CNS Delivery Via Adsorptive Transcytosis

Adsorptive-mediated transcytosis (AMT) provides a means for brain delivery of medicines across the blood-brain barrier (BBB). The BBB is readily equipped for the AMT process: it provides both the potential for binding and uptake of cationic molecules to the luminal surface of endothelial cells, and then for exocytosis at the abluminal surface. The transcytotic pathways present at the BBB and its morphological and enzymatic properties provide the means for movement of the molecules through the endothelial cytoplasm. AMT-based drug delivery to the brain was performed using cationic proteins and cell-penetrating peptides (CPPs). Protein cationization using either synthetic or natural polyamines is discussed and some examples of diamine/polyamine modified proteins that cross BBB are described. Two main families of CPPs belonging to the Tat-derived peptides and Syn-B vectors have been extensively used in CPP vector-mediated strategies allowing delivery of a large variety of small molecules as well as proteins across cell membranes in vitro and the BBB in vivo. CPP strategy suffers from several limitations such as toxicity and immunogenicity—like the cationization strategy—as well as the instability of peptide vectors in biological media. The review concludes by stressing the need to improve the understanding of AMT mechanisms at BBB and the effectiveness of cationized proteins and CPP-vectorized proteins as neurotherapeutics

Transcytosis of Protein through the Mammalian Cerebral Epithelium and Endothelium. I. Chorioid Plexus and the Blood-cerebrospinal Fluid Barrier

The potential for transcytosis (endocytosis → intracellular transport → exocytosis) of protein and membrane events associated with fluid phase and adsorptive endocytic processes within epithelia of the choroid plexus [blood-cerebrospinal fluid (CSF) barrier] were investigated in mice injected intravenously or into the lateral cerebral ventricle with native horseradish peroxidase (HRP) or the lectin wheatgerm agglutinin (WGA) conjugated to HRP. WGA binds to specific cell surface oligosaccharides and enters cells by the process of adsorptive endocytosis; native HRP is taken into cells non-specifically by fluid phase endocytosis. The lysosomal system of organelles and the endoplasmic reticulum, identified by enzyme cytochemical markers applied to choroid epithelia, were analysed for possible participation in transcytosis and compared to epithelial organelles harbouring the exogenous tracer proteins. Blood-borne native HRP was endocytosed readily by choroid epithelia whereas WGA-HRP was not, perhaps because WGA-HRP does not escape fenestrated endothelia as easily as native HRP. The blood-borne proteins incorporated within endocytic vesicles by choroid epithelia were directed to endosomes (prelysosomes) and secondary lysosomes (e.g. tubules, multivesicular/dense bodies) for eventual degradation and did not reach the apical/microvillus surface. Both CSF-borne native HRP and WGA-HRP entered choroid epithelia within endocytic vesicles derived from the microvillus border. Native HRP, ultimately sequestered within endosomes and secondary lysosomes, failed to undergo transcytosis through the epithelia into the basolateral clefts. Conversely, CSF-borne WGA-HRP was transported through the epithelia and released into the basolateral clefts within 10 min post-injection. The lectin conjugate labelled epithelial vesicles, endosomes, secondary lysosomes and, at 30 min post-injection, the transmost saccule of the Golgi complex which exhibits acid hydrolase activity. Tubular profiles, related either to the endosome apparatus or to the lysosomal system, and the endoplasmic reticulum did not appear involved in the transcytotic pathway. The data suggest that CSF-borne protein entering the choroid epithelium by adsorptive endocytosis can undergo rapid transcytosis through the cell. The results provide insight to transcytotic pathways utilizing vesicles, the endosomal apparatus, and the Golgi complex within the choroid epithelium for circumventing the blood-CSF barrier. Hypothesized membrane events and morphological associations among constituents of the endomembrane system within the choroid epithelium are summarized diagrammatically

Endocytic and Exocytic Pathways of the Neuronal Secretory Process and Trans-Synaptic Transfer of Wheat Germ Agglutinin-Horseradish Peroxidase in vivo

