational modifications control association of the tau protein to microtubules, most notably phosphorylation, which in turn regulates the trafficking activity of the protein (Konzack et al., 2007). Under pathological conditions (tauopathy), tau protein becomes hyperphosphorylated and completely disassociates from the microtubule. This has a destabilising effect on the cytoskeleton which impairs cell morphology and disrupts cellular trafficking, eventually causing synapse dysfunction and potentially apoptosis (Spires-Jones et al., 2009). Furthermore, hyperphosphorylated tau becomes intertwined and forms the 33

35 aforementioned NFTs observed in AD (Spires-Jones et al., 2009). Although NFTs have only been demonstrated as toxic in humans in vitro (Mookherjee & Johnson, 2001), their spread remains the only histological marker correlated with progression of AD (Braak & Braak, 1991). As with Aβ42 toxicity, there is substantial research to suggest a relationship between excitotoxicity and tauopathy (Reviewed by Ittner & Götz, 2011). Specifically, studies have found that excitotoxicity results in a short, rapid increase in non-phosphorylated tau, followed by a delayed but sustained period of hyperphosphorylated tau (Mattson, 1990; Pooler et al., 2013; Crespo-biel et al., 2007; Liang et al., 2009). Following a review of several studies, Cowan & Mudher (2013) suggest that the soluble non-phosphorylated tau may be the most toxic, while the NFTs are of low toxicity or not toxic at all. In particular, this is supported by antisense studies which found that inhibiting excitotoxicitytriggered tau upregulation was associated with an increase in cell viability (Pizzi et al., 1994). As well as the initial impact, non-phosphorylated tau is also exported from neurons by excitotoxic stimulation, where it may act as a signalling protein or spread tauopathy to neighbouring cells (Pooler et al., 2013). Indeed, in terms of signalling, tau is able to activate muscarinic acetylcholine receptors, particularly M1 and M3, resulting in an increase in intracellular Ca 2+ concentration (Gomez-Ramirez et al., 2008). As tau is upregulated following an increase in intracellular Ca 2+ concentration (as in excitotoxicity), this may lead to outward cell-to-cell spread of extracellular tau as a chain reaction (Pooler et al., 2013; Mohamed et al., 2013). The pathway by which tau is released from the cytoskeleton and forms NFTs has also been determined. It has been found that excitotoxicity first activates protein phosphatase 2A (PP2A) which dephosphorylates the tau proteins, stimulating release from the microtubules (Liang et al., 2009). The tau proteins are then truncated by Ca 2+ activated caspase-3 to a more toxic form (Crespo-biel et al., 2007; Zilka et al., 2006). After a delay of approximately an hour, PP2A is subsequently inhibited and tau becomes 34

36 hyperphosphorylated by kinases such as GSK3β and Cyclin-dependent kinase 5 to form NFTs (Liang et al., 2009; Crespo-Biel et al., 2007; Zilka et al., 2006). Furthermore, inhibition of PP2A and activation of GSK3β is known to inhibit autophagy and as such excitotoxicity may reduce cellular capacity to degrade NFTs (Bánréti et al., 2012; Krüger et al., 2012; Caccamo et al., 2013) Protein synthesis and autophagy. Cellular health depends on the correct balance of protein turnover. Although there are many ways to activate gene transcription and protein synthesis, proteolysis falls mainly between two pathways; the ubiquitin pathway for short-lived or misfolded proteins, and macroautophagy for amino acid recovery in response to stress (Reviewed by Larsen & Sulzer, 2002). There are a significant number of studies which suggest that the BCAAs have an anabolic effect on cells and may stimulate protein synthesis while inhibiting macroautophagy. This is considered to be mediated primarily through the mammalian target of rapamycin (mtor) pathway (Blomstrand et al., 2006; Rennie et al., 2006). The mtor enzyme is a serine/threonine kinase which forms part of the highly regulated mtor complex 1 and 2, important for signalling cell survival and growth via upstream sensing proteins (Figure 1.9). It is considered that in nutritionally favourable conditions, the mtor complex 1 is activated by these sensors and will phosphorylate substrates such as eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) and p70s6 kinase, to stimulate protein synthesis (Reviewed by Laplante & Sabatini, 2012) (Figure 1.9). Furthermore, phosphorylation of Unc-51 Like Autophagy Activating Kinase 1 protein leads to a reduction in macroautophagy by inhibiting formation of the autophagosome (Kim et al., 2001) (Figure 1.9). On the other hand, during stress or starvation the mtor complex 1 is not activated, leading to a net protein synthesis inhibition and macroautophagy activation (Laplante & Sabatini, 2012). The amino acids released from macroautophagy may activate the mtor1 complex as a negative 35

