American Journal of Scientific Research ISSN 1450-223X Issue 6 (2009), pp.79-85 © EuroJournals Publishing, Inc. 2009 http://www.eurojournals.com/ajsr.htm
Brain Lactoferrin: An Endogenous Peptide or Merely an Intruder H.I. Marrif Dep. of Pharmacology and Toxicology, College of Pharmacy, Qassim University Qassim University, P.O. Box 6800, Buraidah 51452, Saudi Arabia E-mail:
[email protected] Naser. A. Alwabel Dep. of Pharmacology and Toxicology, College of Pharmacy, Qassim University Qassim University, P.O. Box 6800, Buraidah 51452, Saudi Arabia E-mail:
[email protected] H.M. Mousa Dep. of Food Science and Human Nutrition, College of Agriculture & Veterinary Medicine Qassim University, P.O. Box 6800, Buraidah 51452, Saudi Arabia E-mail:
[email protected] Abstract The focus of this review is on literature that deals with the effects of lactoferrin on the brain. This intriguing peptide has a large array of biological effects. This peptide is considered as part of the innate immune system with ipso facto defense properties. Produced from degranulation of neutrophils and other cells, it travels through circulation and crosses the blood brain barrier. Lactoferrin protein and mRNA are also express in key brain immune cells, the microglia. It is clearly established that upregulation of lactoferrin in the brain is usually associated with neuropathology. Considerable efforts have been directed toward studying the level of lactoferrin expression in different neurological diseases and aging. Nonetheless, pieces of the puzzle are still missing for a possible therapeutic use of this peptide in brain pathogenesis and repair.
Introduction Lactoferrin is a tiny cationic iron-binding glycoprotein of about 80 KD. The peptide has a potential intrinsic activity and it is believed to be fortuitous to the body’s defense system. Lactoferrin was first isolated in 1939 by Sorensen and Sorensen [1]. Earlier, research has delineated its existence in milk of human and animal sources [2,3]. Later however, the peptide was detected in different body fluids, exocrine secretions and blood [4,5]. The peptide was also localized in different brain structures. This observation instigates a legitimate question; where does this peptide come from? Lactoferrin was detected in secretary epithelial cells and in neutrophils [6,7]. The deregulation of neutrophils is believed to be the main source of the circulating lactoferrin which was eventually found in brain structure [8]. The peptide is synthesized and stored in the neutrophiles secondary granules [6]. Lactoferrin exists in three isoforms: alpha, beta and gamma forms and it is a single chain of about 700 amino acid residues [9]. It is composed of two globular proteins bridged by an alpha helix. The protein
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could be glycosylated at different sites. The peptide has iron binding sites much like the other family of transferrin, but with the ability to bind to other metal ions in blood [10]. In humans, lactoferrin gene is localized at chromosome three. Different lactoferrin transcripts were reported in different tissues and believed to be a result of alternative splicing of the same gene [11]. The promoters region of the gene encompasses constitutive and inducible transcription factors such as lactoferrin expression upregulated by steroids through the regulatory elements COUP/ERE, located 365 bp from the initiation start site [12,13]. Couples of other crucial regulatory elements have been detected in the promoter region including mitogen response module, and inflammatory response element [14]. The gene itself is highly conservative among species with an identical organization (17 exons with 15 encoding Lf) and conserved codon interruptions at the intron-exon splice junctions [14]. Sixteen single-nucleotide polymorphisms (SNP) have been reported in the gene which could produce peptides with different properties [15]. Methylation of MspI sites (455, 54, and 22) of the lactoferrin gene revealed a crucial result. The gene expression was very high in the uterus and spleen, low in lung, medium in liver and a trace expression was observed in the brain and kidney [16]. Lactoferrin receptors were localized in a number of cells including the immune cells, lymphocytes, macrophages, monocytes, hepatic and epithelial cells, fibroblasts, brain glia and neurons. Lactoferrin receptors were also detected in bacteria [17]. In the mouse brain, lactoferrin has been detected in different area of the brain [18]. Lactoferrin receptors are noted to mediate anti-nociception. PEP1261, a correspondent to residues 39–42 of human lactoferrin, was reported to have antipyretic and antinociceptive effects in rats [19]. Previous work has noted that the antinociceptive effects of lactoferrin are mediated by potentiating the peripheral μ-opioid receptors and nitric oxide. [20,21].
