K.M. Dziegielewska

Synthesis and localization of plasma proteins in the developing human brain: Integrity of the fetal blood-brain barrier to endogenous proteins of hepatic origin☆

The distribution and possible origins of plasma proteins in the human embryonic and fetal brain at different stages of development have been investigated by a combination of isolation and translation of mRNAs and immunocytochemistry using specific antisera. As many as 23 plasma-like proteins have been identified using immunocytochemical methods at the light microscopical level. The presence of mRNAs for 13 of the immunocytochemically positive plasma proteins was demonstrated by in vitro and in ovo translation followed by crossed immunoelectrophoresis and autoradiography; this indicates in situ synthesis of these proteins (e.g., alpha-fetoprotein, alpha 1-antitrypsin, GC-globulin, alpha 2-macroglobulin, pseudocholinesterase, and transferrin) in some brain regions. The regional distribution of some proteins and the absence of some mRNAs suggest that the presence of certain plasma proteins in developing brain may be accounted for by uptake from csf or via nerve processes extending beyond the blood-brain barrier. In several cases, specific proteins appear to be associated with defined cell types, e.g., alpha-fetoprotein, GC-globulin, and ceruloplasmin with neurons, alpha 2-macroglobulin with endothelial cells, and ferritin with glial cells. Some proteins were associated with two or three cell types, e.g., alpha 1-antitrypsin with neurons and glia, and transferrin and alpha 2HS-glycoprotein with neurons, glia, and endothelial cells. Comparison of the expression of mRNAs from fetal brain and liver injected into Xenopus oocytes showed that a few proteins (transferrin and ceruloplasmin) were secreted when liver mRNA was injected, but not when brain mRNA was injected. This suggests that there may be an important difference in the structure and/or processing of these proteins in the brain which may reflect a function different from that associated with them when they originate from the liver. Staining was generally intracellular rather than extracellular; plasma proteins were not associated with the areas immediately around blood vessels although there was a strong immunoprecipitation for each protein within the lumen of cerebral blood vessels. These immunocytochemical findings together with the identification of mRNAs for a large number of plasma proteins in immature brain are discussed in relation to animal experimental work which suggests that the blood-brain barrier to protein is present even at very early stages of brain development.

Studies of the development of brain barrier systems to lipid insoluble molecules in fetal sheep.

1. The development of the blood‐brain and blood‐c.s.f barriers to lipid insoluble substances of different molecular radii has been studied in fetal sheep, early (60 days) and late (125 days) in gestation, using labelled erythritol (C14), sucrose (3H or 14C), inulin (3H or 14C) and albumin (125I), or albumin and IgG detected by immunoassay. 2. Morphological studies of fetal brain and choroid plexus at the same gestational stages were carried out using thin section electron microscopy and the freeze fracture techniques. 3. Penetration of markers into c.s.f. was substantially greater at 60 days than at 125 days, but at both ages the steady‐state level achieved appeared to be related to molecular size. 4. A simple model describing penetration from blood into c.s.f. at 60 days is proposed. It involves the assumption that c.s.f. and brain extracellular fluid are effectively separate compartments; morphological and permeability data which supports this assumption is presented. The data for c.s.f. at 60 days are consistent with the suggestion that the markers penetrate into c.s.f. by diffusion and are not restricted by small pores in the interface between blood and c.s.f. 5. The reduction in penetration which occurred by 125 days for all markers except erythritol appears to be accounted for by an increase in the sink effect and a decrease in the effective surface area for exchange between blood and c.s.f. 6. Intercellular tight junctions of both cerebral endothelial cells and choroid plexus epithelial cells were well formed at 60 days gestation. There was no change in junctional characteristics previously thought to correlate with transepithelial permeability (tight junction depth and strand number) between the two ages studied, although there were marked changes in permeability. 7. Evidence is advanced in support of the hypothesis that in the fetus much of the penetration of lipid insoluble non‐polar substances across the blood‐c.s.f. barrier and perhaps across the blood‐brain barrier occurs via a transcellular route consisting of a system of tubulo‐cisternal endoplasmic reticulum. Penetration via the choroid plexus appears to be the dominant route for penetration from blood into c.s.f. in the 60 day fetus.

Proteins in cerebrospinal fluid and plasma of fetal sheep during development

1. The concentration of total protein in c.s.f. and plasma has been measured in fetal sheep of different gestational ages and in the adult. In c.s.f. it was highest (approximately 840 mg/100 ml.) in the youngest fetuses (35 days) and declined steeply by 60 days (260 mg/100 ml.). It decreased less markedly in the last half of gestation to reach about 50 mg/100 ml. at 125 days which is twice the adult value. Protein concentration in plasma was lowest in the youngest fetuses and did not rise much until the second half of gestation during which time it doubled. There was a further rather larger increase between the late fetal (125 days) stage and the adult.