The lectin wheat germ agglutinin (WGA) conjugated to horseradish peroxidase (HRP) was employed to study the endocytic and exocytic pathways of the secretory process in neurons and the potential for trans-synaptic transfer of molecules within the CNS. WGA-HRP binds to surface membrane oligosaccharides and enters cells by adsorptive endocytosis. The lectin conjugate was administered intranasally or into the cerebral ventricles of mice; postinjection survival times ranged from 5 minutes to 6 days. Due to binding of the lectin to ependymal cells subsequent to an intraventricular injection, only select populations of neurons (i.e., hippocampal formation; paraventricular nuclei; midbrain raphe; VI, X, XII motor nuclei; among others) were exposed extracellularly to WGA-HRP and became labeled by retrograde axoplasmic transport from axon terminals or by direct cell body/ dendritic uptake. WGA-HRP delivered intranasally was endocytosed by first-order olfactory neurons and transported by anterograde axoplasmic flow to the terminal field within the glomerular layer of the main olfactory bulb; eventually perikarya of the mitral cell layer were labeled, presumably by anterograde trans-synaptic transfer of the lectin conjugate. In the variety of neurons analyzed ultrastructurally following exposure to WGA-HRP, the proposed sequence of intracellular pathways through which peroxidase reaction product was traced over time was: cell surface membrane endocytic structures endosomes (presecondary lysosomes) transfer vesicles transmost Golgi saccule vesicles, vacuoles, and/or dense core granules. WGA-HRP also labeled vesicles and tubules that were channeled to and/or derived from spherical endosomes, dense bodies, and multivesicular bodies. The peroxidase-positive, membrane-delimited products of the trans Golgi saccule contributed to anterograde axonal transport vectors and accumulated within axon terminals. A second contribution to these vectors was provided by peroxidase-labeled tubules and dense bodies believed to represent components of the lysosomal compartment. Profiles of the axonal reticulum comparable to those that stained cytochemically for glucose-6-phosphatase activity, a marker for the endoplasmic reticulum, were not associated with the transport of WGA-HRP. Trans-synaptic transfer of WGA-HRP from primary olfactory neurons to postsynaptic cells in the olfactory bulb was reflected in peroxidase-positive endocytic vesicles, endosomes, dense bodies, and the trans Golgi saccule. Native HRP, which is taken into cells by fluid phase endocytosis, served as a control and was delivered into the CNS by intranasal, intravenous, or intraventricular injection. Organelles that contained native HRP were identical to those labeled with WGA-HRP, excluding the Golgi complex and its membrane-delimited products; exocytosis and trans-synaptic transfer of native HRP were not evident. The results suggest that in the neuron: (1) Macromolecules processed and packaged for export by the Golgi complex are transported independently of the axonal endoplasmic reticulum. This transport may be directed throughout the neuron and is similar to that which occurs in non-neural cells; (2) Populations of vesicles within the axon terminal are derived from the Golgi complex and/or endocytosis; and (3) WGA-HRP molecules or fragments thereof packaged within Golgi-derived vesicles, vacuoles, and dense core granules are exocytosed from axon terminals for adsortive endocytosis and possibly fluid-phase endocytosis by postsynaptic neurons. The trans-synaptic transfer of macromolecules processed and packaged in the neuron is dependent upon the Golgi complex and the exocytic/endocytic pathways of the secretory process

Lectin-Labeled Membrane is Transferred to the Golgi Complex in Mouse Pituitary Cells in vivo

Labeling of the Golgi complex with the lectin conjugate wheat germ agglutinin-horseradish peroxidase (WGA-HRP), which binds to cell surface membrane and enters cells by adsorptive endocytosis, was analyzed in secretory cells of the anterior, intermediate, and posterior lobes of mouse pituitary gland in vivo. WGA-HRP was administered intravenously or by ventriculo-cisternal perfusion to control and salt-stressed mice; post-injection survival times were 30 min-24 hr. Peroxidase reaction product was identified within the extracellular clefts of anterior and posterior pituitary lobes through 24 hr but was absent in intermediate lobe. Endocytic vesicles, spherical endosomes, tubules, dense and multivesicular bodies, the trans-most saccule of the Golgi complex, and dense-core secretory granules attached or unattached to the trans Golgi saccule were peroxidase-positive in the different types of anterior pituitary cells and in perikarya of supraoptico-neurohypophyseal neurons; endoplasmic reticulum and the cis and intermediate Golgi saccules in the same cell types were consistently devoid of peroxidase reaction product. Dense-core secretory granules derived from cis and intermediate Golgi saccules in salt-stressed supraoptic perikarya likewise failed to exhibit peroxidase reaction product. The results suggest that in secretory cells of anterior and posterior pituitary lobes, WGA-HRP, initially internalized with cell surface membrane, is eventually conveyed to the trans-most Golgi saccule, in which the lectin conjugate and associated membrane are packaged in dense-core secretory granules for export and potential exocytosis of the tracer. Endoplasmic reticulum and the cis and intermediate Golgi saccules appear not to be involved in the endocytic/exocytic pathways of pituitary cells exposed to WGA-HRP

Tubular Profiles do not form Transendothelial Channels through the Blood-brain Barrier