37 Figure 1.9 mtor1 pathway. The mtor1 pathway is involved in the regulation of autophagy and protein synthesis in response to a cellular stress and nutrient signals. In particular, mtor1 is activated by amino acids and insulin. L-leucine has been demonstrated as the strongest amino acid activator of mtor1 and activation of mtor1 inhibits protein degradation and recycling by macroautophagy. Conversely, while in a starvation state mtor1 is inhibited and macroautophagy becomes active. Release of amino acids following autophagy deactivates mtor1 as a negative feedback loop (Zarogoulidis et al., 2014). Abbreviations: 4EBP1 Eukaryotic translation initiation factor 4E-binding protein 1. AKT RAC-alpha serine/threonine-protein kinase. IRS1 Insulin receptor substrate 1. PI3K Phosphoinositide 3-kinase. PP2A Protein phosphatase 2A. Rheb Ras homolog enriched in brain. TSC1 Tuberous sclerosis 1. 36

38 feedback loop (Laplante & Sabatini, 2012) (Figure 1.9). Studies have shown that mtor is activated by L-leucine more than by any other amino acid (Shigemitsu et al., 1999), although the BCKA, KIC, was also a strong activator of mtor indicating that BCAT transamination may be important in mtor activation (Xu et al., 2001). With this considered, inhibitory compounds or knock-down studies could be used to examine the importance of hbcat in L-leucine signalling; specifically, to investigate if L-leucine mediated mtor activation is dependent on hbcat activity. There is strong evidence in animal models to support a relationship between activation of mtor and the presence of the histological features of AD (Caccamo et al., 2010; Li et al., 2005). Increased activation of mtor is correlated with a rise in the number and severity of Aβ plaques and NFTs, while reduced mtor activity resulted in fewer Aβ plaques and NFTs. It has been proposed that the former is due to an increase in autophagy activity, which could degrade plaques and NFTs within cells (Caccamo et al., 2010; Hamano et al., 2008) (Figure 1.11) Protein misfolding and redox pathology. Increases in misfolded proteins and oxidative stress are well described in the pathology of AD (Reviewed by Sekoe, 2004 and Butterfield et al., 2001; Wang et al., 2014). Indeed, studies have determined that misfolding can both be caused by, and amplify, Aβ toxicity and tau pathology, which are intrinsically linked to AD (Stamer et al., 2002; Butterfield et al., 2001). An excess of misfolded proteins may cause oxidative stress, while conversely, oxidative stress may lead to misfolded proteins, indicating that a fine balance is required for healthy physiological conditions (Reviewed by Malhotra & Kaufman, 2007b). Several proteins involved in the response to oxidative stress and unfolded proteins are dysregulated or dysfunctional in AD (Reviewed by Sekoe, 2004 and Butterfield et al., 2001). Of these, the enzymes PDI, thioredoxin (TRx), and glutaredoxin (GRx) are of particular interest for this study because, similar to hbcat, they contain a 37

39 CXXC motif critical to their activity (Reviewed by Carvalho et al., 2006). Functionally, TRx, and GRx reduce substrate protein disulphide bonds to thiols using a co-enzyme or cofactor; glutathione for PDI and GRx, and the protein TRx reductase for TRx (Reviewed by Fernandes & Holmgren, 2004, Arnér & Holmgren, 2000, and Wilkinson & Gilbert, 2004). As previously described, the hbcat enzymes have a chaperone activity which can be enhanced by PDI and glutathione through the CXXC motif (El Hindy et al., 2014). Because of these similarities in redox sensitive structure and chaperone activity, it is an aim of this thesis to determine if hbcat has an impact on the redox status of the cell, either by regulating other redox proteins or by modifying glutathione concentration. In AD pathology studies, it has been demonstrated that TRx is downregulated in neurons, while reports of GRx expression are inconsistent (Lovell et al., 2000; Akterin et al., 2006; Ginsberg et al., 2006). A study of 120 cases found that both GRx and TRx were significantly increased in the cerebrospinal fluid of patients with AD compared to controls; this was unrelated to cell death, suggesting that the proteins were exported by cells (Arodin et al., 2014). Expression of PDI in the AD brain also found that the protein is colocalised with NFTs and inactivated by S-nitrosylation, leading to an accumulation of misfolded proteins (Uehara et al., 2006; Honjo et al., 2014) The blood-brain barrier. The BBB controls the flow of molecules, macromolecules, or cells between the cerebral tissue and the blood supply of the body. Within the brain, the vasculature is comprised primarily from endothelial cells, astrocytes, pericytes, and extracellular matrix (Reviewed by Abbott et al., 2010). The endothelial cells which constitute peripheral vasculature form a relatively permeable layer, allowing for the free flow of nutrients, macromolecules, and for invasion by cells. However, distinct characteristics of the cells which compose the BBB prevent the free flow of these entities into the brain, and as such the environment is extremely highly regulated (Ehrlich, 1885; reviewed by Rubin & Staddon, 1999). The 38

