Transglutaminases and neurodegeneration

Thomas M. Jeitner*, John T. Pinto†, Boris F. Krasnikov†, Mark Horswill‡, and Arthur J. L.Cooper†*Red Anvil, LLC, Milwaukee, Wisconsin, USA†Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NewYork, USA‡Developmental Vascular Biology Program, Medical College of Wisconsin, Milwaukee, Wisconsin,USA

AbstractTransglutaminases (TGs) are Ca2+-dependent enzymes that catalyze a variety of modifications ofglutaminyl (Q) residues. In the brain, these modifications include the covalent attachment of a numberof amine-bearing compounds, including lysyl (K) residues and polyamines, which serve to eitherregulate enzyme activity or attach the TG substrates to biological matrices. Aberrant TG activity isthought to contribute to Alzheimer disease, Parkinson disease, Huntington disease, and supranuclearpalsy. Strategies designed to interfere with TG activity have some benefit in animal models ofHuntington and Parkinson diseases. The following review summarizes the involvement of TGs inneurodegenerative diseases and discusses the possible use of selective inhibitors as therapeutic agentsin these diseases.

Keywordscystamine; neurodegeneration; polyamines; transglutaminase; γ-glutamylpolyamines; γ-glutamyl-ε-lysine

Cerebral transglutaminasesEight active transglutaminases (TGs) (TGs 1–7 and factor XIIIa) are expressed in mammals,of which TGs 1–3 (Kim et al. 1999)1 and 6 (Hadjivassilou et al. 2008) are present in humanbrain. The major reaction thus far attributed to the cerebral TGs is transamidation. In thisreaction the carboxamide moiety of a Q residue [-C(O)NH2] is converted to a substitutedcarboxamide [-C(O)NHR] by nucleophilic attack of an amine [RNH2] such as various mono-,di-, and polyamines or the ε amino group of a K residue (Lorand and Graham 2003). Of thepossible transamidation linkages, the γ-glutamyl-ε-lysine [Nε-(γ-L-glutamyl)-L-lysine] (GGEL)isopeptide linkage formed between Q and K resides, is the most commonly studied. GGELbonds occur both within and between polypeptide chains, and thereby contribute to theformation of stable soluble and insoluble polymers. TGs also cross-link proteins via bis-γ-glutamylpolyamine bridges between Q residues (Piacentini et al. 1988). These linkages areformed by two successive transamidations: the first utilizes a free polyamine to generate a γ-glutamylpolyamine residue, which becomes the amine-bearing substrate for a second

Address correspondence and reprint requests to Dr Arthur J. L. Cooper, Department of Biochemistry and Molecular Biology, New YorkMedical College, Valhalla, NY 10595, USA. arthur_cooper@nymc.edu.Conflicts of interest: All authors declare no conflicts of interests.1Given the need for brevity, the number of citations has been restricted/limited.

NIH Public AccessAuthor ManuscriptJ Neurochem. Author manuscript; available in PMC 2009 September 28.

Published in final edited form as:J Neurochem. 2009 May ; 109(Suppl 1): 160–166. doi:10.1111/j.1471-4159.2009.05843.x.

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transamidation. bis-γ-Glutamylpolyamine cross-links are formed at least as frequently as thoseinvolving GGEL (Piacentini et al. 1988). Under physiological conditions, the majority of cross-linking bonds are generated outside of cells where the concentrations of Ca2+ are sufficientlyhigh enough to stimulate the catalysis of these bonds by TGs. TG 2 and 3 have three Ca2+

binding sites and the current indications are that the catalysis of GGEL bonds requires theoccupation of all three sites (Datta et al. 2006).

Intracellular Ca2+ concentrations rarely match the extracellular concentrations. Moreover, GTPacts in cells as an endogenous inhibitor of TGs (Bergamini et al. 1987). Nevertheless,intracellular TG-catalyzed reaction products can be detected in normal cells, especially thoseproducts related to polyamination (Piacentini et al. 1988). The consequences of polyaminationin the brain, however, are poorly understood as only a limited number of polyaminated proteinshave been identified (Tucholski et al. 1999). Of these, phospholipase A2 (PLA2) is especiallyinteresting since the polyamination of this enzyme may contribute to the inflammationassociated with neurodegeneration. PLA2 produces two groups of pro-inflammatorymediators: leukotrienes and prostaglandins. Polyamination of PLA2 results in a 3-fold increasein activity (Cordella-Miele et al. 1993) that may persist for the life of the protein given theinability of most peptidases to hydrolyze γ-glutamylamine (GGEL, γ-glutamylpolyamine andbis-γ-glutamylpolyamine) linkages (Fink and Folk 1981). Thus, polyamination may representa unique post-translational modification of enzymes that permanently affects activity. Thissituation contrasts with other types of covalent post-translational modifications, such asphosphorylation that typically are transient.