Brain Lactoferrin lactoferrin was detected in cerebrospinal fluid in rat brain after 30 minutes of intravenous infusion of a dose of 30mg/kg lactoferrin. It is believed that choroid plexus is one of the main routes for transportation of lactoferrin into brain structure [22]. Brain structure is protected by the blood brain barrier (BBB) and it is not permeable to molecules unless the BBB is altered by a pathological conditions or aging [23]. The mechanism of the lactoferrin transportation to the brain, using an in vitro model of the blood brain barrier of bovine capillary, was studied by the same group that reported that lactoferrin is transported intact into the cerebrospinal fluid via a receptor mediated endocytotic mechanism [24]. They also reported that capillary endothelial cells have two systems of transportation. One is described as specific with high affinity binding sites (Kd 5 37.5 nM; n 5 90,000/cell) and the other with low affinity binding sites (Kd 5 2 mM; n 5 900,000 sites/cell) [25]. After intravenous infusion, the peptide reported to accumulate in organs such as liver, spleen, and kidney [26]. Movement of the lactoferrin in the extracellular space of the brain matrix (between brain cells) was observed using confocal imaging, binding assays and florescent labeled lactoferrin. It has been observed that lactoferrin binds to heparan sulfate proteoglycans, a prominent component of the brain matrix. This binding with heparan decreases the diffusion coefficient of the peptide dramatically and the significance of this interaction is still to be investigated [27]. Conversely, immunohistochemistry studies of lactoferrin revealed the presence of lactoferrin and lysozymes in Browman's Glands and the mucociliary complex [28]. An earlier immunohistochemistry study of human brain revealed that lactoferrin was upregulated in neurons and glia of Alzheimer's Disease patients [29]. Strong signals were detected around the lesions of amyloid deposits and extracellular neurofibrillary tangles, minor signals were detected in intracellular neurofibrillary tangles, neuropil threads, and degenerative neuritis [29]. In a study of lactoferrin levels in cerebrospinal fluid of stroke patients (cerebral bleeding and hemorrhagic infarction patients), the level of lactoferrin peaked at 2-3 days after the onset of stroke symptoms [30]. It was found that a normal level of lactoferrin associated with viral brain infections and an elevation of the peptide was observed in patients with brain bacterial infection [31]. The author attributed this elevation to an ongoing inflammatory process and activation of granulocytes as a source of the peptide upregulation.
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Pharmacology of Lactoferrin After binding to its receptors, lactoferrin elicits its effects on cells by different mechanisms of action. It has a wide range of effects against bacteria, viruses, protozoa, and fungi [32]. The bacteriostatic effect is mediated by sequestering iron which is essential for bacterial growth [33]. The antiviral effect is thought to be mediated by two mechanisms; the ability to adhere to the cell membrane glycosaminoglycans component of the viral membrane [34] and lactoferrin manipulation of RNA and DNA strands [35]. The anti parasitic effect seems to be mediated by the ability of lactoferrin to compromise the parasitic membrane integrality [36]. Anti-inflammatory and immune mediating effects are believed to be mediated by its ability to reduce synthesis and release of inflammatory mediators and cytokines which regulate immune cells [37, 38]. Anti-tumor properties and ability to interfere with protein synthesis is though to be mediated by its ability to act as a transcription factor [39]. Lactoferrin antinociceptive effects are mediated by potentiating the peripheral μ-opioid receptors and nitric oxide. [20,40].