Proteins in cerebrospinal fluid and plasma of fetal rats during development.

The concentrations of total protein, albumin, and α -fetoprotein have been measured in the cerebrospinal fluid (csf) and plasma of fetal (12 to 22 days gestation) and neonatal (0 to 10 days postnatal) rats. Total protein concentration in cisternal csf increased from about 140 mg/100 ml at 12 days to reach a peak of over 300 mg/100 ml around the time of birth. In the postnatal period the total protein concentration declined to about 100 mg/100 ml at 10 days; the adult value was 24±8 mg/100 ml. There was substantially more α -fetoprotein than albumin in csf at 12 days gestation. Both increased in concentration toward the end of gestation; α -fetoprotein reached a plateau of about 100 mg/100 ml at 17–19 days after which it declined markedly to about 5 mg/100 ml at 10 days postnatal; albumin reached a plateau in csf of about 90 mg/100 ml around the time of birth and declined subsequently. Albumin and α -fetoprotein constituted over 50% of the total protein concentration in csf at all fetal ages studied. In plasma these two proteins made up only 35% of the total protein at 17 days gestation but by the time of birth their contribution had doubled. Total protein concentration in plasma increased throughout the developmental period studied as did that of albumin. α -Fetoprotein was at its highest concentration (440 mg/100 ml) at 19 days gestation; it declined markedly in the postnatal period. Other proteins identified in csf and plasma were: transferrin, α 1 -antitrypsin, and IgG. Evidence is discussed which suggests that the high concentration of protein in fetal csf results from specific transfer of plasma protein across the developing choroid plexus rather than from immaturity of the blood-csf barrier.

Blood—cerebrospinal fluid transfer of plasma proteins during fetal development in the sheep

1. The penetration of human and sheep plasma proteins from blood into c.s.f. of sheep fetuses (57‐86 days gestation) has been studied. The proteins were injected intravenously via cotyledonary vessels. After different time periods the c.s.f. concentrations of marker proteins were estimated by radioactive counting of iodinated human or sheep proteins or by immunoassay of human proteins.

Postnatal development of the telencephalon of the tammar wallaby (Macropus eugenii). An accessible model of neocortical differentiation.

SummaryThe sequence of development of cell layers in the neocortex of the tammar has been followed from 24 days gestation to 213 days postnatal. The tammar is born at 27 days gestation and the major period of its development occurs during the subsequent 250 days, most of this time being spent within the pouch. Although the pattern of differentiation of the cell layers appears to resemble that described for many Eutherian mammals, the neocortex is at an embryonic 2 layered stage at birth and a cortical plate is not present throughout the telencephalon until 10–15 days postnatal. A transient subplate zone, presenting a characteristic appearance with widely spaced rows of cells aligned parallel to the cortical surface, develops between 20 and 70 days postnatal, but no secondary proliferative region is seen in the subventricular zone of the dorso-lateral wall.Preliminary experiments with (3H)-thymidine injections indicate that the cortical plate follows the “inside-out” pattern of development described in many Eutherian mammals and that the oldest neurons are found in the parallel cell rows of the subplate zone. The importance of the late differentiation of the neocortex in relation to the time of birth and the resulting usefulness of the tammar as an experimental model of cortical development is discussed.

Synthesis of plasma proteins in fetal, adult, and neoplastic human brain tissue☆

The synthesis of plasma proteins directed by mRNA from human brain tissues was studied by combining in vitro or in ovo translation of mRNAs with crossed immunoelectrophoresis of the mRNA-directed labeled polypeptides, followed by autoradiography of the washed plates. Poly(A)-containing mRNA was prepared from different developmental stages of fetal and postnatal human brain and also from primary glioblastomas and meningiomas. Several plasma protein-like polypeptides were identified in the autoradiographs by their migration coordinates in the two-dimensional gels, compared with immunoprecipitates formed by mature, unlabeled, stainable proteins. These included polypeptides migrating like Gc globulin, haptoglobin, fibrinogen, alpha-fetoprotein, transferrin, cholinesterase, and alpha 2-macroglobulin; other, yet unidentified plasma proteins, were also observed. In general, the synthesis of these plasma proteins appeared to be more pronounced in fetal and neoplastic brain tissues than in postnatal tissues. However, clear immunoprecipitates for some of these plasma proteins could also be detected in products directed by mRNA from particular regions of mature, normal brains, indicating that some synthesis of plasma proteins takes place in the human brain even as late as 40 years of age. mRNAs for several proteins were also identified in samples of neoplastic brain. mRNA for transferrin was identified in normal fetal and adult brain but not in either the glioblastomas or meningiomas studied. Microinjected Xenopus oocytes, in which post-translational processing occurs as well, were also used to translate fetal brain mRNA. Several plasma proteins could be detected in the translation products which were induced and stored in the oocytes. These included hemopexin, which could not be detected in the in vitro system. Others, such as cholinesterase, were found to be secreted by the oocytes. These findings indicate that different cell types in the human brain may produce and either store or secrete particular plasma proteins at defined stages in their development.