40 brain is a sterile immune-privileged environment and both pathogenic organisms and most leukocytes are prevented access under healthy conditions (Wekerle et al., 1986; Scholz et al., 2007). However, there are conditions which may lead to a reduction in integrity and function of the BBB such as acute inflammatory response, drug administration, and oxidative stress (Andersson et al., 1992; Scholz et al., 2007; Cosolo et al., 1989; Lochhead et al., 2010). This permeability may lead to an influx of peripheral macromolecules from the plasma such as albumin and plasmin, resulting in oedema and neuronal injury (Zhong et al., 2009; Chen & Strickland, 1997). Additionally, an influx of erythrocytes can cause accumulation of iron, generating ROS (Zhong et al., 2009), while impaired metabolite and nutrient exchange is also reported (Reviewed by Zlokovic, 2008). Finally, diseases such as multiple sclerosis are characterised by leukocyte invasion and may be further compounded by existing inflammatory cytokines and oxidative stress (Daneman, 2012; Mracsko et al., 2014). Initially, it was proposed that the BBB was a unique vascular organ formed by sheets of astrocytes creating a second barrier (Dempsey & Wislocki, 1955), however, experiments with tissue transplantation and hormone treatments found that morphological changes in endothelial cells were induced by the local environment (Stewart & Wiley, 1981). Primary mediators of these morphological changes are the glucocorticoids and astrocyte paracrine signalling (Janzer & Raff, 1987; Luthert et al., 1986) which stimulate an upregulation of proteins which transport molecules between the luminal and abluminal compartments or vice versa. Additionally, these signals increase expression of structural proteins, which pull neighbouring endothelial cells into closer adhesion. There are several classes of the structural proteins, but the most-well characterised are the tight junction (TJ) and adherens junction (AJ) proteins, both of which anchor themselves intracellularly to the actin skeleton and pull cells closer together to create an impermeable junction in a manner similar to muscle cell contraction (Reviewed by Rubin & Staddon, 1999). 39

41 In TJs, the occludin and claudin family of proteins act as extracellular hooks which bind tightly between cells. These are connected to zonula occludens (ZO) family proteins which move along the actin cytoskeleton to form a contraction between the two cells (Figure 1.10; A) (Reviewed by Anderson & Van Itallie, 1995). While the TJs are considered central to reducing paracellular permeability, the AJs appear also to have more of a role in maturation and signalling. The AJ proteins are Ca 2+ dependent cadherins and are bound to the actin skeleton via catenins which again pull the endothelial cells into closer adhesion (Reviewed by Anderson & Van Itallie, 1995; Hartsock & Nelson, 2008). Because of the close adhesion, molecules are unable to pass between the endothelial cells and they must instead pass through them. Passively this may occur if a molecule is highly lipophilic, indeed many drugs are designed to be as lipophilic as possible while maintaining solubility in order to pass through the BBB in this manner. Alternatively, if the molecule is (or is bound to) a polycationic molecule it may become enveloped and pass through the cell by adsorptive transcytosis (Rapoport et al., 1979). Molecules may also be transported through facilitated or active transport. There are a wide variety of pumps or transporters for active transport of molecules, such as glucose transporter 1 (GLUT1), LNAAT, ATA2, excitatory amino acid transporter (EAAT) 1, and monocarboxylic acid transporter (Mct) 1 for α-keto acid transport (Ohtsuki & Terasaki, 2007; Smith et al., 2012). Although transporters may be utilised for drug delivery, they may also cause significant resistance to drugs targeting the brain, as efflux pumps such as p-glycoprotein (P-gp) may transport compounds out of the brain (Figure 10; [B]) (Thiebaut et al., 1987). Additionally, enzymes such as monoamine oxidase B are present in the endothelial cells and may degrade many substrate molecules, providing a metabolic barrier (Kalaria & Harik, 1987). To support these active functions, there is an increased mitochondrial density in the endothelial cells characteristic of the BBB (Oldendorf et al., 1977). Impaired BBB integrity and poor cerebral blood flow (CBF) are widely reported in AD (Alsop et al., 2000; Ujiie et al., 2003; Reviewed by Nelson et al., 2016). This is consistent 40

