In vivo diffusion of lactoferrin in brain extracellular space is regulated by interactions with heparan sulfate

Abstract

The intercellular spaces between neurons and glia contain an amorphous, negatively charged extracellular matrix (ECM) with the potential to shape and regulate the distribution of many diffusing ions, proteins and drugs. However, little evidence exists for direct regulation of extracellular diffusion by the ECM in living tissue. Here, we demonstrate macromolecule sequestration by an ECM component in vivo, using quantitative diffusion measurements from integrative optical imaging. Diffusion measurements in free solution, supported by confocal imaging and binding assays with cultured cells, were used to characterize the properties of a fluorescently labeled protein, lactoferrin (Lf), and its association with heparin and heparan sulfate in vitro. In vivo diffusion measurements were then performed through an open cranial window over rat somatosensory cortex to measure effective diffusion coefficients (D*) under different conditions, revealing that D* for Lf was reduced Ϸ60% by binding to heparan sulfate proteoglycans, a prominent component of the ECM and cell surfaces in brain. Finally, we describe a method for quantifying heparan sulfate binding site density from data for Lf and the structurally similar protein transferrin, allowing us to predict a low micromolar concentration of these binding sites in neocortex, the first estimate in living tissue. Our results have significance for many tissues, because heparan sulfate is synthesized by almost every type of cell in the body. Quantifying ECM effects on diffusion will also aid in the modeling and design of drug delivery strategies for growth factors and viral vectors, some of which are likely to interact with heparan sulfate.drug delivery ͉ extracellular matrix ͉ integrative optical imaging ͉ somatosensory cortex ͉ transferrin T he spread of diffusible signals in brain extracellular space (ECS) is influenced by the local environment, with clearance mechanisms and the ECS volume fraction, tortuosity, and width among the best appreciated factors (1-3). The role that brain extracellular matrix (ECM) components play in modulating diffusion is less understood. Normal brain ECM is composed mostly of hyaluronic acid, a nonsulfated glycosaminoglycan, and proteoglycans carrying either chondroitin sulfate (CSPG) or heparan sulfate (HSPG) glycosaminoglycan side chains, along with the more recently identified reelin and tenascin glycoproteins (4, 5). Although aging (6-8), pathological insults (9, 10), or genetic modifications (11) resulting in altered brain ECM content are often associated with changes in ECS volume fraction, the ability of ECM components to specifically bind and slow the migration of diffusing substances in the ECS remains an open question. A direct ECM effect on extracellular diffusion (e.g., sequestration or slowing of a diffusing substance) has been postulated for proteins capable of binding HSPG (12, 13), but little evidence exists for this phenomenon in vivo.HSPGs are thought to play essential roles in the physiology of all organ systems (14). They compris...

The role of brain barriers in fluid movement in the CNS: is there a ‘glymphatic’ system?

“…A great many studies using a wide variety of tracer substances and multiple different ex vivo/in vivo methods have concluded that the local transport of small and large molecules through the brain ECS of the neuropil is predominantly diffusive in nature; such studies have included ventriculo-cisternal or subarachnoid-cisternal perfusion of radiolabelled tracers [111,135,144], real-time iontophoresis of the small tetramethylammonium ion [126], and integrative optical imaging (IOI) of pressure-injected fluorescently labelled molecules [127,[178][179][180]191]. Electron microscopy studies focused on neuropil ultrastructure have long indicated a brain ECS width of approximately 10-20 nm [21,75,81,136] and, importantly, that these narrow spaces could be accessed with tracers such as HRP (40 kDa) following intracerebroventricular injection [20,21].…”

“…The polyanionic nature and binding capacity of the ECM may significantly impact the diffusion of certain ions and other molecules in the brain ECS (e.g., confinement or binding of ions by perineuronal nets [76] or chondroitin sulphate proteoglycans [83], sequestration of growth factors [173], and protein binding to heparan sulphate proteoglycans [179]). CSF-ISF exchange of molecules is, therefore, expected to be limited by (1) molecular size, (2) ECM interactions, (3) receptor binding, (4) aggregation state (e.g., certain pathogenic proteins), (5) permeability characteristics (e.g., clearance across the BBB), and (6) distance from the brain-CSF interface, and likely other factors.…”

Morphogen transport

“…In addition to geometric hindrance mediated by cellular obstacles, morphogen movement might be further hindered by transient binding to extracellular molecules, such as morphogen receptors or extracellular matrix components (Crank, 1979;Lander et al, 2002;Baeg et al, 2004;Belenkaya et al, 2004;Han et al, 2004;Lin, 2004;Han et al, 2005;Callejo et al, 2006;Hufnagel et al, 2006;Thorne et al, 2008;Wang et al, 2008;Miura et al, 2009;Yan and Lin, 2009;Müller and Schier, 2011;Müller et al, 2012;Sawala et al, 2012). In the drunken sailor analogy, sailors frequently linger in pubs along the way (Fig.…”

“…In contrast to the free diffusion model, which posits that geometric effects from cells on diffusion are negligible or that movement occurs outside of the cell field, hindered diffusion postulates that cell packing, and hence tortuosity (see Box 2), strongly influences the movement of extracellular molecules. Extracellular morphogens must go around cells and this reduces their overall dispersal (Nicholson and Syková, 1998;Rusakov and Kullmann, 1998;Nicholson, 2001;Tao and Nicholson, 2004;Thorne and Nicholson, 2006;Thorne et al, 2008). In the drunken sailor analogy, sailors perform random walks in a region containing buildings, not in a large empty lot (Fig.…”

The Iron-Binding Protein Lactoferrin Protects Vulnerable Dopamine Neurons from Degeneration by Preserving Mitochondrial Calcium Homeostasis

“…It also exhibits antioxidant activities and has both anticarcinogenic and anti-inflammatory properties (Garcia-Montoya et al, 2012). While iron chelation is directly responsible for some of the biological functions of lactoferrin, other activities require interactions of lactoferrin with cell-specific receptors located on target cells (Suzuki et al, 2005;Pierce et al, 2009) or with molecular and cellular components of both hosts and pathogens, including heparan sulfate proteoglycans (HSPGs) (Thorne et al, 2008;Lang et al, 2011) and bacterial lipopolysaccharides (Drago-Serrano et al, 2012).…”