Increased TG(s) in neurodegenerative diseasesTransglutaminase activity is widespread in brain (Kim et al. 1999) and is present in primarycultures of neurons and astrocytes (Perry et al. 1995; Caccamo et al. 2004). TG 2 is associatedwith the extracellular matrix, cell membranes and cytosol of neurons, and TG activity has beenidentified in synaptosomes (Pastuszko et al. 1986), mitochondria (Krasnikov et al. 2005), andnuclei (Lesort et al. 1998). The activity, expression and amounts of individual TG enzymesare increased in a variety of neurodegenerative diseases. TG activity is significantly elevatedin the affected cerebral regions in Alzheimer disease (AD) (Johnson et al. 1997; Kim et al.1999), Huntington disease (HD) (Karpuj et al. 1999; Lesort et al. 1999), and supranuclear palsy(Zemaitaitis et al. 2003). These increases in activity are accompanied by gains in the amountof TG 1 and TG 2 proteins in AD brain (Kim et al. 1999; Bonelli et al. 2002), and also of TG2 protein in the brains of HD (Lesort et al. 1999) and supranuclear palsy (Zemaitaitis et al.2003) patients. Increased TG 2 protein is also found in the CSF of AD (Bonelli et al. 2002)and Parkinson disease (PD) (Vermes et al. 2004) patients.

Not only are the amounts of TG increased in AD, HD and supranuclear palsy, but the conditionsfavoring the activation of these enzymes are also enhanced in these diseases. These conditionsinclude elevations in intracellular Ca2+ due to glutamate-mediated excitotoxity (Caccamo etal. 2004) and other perturbations in Ca2+ homeostasis (Mattson 2007) as well as decreases inGTP concentrations following from losses in energy production (Lin and Beal 2006).

The number of TG 2 transcripts is also increased in HD (Lesort et al. 1999) and supranuclearpalsy (Zemaitaitis et al. 2003), and a shortened alternate transcript of TG 2 encoding a formof enzyme missing the GTP binding domain is expressed in AD brain (Festoff et al. 2002). Anumber of mechanisms may account for the increased transcription and translation of TGs inneurodegenerative disorders. The TG 1 promoter has Ca2+ (Kawabe et al. 1998), retinoid(Polakowska et al. 1999), cAMP, Sp1, and AP1 responsive elements (Medvedev et al. 1999),while the TG 2 promoter contains elements that respond to retinoids (Nagy et al. 1996; Yanet al. 1996), interleukin 6, transforming growth factor β1 (Ritter and Davies 1998), and tumor

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necrosis factor-α (Kuncio et al. 1998). The inflammatory mediators are likely to act via theNF-κB (Nuclear factor κB) binding region in the TG 2 promoter (Kuncio et al. 1998; Kim etal. 2008). NF-κB translocation and DNA binding are stimulated by tumor necrosis factor-αand glutamate, both of which have been shown to increase TG 2 expression in microglia andastrocytes (Campisi et al. 2004; Park et al. 2004). As noted earlier, inflammation accompaniesneurodegeneration and TGs may contribute to this response via the sustained activation ofpolyaminated PLA2. The possibility that TGs may contribute to their continued activationhighlights the necessity of limiting the activity of these enzymes in neurodegenerativedisorders.

Increased TG-catalyzed products in neurodegenerative diseasesIncreased TG activity in neurodegenerative disorders is accompanied by an increase in TG-catalyzed products. Selkoe et al. (1982a,b) demonstrated that cerebral TGs catalyze the invitro polymerization of cytoskeletal elements, and hypothesized that TGs might facilitatepaired helical formation in AD tangles. TG 2 and GGEL cross-links were subsequently shownto co-localize with the tangles (Miller and Anderton 1986; Johnson et al. 1997) and TG2 wasshown to co-localize with plaques (Zhang et al. 1998) in AD brain. Components of the plaquesor tangles, including β-amyloid (Aβ) (Ikura et al. 1993; Dudek and Johnson 1994; Ho et al.1994; Rasmussen et al. 1994), the Dutch mutation of Aβ (Q22 → E22) (Dudek and Johnson1994), tau (Miller and Anderton 1986; Dudek and Johnson 1993; Miller and Johnson 1995;Appelt and Balin 1997; Murthy et al. 1998; Tucholski et al. 1999), and the non-Aβ componentderived from α-synuclein (Jensen et al. 1995) are TG substrates. The in vitro products of thereaction of these substrates with TG bear a striking resemblance to the insoluble polymersfound in AD brain (Jensen et al. 1995; Appelt and Balin 1997; Hartley et al. 2008).