42 Figure 1.10 Mechanisms of the BBB. Transport between the blood and the brain is tightly regulated by endothelial cells of the cerebral microvasculature. [A] Structural proteins bring cells into close adhesion, preventing flow of entities through the paracellular cleft. [B] Many entities may enter the brain by travelling through the endothelial cells. If a molecule is sufficiently lipophilic it may diffuse through the cell, however there are specific transporters for many vital nutrients. The P-gp transporter can also remove unrequired compounds from the brain. Furthermore, compounds may be catabolised in the endothelial cells before being transported. Abbreviations: ZO Zonula occludens. LNAAT Large neutral amino acid transporter. EAAT1/2/3 Excitatory amino acid transporter 1/2/3. P-gp P-glycoprotein. GLUT1 Glucose transporter 1. ATA2 Amino acid transporter. Mct2 Monocarboxylic acid transporter. 41

43 with accumulation of albumin, fibrogen, plasminogen, and iron in AD brains due to increased permeability (Blennow et al., 1990; Ryu & McLarnon, 2009; Hultman et al., 2013; Deibel et al., 1996). Additionally, decreased CBF precedes cognitive impairment and neuronal dysfunction in aged individuals at risk of developing AD (Ruttenberg et al., 2005; Carmeliet & De Strooper, 2012). Reduced CBF leads to a reduced ability of the brain to clear Aβ, causing accumulation of the toxic peptide along the vascular walls (Thal et al., 2009). Critically, it is hypothesised that a decrease in Aβ clearance is the initial cause of Aβ accumulation, rather than the increased production (Mawuenyega et al., 2010). This Aβ accumulation is termed cerebral amyloid angiopathy (CAA) and is linked to brain microbleeds, vascular inflammation and oxidative stress; indeed, CAA is present in 90% of AD patients (Thal et al., 2009; Zlokovic et al., 2011). A vascular twohit hypothesis of AD has been proposed (Zlokovic, 2011) (Figure 1.11). This proposes that early in the disease, vascular insult increases BBB permeability, allowing entry of peripheral Aβ into the brain and hypoperfusion (leading to hypoglycaemia and hypoxia), with the end result of neuronal injury (Reviewed by Zlokovic, 2011). The vascular insult leads to increased BBB disorder present in early AD, and worsens as the diseases progresses (de la Torre & Mussivand, 1993; Skoog et al., 1998) in correlation with Braak staging (Jellinger et al., 2010). As described, CAA increases oxidative stress (Thomas et al., 1996), further impacting the vascular disorder. This has been demonstrated to decrease BBB integrity by inducing a dysfunctional rearrangement of tight junction proteins (Schreibelt et al., 2007), while the antioxidant glutathione was able to ameliorate this effect, potentially through the oxidative response pathway (Song et al., 2014). Furthermore, ROS and NO have been demonstrated to activate matrix metalloproteinases which can also degrade TJ proteins and disrupt BBB integrity (Haorah et al., 2007; Gu et al., 2002). Efficient mitochondrial function is required for maintenance of the BBB, and dysfunction has been demonstrated to increase permeability (Doll et al., 2015), additionally mitochondrial 42

44 Figure 1.11 The vascular two-hit hypothesis of AD. Vascular genetic risk factors or diseases may promote cerebrovascular dysfunction. This is hypothesised to be a first hit in AD, causing neuronal insult, increases in Aβ production, and inhibition of Aβ clearance. The second hit is the resulting Aβ accumulation, which can exasperate neuronal insult, leading to synapse dysfunction, neurodegeneration, and eventually dementia. (Image reprinted with permission; Nelson et al., 2016) 43