The degeneration of nigral dopaminergic neurons in Parkinson disease is believed to be associated with oxidative stress. Since iron levels are increased in the substantia nigra of parkinsonian patients and this metal catalyzes the formation of free radicals, it may be involved in the mechanisms of nerve cell death. The cause of nigral iron increase is not understood. Iron acquisition by neurons may occur from iron-transferrin complexes with a direct interaction with specific membrane receptors, but recent results have shown a low density of transferrin receptors in the substantia nigra. To investigate whether neuronal death in Parkinson disease may be associated with changes in a pathway supplementary to that of transferrin, lactoferrin (lactotransferrin) receptor expression was studied in the mesencephalon. In this report we present evidence from immunohistochemical staining of postmortem human brain tissue that lactoferrin receptors are localized on neurons (perikarya, dendrites, axons), cerebral microvasculature, and, in some cases, glial cells. In parkinsonian patients, lactoferrin receptor immunoreactivity on neurons and microvessels was increased and more pronounced in those regions of the mesencephalon where the loss of dopaminergic neurons is severe. Moreover, in the substantia nigra, the intensity of immunoreactivity on neurons and microvessels was higher for patients with higher nigral dopaminergic loss. These data suggest that lactoferrin receptors on vulnerable neurons may increase intraneuronal iron levels and contribute to the degeneration of nigral dopaminergic neurons in Parkinson disease.

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Changes in the distribution of the iron-binding protein lactotransferrin have recently been described in the central nervous system during a variety of neurodegenerative disorders. To investigate whether lactotransferrin is associated with the neuropathological changes that characterize Parkinson's disease, we analyzed the distribution of this protein in the mesencephalon of neurologically normal individuals and patients affected with Parkinson's disease using quantitative immunohistochemical methods. High levels of lactotransferrin were observed in a large population of neurons in the substantia nigra of control cases. Lactotransferrin-positive neurons were severely affected by the neurodegenerative process that occurs in Parkinson's disease as indicated by a severe decrease in the number of immunolabeled neurons in all of these cases. Quantitative analysis also demonstrated higher immunolabeling levels of lactotransferrin in the surviving neurons in the substantia nigra and ventral tegmental area of Parkinson's disease cases compared to control cases. These results suggest that lactotransferrin may participate actively in the mechanism of neuronal degeneration in Parkinson's disease.

A fluorescent lactotransferrin probe was prepared by coupling 5-( { [2-(carbhydrazino)methyl]-thio) acety1)amino fluorescein to aldehyde groups that were produced by a mild periodic-acid oxidation of the glycan moieties of lactotransferrin. In this manner, the receptor-binding site of the lactotransferrin remains active in contrast to the binding site of the lactotransferrin derivatized with fluorescein isothiocyanate. The fluorescent probe allowed us to characterize, by flow cytometry, the binding of lactotransferrin to non-activated human platelets. The putative lactotransferrin platelet receptor was purified and its immunological and physico-chemical properties were found to be very similar to those of the receptor previously isolated from activated human lymphocytes. Lactotransferrin inhibits ADP-induced platelet aggregation at concentrations down to 5 nM, which can be reached in the plasma after leukocyte degranulation. Inhibition of platelet aggregation was also observed with the N-terminal fragment of lactotransferrin (residues 3 -281 ; 50% inhibition = 2 pM) and with CFQWQRNMRKVRGPPVSC synthetic octodecapeptide (residues 20 -37 ; 50% iahibition = 20 pM) corresponding to one of the two external loops (residues 28-34 and 39-42) where we recently located the receptor-binding site. The activity (50% inhibition = 500 pM) of the tetrapeptide KRDS (residues 39-42), which has already been described, was at least 25-times and 16000-times lower than the activity of the octodecapeptide and of the lactotransferrin molecules, respectively. Finally, the inhibition was demonstrated to be mediated by a mechanism which requires the binding of lactotransferrin to its putative receptor and not to platelet glycoprotein IIb-IIIa.Lactotransfemn (also called lactoferrin), discovered by Montreuil et al. in human milk [l, 21 was also found in human granulocytes [3] and numerous epithelial secretions [4, 51. Major progress has been made in the determination of its glycan [6] and peptide [7,8] sequences, three-dimensional structure and the understanding of iron capture [9, 101. In contrast, little is known about either its biological activity or target cells. Lactotransferrin has been proposed to be involved in iron absorption [2, 11, 121, growth-factor activity [13, 141, antibacterial activity [15, 161 and immunological processes [17], but none were definitively established. The nature of the target cells was investigated and binding sites were described for enterocytes [ l l , 12, 18, 191, liver cells [20, 213 and leukocytes [22]. However, no platelet lactotransferrin receptor was found [22] cyte-membrane receptor [23 -251. It has been recently shown that the KRDS tetrapeptide, which corresponds to loop 39-42 of lactotransferrin molecule and is thus an integral part of the lactotransferrin receptor binding site, inhibits platelet aggregation [26 -281. We have therefore investigated the presence of the lactotransferrin receptor on non-activated human platelets. In order to achieve this and because the fluorescein-isothiocyanat...