Huntington disease is caused by a CAG expansion in the huntingtin (htt) gene that encodes alength of contiguous Q residues [polyglutamine (Qn)] in the N-terminus of the expressedprotein. Green (1993) hypothesized that the expanded Qn region would favor the formation ofTG-catalyzed GGEL linkages and lead to the formation of htt-containing aggregates. In supportof this hypothesis, expanded Qn domains are excellent TG substrates (Kahlem et al. 1996;Cooper et al. 1997a; Gentile et al. 1998; Lesort et al. 1999; Zainelli et al. 2005) and mutanthtt is present in HD aggregates (DiFiglia et al. 1995) as are GGEL crosslinks (Zainelli et al.2003).

The increased cerebral aggregation seen in HD, PD, and supranuclear palsy is also associatedwith a comparable increase in GGEL immunoreactivity within the polymers (Zemaitaitis etal. 2000; Zainelli et al. 2003; Andringa et al. 2004). Although some concerns have been raisedabout the specificity of GGEL antibodies in immunoblots (Johnson and LeShoure 2004), theincrease in protein-associated GGEL in AD brain has been unequivocally confirmed usingmass spectrometric techniques (Kim et al. 1999; Nemes et al. 2004).

As noted earlier, the isopeptide bonds in γ-glutamylamine linkages are resistant to proteolysis(Fink and Folk 1981). Moreover, the ability to metabolize free γ-glutamylamines in brain islimited. Consequently, γ-glutamylamines are excised intact during proteolysis and are presentin brain and CSF (Jeitner et al. 2001, 2008; Dedeoglu et al. 2002). A several-fold increase infree GGEL has been measured in the brains of HD patients (Dedeoglu et al. 2002), and theamount of GGEL in the CSF of patients with AD, PD (Sárvari et al. 2002), or HD (Jeitner etal. 2001, 2008; Dedeoglu et al. 2002) is also increased relative to control CSF. The increasein CSF GGEL reported in HD is also matched by comparable increases in the amounts of CSFγ-glutamylspermidine, γ-glutamylputrescine and bis-γ-glutamylputrescine (Jeitner et al.2008). CSF contains higher (μM) quantities of γ-glutamylspermidine than GGEL (< μM) in

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accord with the suggestion noted above that TGs predominantly catalyze polyamination overQ → K cross-linking within the brain.

Possible mechanisms for TG-mediated neurotoxicityAlthough the formation of insoluble protein aggregates has been proposed to account for thetoxic actions of TGs, the role of such aggregates in the etiology of diseases such as HD iscontroversial (Kuemmerle et al. 1999; Sieradzan and Mann 2001). Indeed, mice that lackedTG 2 and over-express mutant htt had 30% more brain aggregates and still lived longer thantheir TG 2- and mutant htt-expressing littermates (Mastroberardino et al. 2002). The increasedaggregation is unlikely to have been due to compensatory cross-linking by TG 1 and 3, sincethe TG 2-deficient mice also exhibited a 10-fold decrease in the number of GGEL linkages. Itwas subsequently shown that GGEL cross-links serve to produce soluble Qn aggregates,whereas polyaminated or unmodified Qn domains spontaneously aggregate to form insolublepolymers (Lai et al. 2004; Konno et al. 2005). These observations suggest that TG 2-catalyzedGGEL bond formation generates soluble aggregates that may be neurotoxic.

Another possibility is that rather than being toxic per se, the soluble and insoluble polymerscause neuronal death by sequestering critical proteins within the aggregates. These proteinscould include, for example, glyceraldehyde 3-phosphate dehydrogenase, α-ketoglutaratedehydrogenase, and histones (Cooper et al. 1997b, 2000; Gentile et al. 1998) in HD, andubiquitin, HSP27, parkin, and α-synuclein in AD (Nemes et al. 2004). It has also been suggestedthat congestion of proteasomes may contribute to CAG-expansion and other neurodegenerativediseases (Cooper et al. 2002; Wang et al. 2008). In support of this hypothesis, components ofthe ubiquitin proteasome system are found in HD aggregates (Bennett et al. 2007).