Synthesis of plasma proteins by rat fetal brain and choroid plexus.

Several plasma proteins have previously been demonstrated to be within cells (presumed neurons) in the developing brain of various species. The possibility that the plasma proteins α‐fetoprotein (AFP) albumin and transferrin may be synthesized by developing brain and choroid plexus has been investigated in fetal rats of 18 to 22 days gestation. Samples of these tissues and of liver were incubated in Krebs solution containing [3H]leucine at 37°C for 1 h. Radioactively labelled AFP, albumin and transferrin were extracted and separated by immunoprecipitation. Incorporation of [3H]leucine into the plasma proteins was demonstrated in both fetal brain and choroid plexus. Incorporation was completely blocked by cycloheximide. It is concluded that fetal brain and choroid plexus synthesize AFP, albumin and transferrin and that secretion of these proteins by developing brain and choroid plexus cells probably contributes to the high concentration of plasma proteins in fetal csf.

A fetuin-related glycoprotein (α2HS) in human embryonic and fetal development

SummaryThe human plasma protein, α2HS glycoprotein, has an amino acid composition very similar to that of fetuin, the major protein in fetal calf and lamb serum. Immunohistochemical studies of human fetuses (6–33 weeks gestation) showed that α2HS glycoprotein and fetuin have similar distributions in developing brain and several other tissues, e.g., bone, kidney, gonads, gastrointestinal tract, respiratory and cardiovascular systems. There were notable differences in the liver and thymus in the distribution of the two proteins. Fetuin and α2HS glycoprotein are present in plasma and cerebrospinal fluid of both human and sheep fetuses; their concentrations are reciprocally related: in human plasma and cerebrospinal fluid α2HS glycoprotein concentration is high and fetuin low; the reverse is the case in sheep fetuses.Estimates of the concentration of α2HS glycoprotein in human fetal cerebrospinal fluid and plasma were obtained. It is suggested that α2HS glycoprotein may play a role in developing tissues, especially in the human fetus, similar to that of fetuin in other species.

Plasma proteins in fetal sheep brain: blood-brain barrier and intracerebral distribution.

1. Plasma proteins have been demonstrated to be present in early fetal sheep brain in amounts which cannot be accounted for by blood contamination. 2. The distribution of alpha‐fetoprotein, albumin, fetuin, alpha 1‐antitrypsin and transferrin has been studied by immunoassay of extracts from brain homogenates and by immunoperoxidase histochemistry of fetal brains between 37 and 125 days gestation (term is 150 days). 3. At 35 days gestation fetuin and albumin were quantitatively the most important proteins in fetal brain, both as estimated by extraction and by immunohistochemistry. Both of these proteins, and also alpha‐fetoprotein and alpha 1‐antitrypsin, declined considerably in amount by 60 days gestation. After 60 days the concentrations of albumin and alpha‐fetoprotein were not significantly different from that due to blood contamination and only occasional cells could be demonstrated by immunohistochemistry. Fetuin and alpha 1‐antitrypsin were present in reduced but significant amounts at least up to 125 days gestation. 4. The immunohistochemical results showed that considerable numbers of immature neurones were stained for some plasma proteins. Fetuin positive cells predominated both in terms of the larger number of cells which stained at 35‐40 days gestation and in the persistence of positive cells up to 125 days gestation. Numerous cells in the neuroependymal layer and in several layers of the developing cortical plate were positive, especially early in gestation. Only a few transferrin or alpha 1‐antitrypsin positive cells were observed. 5. Permeability of the blood‐brain barrier to sheep or human serum albumin (labelled with 125I) was tested in 60 day fetal sheep by intravenous injection and estimation of brain radioactivity at 3 or 6 hr with allowance for blood contamination. Only a very small (but significant) penetration of protein was detected. Unlike penetration of protein into c.s.f. at the same age, it did not reach its natural steady state in brain. 6. It is concluded that the blood‐brain barrier to protein is well developed in the immature fetal sheep but that developing neurones probably acquire certain plasma proteins directly from the c.s.f. when they are differentiating in the neuroependyma. The subsequent distribution of plasma protein positive cells in different brain regions is suggested to be due to the migration of developing neurones for the neuroependyma although the possibility of local synthesis of plasma proteins has not been excluded.