Elsevier

Neurobiology of Aging

Volume 24, Issue 8, December 2003, Pages 1147-1154
Neurobiology of Aging

Decreased plasma membrane expression of striatal dopamine transporter in aging

https://doi.org/10.1016/S0197-4580(03)00129-5Get rights and content

Abstract

Aging in rodents, monkeys, and man is correlated with a reduction in dopamine transporter (DAT) ligand binding and DAT function. Using Western blot techniques, we investigated whether the source of these age-related changes in DAT was correlated with decreases in DAT protein levels in the striatum, substantia nigra (SN), nucleus accumbens (NAc), and ventral tegmental area (VTA) of 6, 18, and 24-month-old male Fischer 344 rats. The relative levels of tyrosine hydroxylase (TH) were also determined in each region. In the striatum, we also assessed [3H]-DA uptake and DAT plasma membrane expression using a membrane-impermeant biotin analog in crude synaptosomes prepared from these age groups. There was no significant age-related difference in DAT immunoreactivity per total protein or per total TH in striatum, NAc, SN, or VTA. Significant age-related changes in TH were only seen in the VTA of the 24-month-old rats (∼60% decrease). However, [3H]-DA uptake and DAT protein recovered in the biotinylated fraction in 24-month-old rats were significantly decreased (∼30%) compared to 6-month-old animals in the striatal synaptosomes. These data suggest that age-related decreases in striatal DAT function and ligand binding are related to a decrease in plasma membrane expression of DAT and not a decrease in the steady-state levels of DAT protein or loss of dopaminergic neuropil.

Introduction

The hallmark of aging in animals and man is slowing of motor movements (bradykinesia) and impairments in posture and balance. Such impairments resemble those seen in Parkinson’s disease and both conditions are associated with perturbations of dopaminergic circuitry [5], [22], [36]. Unlike Parkinson’s disease, there is not an extensive loss (>50%) of dopamine (DA) containing neurons in the pars compacta of the substantia nigra (SN) in normal aging in humans or in animal models of aging [15], [24], [36]. However, aging is associated with alterations in nigrostriatal DA neuron function. The reported age-related effects include, and are not limited to, decreased basal extracellular DA levels [14], decreased evoked DA release [13], [14], [15], [18], and a decreased clearance capacity for DA [13], [19].

The clearance of released DA is mediated by the plasma membrane-associated dopamine transporter (DAT), a twelve transmembrane domain protein that recycles DA back into DA neurons [6], [41]. Age-related decreases in DAT function may be related to the availability of DAT protein. Aging is associated with decreases in DAT ligand binding in animals and man [1], [10], [20], [27], [30], [39]. Clearly, the decreases in DAT function and ligand binding in aged animals and man could be attributed to decreases in the synthesis of DAT protein, numbers of DA neurons, a functional change in DAT that alters affinity for DA, or a change in DAT plasma membrane expression. In the present study, we determined DAT protein levels in the striatum, nucleus accumbens (NAc), SN, and ventral tegmental area (VTA) in Fischer 344 rats of ages 6, 18, and 24 months (approximately equivalent to human ages of 18, 54, and 72 years, respectively). In addition, we measured tyrosine hydroxylase (TH) (the rate-limiting enzyme in DA biosynthesis) levels to normalize DAT protein against TH-synthesizing dopaminergic neuropil. Moreover, we determined DA uptake and attempted to measure DAT plasma membrane expression in striatal synaptosomes across these age groups by using a membrane impermeant reagent to isolate cell surface DAT. Here we show that the previously established age-related reductions in DAT function and ligand binding may be associated with a decrease in the levels of DAT on the plasma membrane, rather than a change in DAT protein levels or dopaminergic neuropil.

Section snippets

Animals and tissue dissection protocol

Two groups of male Fischer 344 rats of ages 6, 18, and 24 months (n=8 for each age group) were obtained from the National Institute on Aging. The first group of eight was used for blot immunolabeling studies and the second used for [3H]-DA uptake and DAT plasma membrane expression studies. For additional biotinylation technique control studies, two similarly young and old Fischer 344 rats were also obtained from the National Institute on Aging. Animals were housed under controlled environmental

TH and DAT protein levels

Prior to analyzing DAT among the age groups, six antibodies to DAT were tested on blots of the four brain regions. Using our method, the sc-1433 antibody gave the greatest immunoreactivity (at 70–75 kDa, using Bio-Rad pre-stained protein standard as a reference) per μg total protein. Using hippocampus tissue as a negative control, we did not detect immunoreactivity in this molecular weight region with this antibody. To determine any age-related changes in total DAT protein, we first established

Discussion

A major finding from the present studies is that the steady-state protein levels of DAT do not change in aging in the Fischer 344 rat. By contrast, the amount of DAT protein recovered from the membrane fraction that predominantly represents the plasma membrane of crude striatal synaptosomes is significantly decreased in the aged rats (24-month-old) compared to the young (6-month-old) animals. We also found no significant change in total DAT levels either in the cognate cell body region of the

Acknowledgements

This work was supported by USPHS Grants AG06434, AG13494, N39787, P20RR15592, and MH01245.

References (41)

  • N.R. Zahniser et al.

    Chronic and acute regulation of Na+/Cl dependent neurotransmitter transporters: drugs, substrates, presynaptic receptors, and signaling systems

    Pharmacol. Ther.

    (2001)
  • S. Apparsundaram et al.

    Acute regulation of norepinephrine transport: II. PKC-modulated surface expression of human norepinephrine transporter proteins

    J. Pharmacol. Exp. Ther.

    (1998)
  • M.J. Bannon et al.

    Dopamine transporter mRNA content in human substantia nigra decreases precipitously with age

    Proc. Natl. Acad. Sci. U.S.A.

    (1992)
  • M.J. Bannon et al.

    Age-related and regional differences in dopamine transporter mRNA expression in human midbrain

    Neurology

    (1997)
  • D.A. Bennett et al.

    Prevalence of parkinsonian signs and associated mortality in a community population of older people

    N. Engl. J. Med.

    (1996)
  • B.T. Chen et al.

    Novel Ca2+-dependence and time course of somatodendritic dopamine release: substantia nigra versus striatum

    J. Neurosci.

    (2001)
  • S. Doolen et al.

    Protein tyrosine kinase inhibitors alter human dopamine transporter activity in Xenopus oocytes

    J. Pharmacol. Exp. Ther.

    (2001)
  • M.E. Emborg et al.

    Age-related declines in nigral neuronal function correlate with motor impairments in rhesus monkeys

    J. Comp. Neurol.

    (1998)
  • R.R. Gainetdinov et al.

    Monoamine transporters: from genes to behavior

    Annu. Rev. Pharmacol. Toxicol.

    (2003)
  • G.A. Gerhardt et al.

    Changes in somatodendritic but not terminal dopamine regulation in aged Rhesus monkeys

    J. Neurochem.

    (2002)
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