The contribution of tubular profiles within the mammalian cerebral endothelium to the formation of transcellular channels was analysed following exposure of the endothelium to native horseradish peroxidase (HRP) dissolved in saline or dimethyl sulphoxide (DMSO) administered intravenously in mice. Within 5–15 min, but not at 30 min to 2h postinjection, peroxidase-positive extravasations were evident within the parenchyma of the forebrain and brainstem of mice exposed and not exposed to DMSO. The extravasations may be associated with the rupture of interendothelial tight junctions at the level of arterioles as a consequence of the perfusion-fixation process. Ultrastructural inspection of endothelia within and away from areas of peroxidase extravasation revealed the following intraendothelial, peroxidase-positive organelles: presumptive endocytic vesicles, endosomes (a prelysosomal compartment), multivesicular and dense bodies, and tubular profiles. Statistical analysis of the concentration of HRP-labelled presumptive endocytic vesicles, which may coalesce to form tubules, within endothelia from mice injected intravenously with HRP-DMSO compared to mice receiving HRP-saline revealed no significant difference. HRP-positive tubular profiles were blunt-ended, variable in length and width, and appeared free in the cytoplasm or in continuity with dense bodies. Labelled tubules free in the cytoplasm were positioned parallel to the luminal and abluminal plasma membranes and were less frequently oblique or perpendicular to these membranes. Tubular profiles analysed in serial thin sections or with a goniometer tilt stage did not establish membrane continuities with the luminal and abluminal plasma membranes. Peroxidase-positive tubular profiles were similar morphologically to those exhibiting acid hydrolase activity but did not share morphological and enzyme cytochemical similarities with the endoplasmic reticulum that stained for glucose-6-phosphatase (G6Pase) activity. G6Pase-positive profiles of endoplasmic reticulum were not observed to contribute to a transendothelial canalicular network. Our results suggest that: (i) peroxidase-labelled tubules, acid hydrolase-positive tubules, and G6Pase-positive endoplasmic reticulum do not form transcellular channels through the cerebral endothelium; (ii) tubular profiles labelled with blood-borne HRP in the cerebral endothelium are associated with the eridosome apparatus and/or the lysosomal system of organelles; and (iii) DMSOdoes not appear to alter the permeability of the blood-brain barrier to blood-borne protein

Further Studies of the Secretory Process in Hypothalamo neurohypophysial Neurons. An Analysis Using Immunochemistry, Wheat Germ Agglutinin-Peroxidase and Native Peroxidase

The axonal endoplasmic reticulum (ER) and synaptic-like (micro)vesicles within axon terminals of the neurohypophysis and their contribution to the secretory process in hypothalamo-neurohypophysial neurons have been investigated cytochemically in normal mice and in mice given 2% salt water to drink for stimulation of hormone synthesis in and release from these neurons. Cytochemical techniques included the peroxidase-antiperoxidase (PAP) immunocytochemical method for localization of neurophysin, wheat germ agglutinin-horseradish peroxidase (WGA-HRP) as a tracer for the anterograde axonal transport of membrane from within the perikaryon, and blood-borne native horseradish peroxidase (HRP) as a tracer for internalized axon terminal membrane. The primary antiserum employed was directed against neurophysins I and II, the carrier proteins for the peptide hormones oxytocin and vasopressin, respectively. PAP reaction product was observed over neurosecretory granules but never over the endoplasmic reticulum, microvesicles or other organelles in axons and terminals of the neurohypophysis WGA-HRP was delivered extracellularly to cell bodies of paraventricular neurons by cerebral ventriculocisternal perfusion. Internalized perikaryal surface membrane tagged with WGA-HRP was recycled through the innermost Golgi saccule (GERL) from which neurosecretory granules were formed. The anterograde axonal transport of membrane-bound WGA-HRP was manifested within the neurosecretory granules; WGA-HRP did not label the axonal reticulum or terminal microvesicles in the neurohypophysis. Blood-borne native HRP endocytosed into neurohypophysial terminals was associated with a plethora of microvesicles measuring 40–70 nm in diameter and vacuoles similar in size to the 100–300-nm-wide neurosecretory granules. The microvesicles contributed to the formation of numerous vacuoles. The internalization of axon terminal membrane as microvesicles incorporating HRP was quantitatively greater than vacuoles in both salt-stressed and control mice. The results suggest that in the hypothalamo-neurohypophysial system of the mouse the axonal ER and terminal microvesicles are not involved in the transport, storage, and exocytosis of neurosecretory material and perhaps other molecules processed through the innermost Golgi saccule. Nevertheless, a prominent population of the microvesicles within axon terminals of the neurohypophysis does participate in the secretory process. These vesicles are involved directly in the internalization of the terminal surface membrane subsequent to release of secretory granule content. The recapture of neurohypophysial terminal membrane in toto may be represented as endocytic structures ranging in size from 40 through 300 nm in diameter

A Blood-Brain Barrier? Yes and No.

