Inhibition and covalent modification of tyrosine hydroxylase by 3,4-dihydroxyphenylacetaldehyde, a toxic dopamine metabolite

Abstract

Parkinson's disease (PD) is a neurodegenerative disorder marked by the selective loss of dopaminergic neurons, leading to a decrease of the neurotransmitter dopamine (DA). DA is metabolized by monoamine oxidase to 3,4-dihydroxyphenyacetaldehyde (DOPAL). While the mechanism of pathogenesis of PD is unknown, DOPAL has demonstrated the ability to covalently modify proteins and cause cell death at concentrations elevated from physiologic levels. Currently, the identities of protein targets of the aldehyde are unknown, but previous studies have demonstrated the ability of catechols and other DA-catabolism products to interact with and inhibit tyrosine hydroxylase (TH). Given that DOPAL is structurally related to DA and is a highly reactive electrophile, it was hypothesized to modify and inhibit TH.

The data presented in this study positively identified TH as a protein target of DOPAL modification and inhibition. Furthermore, western blot analysis demonstrated a concentration-dependent decrease in antibody recognition of TH. DOPAL in cell lysate significantly inhibited TH activity as measured by decreased l-DOPA production. Inhibition of TH was semi-reversible, with the recovery of activity being time and concentration-dependent upon removal of DOPAL. These data indicate DOPAL to be a reactive DA-metabolite with the capability of modifying and inhibiting an enzyme important to DA synthesis.

Introduction

Parkinson's disease (PD) is a neurodegenerative disorder marked by the selective loss of dopaminergic neurons in the substantia nigra. Such a condition leads to a decrease in the important neurotransmitter dopamine (DA), causing a variety of symptoms including motor impairment. Currently, the mechanism of pathogenesis is unknown; however, studies have revealed a link to both environmental causes, such as pesticides, as well as endogenously produced oxidative stress (Andersen, 2004, Jenner, 2003, Fleming et al., 1994).

DA is metabolized by monoamine oxidase (MAO) to the reactive intermediate 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is then further oxidized by mitochondrial aldehyde dehydrogenase 2 (ALDH2) to 3,4-dihydroxyphenylacetic acid (DOPAC), and in a lesser pathway, by cytosolic aldehyde reductase (ALR) to 3,4-dihydroxyphenylethanol (DOPET) (Burke et al., 2004). While previous work has established DA to be an endogenous neurotoxin, capable of auto-oxidation leading to protein modification (Graham, 1978, Graham et al., 1978, Stokes et al., 1999), the metabolite DOPAL was found to be several orders of magnitude more toxic than DA (Burke, 2003, Burke et al., 2003). Physiological concentrations of DOPAL were measured to be ∼2–3 μM, and it was shown that when levels of DOPAL are slightly elevated (6.6 μM), there is a decrease in TH-positive cells, indicating dopaminergic cell death (Kristal et al., 2001, Mattammal et al., 1995, Burke et al., 2003, Burke, 2003). Furthermore, DOPAL has been implicated in protein modification (Rees et al., 2007, Helander and Tottmar, 1989, Ungar et al., 1973, Nilsson and Tottmar, 1987, LaVoie et al., 2005). Studies have demonstrated the ability of DOPAL to covalently modify Lys residues via the aldehyde (Rees et al., 2009), forming a Schiff-base structure predicted to interfere with normal protein function. Currently, specific protein targets of DOPAL are unknown, but previous studies have revealed that catechols and other DA-metabolism products interact with and inhibit tyrosine hydroxylase (TH), the rate-limiting step in DA synthesis (Laschinski et al., 1986, Xu et al., 1998).

Tyrosine hydroxylase (EC 1.14.13.41) catalyzes the oxidation of l-tyrosine to l-DOPA, and l-amino acid decarboxylase converts l-DOPA to DA (Nagatsu et al., 1964, Elsworth and Roth, 1997). Due to the importance TH plays in the synthesis of DA, a decrease or inhibition of this enzyme is predicted to yield a decrease in DA. Furthermore, studies have shown l-DOPA to have trophic properties, leading to an increase in TH+ neurons in both male and female rats with 6-hydroxydopamine lesions (Datla et al., 2001, Murer et al., 1998). Therefore, inhibition of TH activity may also lead to a decrease in cell function and viability.

It is hypothesized that DOPAL is an endogenously produced neurotoxin that modifies and inhibits enzymes important to DA biosynthesis, such as TH, leading to a decrease in DA production. The work presented here confirms DOPAL toxicity in dopaminergic PC6-3 cells, demonstrates inhibition of TH by DOPAL, and for the first time positively identifies TH as a protein target of DOPAL modification.