Journal of Neurochemistry Raven Press, Ltd., New York 0 1992 International Society for Neurochemistr).

Facilitated Transport of the Neurotoxin, P-N-MethylaminoL-Alanine, Across the Blood-Brain Barrier Quentin R. Smith, Hiroshi Nagura, Yoshiaki Takada, and *Mark W. Duncan Laboratory of Neurosciences, National Institute on Aging, *Intramural Research Program, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, U.S.A.

Abstract: /3-N-Methylamino-L-alanine(BMAA) is a neurotoxic plant amino acid that has been implicated in the pathogenesis of the high incidence amyotrophic lateral sclerosis and related parkinsonism dementia of the western Pacific. Previous studies have demonstrated that BMAA is taken up into brain following intravenous or oral administration. To examine the kinetics and mechanism of brain transfer, BMAA influx across the blood-brain bamer was measured in rats using an in situ brain perfusion technique. BMAA influx was found to be saturable with a maximal transfer rate (V,,,,,) of 1.6 k 0.3 X pmol/s/g and a half-saturation constant (K,) of 2.9 k 0.7 mM based on total perfusate BMAA concentration. Uptake was sodium independent and inhibitable by excess L-leucine, but not by L-lysine, L-glutamate, or

methylaminoisobutync acid, indicative of transfer by the cerebrovascular large neutral amino acid carrier. L-BMAA competitively reduced brain influx of L-[ ''C]leucine, as expected for cross-inhibition. The results demonstrate that BMAA is taken up into brain by the large neutral amino acid camer of the blood-brain bamer and suggest that uptake may be sensitive to the same factors that affect neutral amino acid transport, such as diet, metabolism, disease, and age. Key Words: Neurodegenerative disease-Excitatory amino acid-Amyotrophic lateral sclerosis-Parkinson's diseaseBlood-brain bamer-Transport. Smith Q. R. et al. Facilitated transport of the neurotoxin, P-N-methylamino-L-alanine, across the blood-brain bamer. J. Neurochem. 58, 1330- 1337 (1992).

During the past 50 years three high-incidence foci of amyotrophic lateral sclerosis (ALS) and parkinsonism dementia (PD) have been found among isolated populations of the western Pacific, including the Chamorros of Guam and Rota, the Auyu and Jakai of West New Guinea, and the inhabitants of the northern Kii Peninsula, Honshu Island, Japan (for review, see: Garruto and Yase, 1986; Kurland, 1988). Incidence rates for ALS and PD among these populations have exceeded those in the United States and other developed countries by 50- to 1,000-fold (Spencer, 1987). Epidemiological research into etiologic factors has failed to demonstrate critical genetic (Plato et al., 1986) or infectious (Gibbs and Gajdusek, 1982) links in any of the three foci. As incidence rates in recent years appear to be declining (Garruto et al., 1985), most cur-

rent studies are focussing on potential environmental factors that are disappearing as lifestyles in the regions change. One environmental factor that has been linked to western Pacific ALS/PD is consumption and exposure to toxic cycad plants. Whiting (1963) and Spencer et al. ( 1 9 8 7 ~ - c )have established that seeds of various cycads, in particular members of the genus Cycas, have been used in each of the high-incidence foci for food or as traditional medicine. In Guam, such use is reported to have been especially heavy during and immediately following World War I1 when other foods were less available to the native population (Spencer et al., 1 9 8 7 ~ )Chemical . studies have identified at least two toxins in cycads: P-N-methylamino-L-alanine (BMAA) (Fig. 1) (Vega and Bell, 1967), a nonprotein

Received May 23, 1991; revised manuscript received September 3, 1991; accepted September 9, 1991.

Address correspondence and reprint requests to Dr. Q. R. Smith at Section on Neurochemistry and Brain Transport, Laboratory of Neurosciences, National Institute on Aging, NIH Bldg 10. Room 6C103, Bethesda, MD 20892, U.S.A. Abbreviations used. ALS, amyotrophic lateral sclerosis; BCH. 2aminobicyclo[2.2.1Iheptane-2-carboxylic acid; BMAA, P-N-methylamino-L-alanine; C$, perfusate concentration of BMAA or tracer amino acid; C,,, total perfusate BMAA concentration; C,, perfusate

concentration of competitor; F, cerebral perfusion fluid flow; GC/ MS, gas chromatography-mass spectrometry; Jin, brain influx rate; K , , transport inhibition constant; K,. , unidirectional transfer coefficient for brain uptake; KD, constant of nonsaturable transport; K,, half-saturation constant; MeAIB, methylaminoisobutyric acid; PA, blood-brain barrier permeability-surface area product; PD, parkinsonism dementia; qEl. total quantity of BMAA or tracer amino acid in brain; T, net time of perfusion;,,,'L maximum saturable influx rate: V,, brain vascular volume.

1330

BLOOD-BRAIN BARRIER TRANSPORT OF BMAA

B CH3-

NH-

CH2-

+

COO I

y

3

C

H

COO-

D

I

+

CH3-NHp-CCH~-C-COO

NH

I

-

I H

FIG. 1. Structure of BMAA showing charged groups at physiologic pH (-7.4). A 0-protonated neutral zwitterion of BMAA, the predominant form of BMAA in aqueous solution at neutral pH (-51%). B a-Protonated neutral zwitterion of BMAA (-8%). C. Tripolar cation (- 1O0/o). D a-Carbamate of BMAA. A 0-carbamate can also form. Together the a-and @-carbarnateshave been suggested

to comprise -31% of total BMAA at physiologic HC03 concentrations (Myers and Nelson, 1990). (For more on calculation of fractions, see Discussion).

amino acid, and cycasin, the glycone of the alkylating agent methylazoxymethanol (Nishida et al., 1955). In vivo BMAA produces motor neuron dysfunction and parkinsonian features when fed to monkeys in high doses (2100 mg/kg/day for 2-12 weeks) (Spencer et al., 1987a). In vitro BMAA elicits excitotoxic neuronal degeneration by a mechanism that can be blocked by N-methyl-D-aspartate receptor antagonists (Spencer et al., 1987~;Ross et al., 1987). However, the role of BMAA in the pathogenesis of ALS/PD has been questioned (Garruto et al., 1988; Spencer et al., 1990), as cycad seeds contain relatively low concentrations of BMAA (