Ventriculo-cisternal perfusion of horseradish peroxidase (HRP) in the mouse brain has demonstrated that a brain-blood barrier exists at the microvascular endothelium in brain parenchyma but not in the median eminence of the hypothalamus. The brain-blood barrier is similar to the blood-brain barrier in that: tight junctions prevent the movement of protein between endothelial cells, HRP taken into the endothelial cells is directed to lysosomal dense bodies, and, contrary to the literature, a vesicular transendothelial transport of HRP from brain to blood does not occur under normal conditions. The endocytosis of ventricular injected HRP from the abluminal side of the endothelium is demonstrably less than the endocytosis of intravenous injected HRP from the luminal side; hence, the cerebral endothelium expresses a degree of polarity regarding the internalization of its cell surface membrane and extracellular protein. The passage of cerebrospinal fluid-borne or blood-borne HRP between some ependymal cells of the median eminence is not precluded by tight junctions. These patent extracellular channels offer a direct pathway for the exchange of substances between cerebrospinal fluid in the third ventricle and fenestrated capillaries in the median eminence

Angioarchitecture of the CNS, Pituitary Gland, and Intracerebral Grafts Revealed with Peroxidase Cytochemistry

Blood vessels of the fetal, neonatal, and adult subprimate and primate CNS, including circumventricular organs (e.g., median eminence, pituitary gland, etc.), and of solid CNS and nonneural (anterior pituitary gland) allografts placed within brains of adult mammalian hosts were visualized with peroxidase cytochemistry applied in three ways: (1) to tissues from animals injected systemically with native horseradish peroxidase (HRP) or peroxidase conjugated to the lectin wheat germ agglutinin (WGA) prior to perfusion fixation; (2) to tissues from animals infused with native HRP into the aorta subsequent to perfusion fixation; and (3) to tissues from animals fixed by immersion and incubated for endogenous peroxidase activity in red cells retained within blood vessels. In neonatal and adult animals receiving native HRP intravascularly, non-fenestrated vessels contributing to a blood-brain barrier were outlined with HRP reaction product when tetrarnethyl-benzidine (TMB) as opposed to diaminobenzidine (DAB) was used as the chromogen; fenestrated vessels of circumventricular organs were not discernible due to the density of extravascular reaction product. Fenestrated and nonfenestrated cerebral and ext racer ebral blood vessels exposed to blood-borne WGA-HRP were visible when incubated in TMB and DAB solutions. Native HRP infused into the aorta of fixed animals likewise labeled nonfenestrated vessels throughout the brain upon exposure to TMB or DAB but obscured fenestrated vessels of the circumventricular organs. Endogenous peroxidase activity of red cells, seen equally well with TMB and DAB, outlined blood vessels throughout the cerebral gray and white matter and all circumventricular organs in fetal, neonatal, and adult animals

Avenues for Entry of Peripherally Administered Protein to the Central Nervous System in Mouse, Rat, and Squirrel Monkey

Pathways traversed by peripherally administered protein tracers for entry to the mammalian brain were investigated by light and electron microscopy. Native horseradish peroxidase (HRP) and wheat germ agglutinin (WGA) conjugated to peroxidase were administered intranasally, intravenously, or intraventricularly to mice; native HRP was delivered intranasally or intravenously to rats and squirrel monkeys. Unlike WGA-HRP, native HRP administered intranasally passed freely through intercellular junctions of the olfactory epithelia to reach the olfactory bulbs of the CNS extracelluarly within 45–90 minutes in all species. The olfactory epithelium labeled with intravenouslydelivered HRP, which readily escaped vasculature supplying this epithelium. Blood-borne peroxidase also exited fenestrated vessels of the dura mater and circumventricular organs. This HRP in the mouse, but not in the other species, passed from the dura mater through patent intercellular junctions within the arachnoid mater; in time, peroxidase reaction product in the mouse brain was associated with the pial surface, the Virchow-Robin spaces of vessels penetrating the pial surface, perivascular clefts, and with phagocytic pericytes located on the abluminal surface of superficial and deep cerebral microvasculature. Blood-borne HRP was endocytosed avidly at the luminal face of the cerebral endothelium in all species. WGA-HRP and native HRP delivered intraventricularly to the mouse were not endocytosed appreciably at the abluminal surface of the endothelium; hence, the endocytosis of protein and internalization of cell surface membrane within the cerebral endothelium are vectorial. The low to non-existent endocytic activity and internalization of membrane from the abluminal endothelial surface suggests that vesicular transport through the cerebral endothelium from blood to brain and from brain to blood does not occur. The extracellular pathways through which probe molecules enter the mammalian brain offer potential routes of passage for blood-borne and airborne toxic, carcinogenic, infectious, and neurotoxic agents and addictive drugs, and for the delivery of chemotherapeutic agents to combat CNS infections and deficiency states. Methodological considerations are discussed for the interpretation of data derived from application of peroxidase to study the blood brain barrier