The above observations suggest the following model for the contribution of TGs toneurodegenerative diseases. Early in these diseases, TGs predominantly catalyzepolyamination reactions. As these diseases progress, TGs begin to form more GGEL cross-links, which stabilize soluble toxic protein aggregates, eventually leading to removal of keyproteins and to a fatal congestion of proteasomes.

TGs as potential therapeutic targets in neurodegenerative diseasesSeveral authors have raised the possibility that TG inhibitors may be of therapeutic benefit inneurodegenerative diseases (e.g. Cooper et al. 2002; Gentile and Cooper 2004), and one suchin vitro inhibitor – cystamine – is beneficial in murine models of HD and PD (e.g. Dedeogluet al. 2002; Van Raamsdonk et al. 2005; Stack et al. 2008). We have shown that cysteamine,the reduced form of cystamine, is a competitive inhibitor/alternative substrate of TG 2 (Jeitneret al. 2005). Cysteamine attenuates polyamination by acting as an alternative TG 2 substrate,presumably forming Nβ-(γ-L-glutamyl)-cysteamine linkages (Jeitner et al. 2005). In additionto cysteamine, cystamine is metabolized to hypotaurine and taurine, and cystamine treatmentin mice leads to increased brain cysteine levels (Fox et al. 2004; Pinto et al. 2005). We testedthe ability of hypotaurine, taurine, cysteine, and cysteamine to inhibit TG 2. Of the testedcompounds, only cysteamine was able to inhibit TG 2-catalyzed polyamination (Fig. 1). Asneither cystamine nor cysteamine can be detected (detection limit ≤ 20 μM) in the brains ofcystamine-treated YAC128 (HD) mice (Pinto et al. 2005), the conversion of cystamine tocysteamine, and then to Nβ-(γ-L-glutamyl)-cysteamine, is likely to be rapid.

Prolonged cystamine treatment results in decreased TG activity (Dedeoglu et al. 2002; VanRaamsdonk et al. 2005), even though cysteamine is not an irreversible TG inhibitor (Jeitneret al. 2005). Thus, another mechanism must account for the diminished TG activity. Wehypothesize that cystamine-derived cysteamine inhibits the binding of transcription factors toTG promoters and thereby limits the transcription of TGs. In support of this hypothesis,

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cysteamine attenuates the DNA binding of AP1 and NF-κB (Goldstone et al. 1995), and bindingsites for these factors are present in the TG 1 and TG 2 promoters (Kuncio et al. 1998;Medvedev et al. 1999; Kim et al. 2008).

As indicated above, cystamine has multiple biological actions in the brain, including raisingthe levels of cysteine (Fox et al. 2004; Pinto et al. 2005). Cystamine also causes elevation ofbrain-derived neurotropic factor (Borrell-Pages et al. 2006) and possibly attenuates apoptosisthrough inhibition of caspase 3 activity (Lesort et al. 2003). Recently, we discovered anotherpotentially beneficial property of cystamine. Cystamine, at concentrations as low as 15 μM,significantly attenuated dopamine-induced macroautophagy in SH SY5Y cells, whereascysteamine had no effect (Fig. 2). Dopamine reduced the viability of SH SY5Y cells by 48 ±3% (mean ± SEM, n = 11) after 24 h, while the combination of dopamine and cystamine (15μM) only reduced viability by 24 ± 2% (p < 0.05, paired t-test). In these experiments, the cellswere pre-treated with cystamine for 2 h, and then treated for a further 24 h (i.e. 26 h).Importantly, the treatment of cells with cystamine for only 4 h prior to the application ofdopamine was as effective as the 26-h cystamine treatment (Fig. 2). This observation suggeststhat cystamine primes the cells against the induction of macroautophagy.

Given the multiplicity of biological activities attributed to cyst(e)amine, it is difficult to assignthe therapeutic benefit of this agent to the inhibition of TGs per se. Moreover, excesscysteamine has been reported to be harmful in at least one setting. Thus, Frankel and Schipper(1999) noted that cysteamine induces the appearance of iron-rich (peroxidase-positive)cytoplasmic inclusions in cultured rat astroglia, which are identical to glial inclusions thatprogressively accumulate in substantia nigra and other subcortical brain regions with advancingage.