Vestiges of Lactoferrin in Neuropathology The study of lactoferrin receptor density in postmortem brains of Parkinson's patients showed that the receptors are found in neuronal structures (perikarya, dendrites and axons), blood capillaries and glia [41]. Receptor density in mesencephalon was high in areas that had suffered the loss of dopamenergic neurons and in substantia nigra. The intensity of immunoreactivity on neurons and microvessels was also higher for patients with higher nigral dopamenergic loss [41,42]. Interestingly, it was reported that the level of lactoferrin in plasma of some Parkinson's patients correlated inversely with Parkinsonism’s Disease severity [43]. Probably, the most intriguing finding in the Parkinson research is the observation of iron accumulation in the lesions which was also association with lactoferrin expression [44]. Using in situ hybridization and immunohistochemistry, lactoferrin mRNA has been localized in reactive microglial cells which were positive to other identifying markers such as the CD68 and the CR1 antigens [45,46]. It has been suggested that the brain immune cells such as the reactive microglia and the circulating monocytes/macrophage cells are the main source of lactoferrin in the brain. Microglial cells are immune cells in nature which are derived from CD45 cells of the bone marrow [47]. When microglias are activated, they are known as macrophages and they play a significant role in brain pathology and immunity. They foster a large array of receptors and can secrete immune mediators [48]. Thirty minutes following deep hypothermic circulatory arrest in children, a significant increase in brain lactoferrin (from a basic of 16 +/- 20 microg/mL to 196 +/- 70 μg/mL) has been observed which could indicate an activation of brain neutrophils [49]. Interestingly, the level of plasma lactoferrin, elastase, and levels of erythrocyte membrane bound hemoglobin are risk factors for cardiovascular events [50]. Lactoferrin protein expression has been investigated in brain tumor cells including astrocytomas, anaplastic astrocytomas and multiforme glioblastomas by immunohistochemistry and it was concluded that a higher immune reactivity of lactoferrin was detected in the cytoplasmic cells of the astrocytomas and less reactivity was observed in glioblastomas. They attributed the difference to a probable changes in lactoferrin receptors in different kinds of tumors [51]. Analysis of the lesions from Alzheimer’s Disease as well as tissues from different neurodegenerative diseases including Down Syndrome, Amyotrophic Lateral Sclerosis/ParkinsonismDementia Complex of Guam, Sporadic Amyotrophic Lateral Sclerosis, or Pick's Disease revealed an accumulation of lactoferrin in the lesions of neuronal damage [52,53]. In an in vitro study of Prion Disease, it has been observed that lactoferrin inhibits the accumulation of amyloidogenic isoform in a dose and time dependent manner. This observation is a breakthrough in Prion research considering the available options for Prion treatment [54]. Study of brain tissues infected with Toxoplasma gondii in mice, revealed that an oral dose of 5mg of lactoferrin was able to increase the survival time of the infected mice while an intraperitoneal dose of 0.1mg lactoferrin was able to prevent death in 100 % of
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the infected mice. The ability of small peptides to resist such an infection is a remarkable event considering the limited accessibility to brain structure [55].
Conclusion Studies of lactoferrin functions in brain are very suggestive of a multimechanstic protective effect [30]. lactoferrin reported to have antimicrobial, anti-inflammatory, antioxidant, anti-tumrogenic and transcription effect [56]. Some of its mechanisms of action seem to be modulated by its ability to donate and sequester iron. Many of these effects were inferred from studies of neurodegenerative and age related brain diseases [30,41,42,29]. Changes in iron metabolism are very evident in diseases such as Parkinson's Disease, Alzheimer's Disease, Huntington's Disease and Amyotrophic Lateral Sclerosis. Change in expression of lactoferrin protein and mRNA were associated with many of the previously mentioned diseases. In short, much direct and indirect evidence point at a basic change in iron metabolism as a causative agent in brain degenerative disease and at lactoferrin as sine qua non in this process [57, 58, 59]. Whether lactoferrin is an endogenous house keeping agent or a circulating peptide responding to an emergency call, it opened the door for brain permeable drugs researches [46].
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