The positive results obtained with a mouse model of HD in which the animals lacked TG 2 area more compelling argument for the involvement of TGs in neurodegeneration(Mastroberardino et al. 2002) than the results obtained with cystamine. In this regard, severalgroups are actively synthesizing more selective TG inhibitors than cystamine as possibletherapeutic agents. These inhibitors include dihydroisoxazole derivatives, peptide-bound1,2,4-thiadiazoles, peptides containing diazo-5-oxo-L-norleucine in place of glutamine, α,β-unsaturated amides and epoxides (Pardin et al. 2008a,b). Pardin et al. have recently focusedtheir attention on trans-cinnamoyl benzotriazole amides and 3-(substituted cinnamoyl)pyridines [azachalcones], which are potent reversible TG inhibitors (Pardin et al. 2008a,b),and have discovered a triazole compound that inhibits guinea pig TG 2 with a Ki value of∼170 nM (Pardin et al. 2008b). Stein and colleagues have discovered another series ofreversible TG inhibitors that are thieno[2,3-d]pyrimidin-4-one acylhyd-razide derivatives(Duval et al. 2005; Case and Stein 2007).

Finally, Sohn et al. (2003) have developed a novel strategy that blocks the polyamination ofPLA2. This group noted that uteroglobin and lipocortin-1 contain common sequences thatantagonize the interaction of TG 2 with PLA2. Synthesized variants of these sequencesprevented both the polyamination of PLA2 and experimentally-induced allergic conjunctivitisin experimental animals. These results suggest that rather than inhibiting TG 2 directly, somebenefit may be derived from targeting the interaction of TGs and specific pathogenic TGsubstrates. Polyaminated PLA2 may be one such target in neurodegenerative disorders.

Conclusion and future prospectsAlthough TGs do not cause neurodegenerative diseases directly, the current evidence suggeststhat this family of enzymes contributes to the neuropathology once the disease process hasbegun. It is anticipated that potent TG inhibitors will soon be evaluated for their therapeutic

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potential in cellular and animal models of HD and other neurodegenerative diseases. Care willbe required to ensure that these inhibitors are sufficiently selective so as not to affect crucialTG reactions critical to normal metabolic processes or to inhibit blood clot formation.

AcknowledgmentsPart of the authors' work cited herein was supported by the National Institutes of Health grant PO1 AG14930.

AbbreviationsAD

Alzheimer disease

Aβ β-amyloid

GGEL γ-glutamyl-ε-lysine [Nε-(γ-L-glutamyl)-L-lysine]

HD Huntington disease

htt huntingtin

PD Parkinson disease

PLA2 phospholipase A2

Qn polyglutamine

TG transglutaminase

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Fig. 1.Effect of cysteamine, cysteine, hypotaurine, and taurine on TG activity. TG activity in thepresence of cysteamine (□), cysteine (■) hypotaurine (○), or taurine (●) was determined bymeasuring the incorporation of radiolabeled putrescine into N,N-dimethylcasein as describedby Jeitner et al. (2005) using tritiated putrescine and his-tagged guinea pig TG 2 (Gillet etal. 2004). The data are depicted as percent of the control (21 910 ± 1731 dpm per tube) andrepresent the mean ± SEM of four separate experiments. The data with cysteamine atconcentrations ≥ 2.5 mM and taurine at 5 and 10 mM were significantly different from that ofthe control (p < 0.05, paired t-test).

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Fig. 2.Effect of cyst(e)amine on dopamine-induced macroautophagy. SH SY5Y cells at 70%confluence in a 75 cm2 flask were detached with 0.05% trypsin : verscene (1 : 1), then collectedinto 50 mL 10% fetal bovine serum, 90% Dulbecco's modified eagle medium prior to seedingonto 24 multi-well plates at 1 mL per well. Twenty-four hours later the cells were treated witheither cystamine (■) or cysteamine (□) for 2 h. The cells were then incubated for an additional24 h together with 10−4 M dopamine to induce macroautophagy as described by Gomez-Santoset al. (2003). The data from these studies are depicted as the mean ± SEM of four separateexperiments. The cells were also treated with cystamine for 4 h then washed three times withHank's balanced salt solution (HBBS) at 37°C, followed by 24 h incubation with 100 μMdopamine (gray open circles). The data from these studies represent the mean of two individualexperiments that did not vary by more than 5% of the mean. At the end of the incubations, thecells were washed twice with HBSS at 37°C then incubated with 250 μL of 1 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide in HBSS for 30 min at 37°C, for thedetermination of viability. The resulting formazan precipitates were then solubilized with 200μL DMSO. The viability of cells treated with cystamine at 15 μM and 100 μM dopamine (■)was significantly different than that of cells treated with dopamine alone (p < 0.05, paired t-test.

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