Developmental immunohistochemistry of bifunctional protein in human brain

Immunohistochemical studies of a peroxisomal enzyme, bifunctional protein, were performed on human brains (occipital cortex, cerebellum, pons) from fetus to young adult. Bifunctional protein-positive neurons appeared at 23–25 weeks of gestation in the facial nuclei of pons, at 27–28 weeks in the occipital cortex and Purkinje cells of vermis, and at 36–38 weeks in the Purkinje cells of the cerebellar hemisphere and pontine nuclei. They then increased in number with gestational age. However, bifunctional protein-positive glia appeared early in the occipital deep white matter at 17–20 weeks of gestation, their appearance shifting from the deep to the superficial white matter with increasing age. These results suggest that bifunctional protein is closely related to neuronal maturation and gliogenesis of premyelination in the human brain during development as other peroxisomal enzymes.     

Developmental immunohistochemistry of the 22 kDa peroxisomal membrane protein in the human brain

We studied immunohistochemically the 22 kDa peroxisomal membrane protein (PMP 22). In the control brain, the immunopositive neurons for PMP 22 appeared 2–3 weeks earlier than those for peroxisomal enzymes. PMP 22-immunopositive glial cells appeared at 26–27 gestational weeks, increased with gestational age, and were rarely recognized after 1 year of age. In the patients with Zellweger syndrome, PMP 22-immunoreactivity was recognized in neurons, glias, liver and kidney cells.     

Developmental changes of epidermal growth factor-like immunoreactivity in the human fetal brain

We investigated the immunohistochemical localization of epidermal growth factor (EGF) in the developing human brain from 6 weeks of gestation to 3 months postpartum. EGF-like immunoreactivity varied in its localization and intensity according to the stage of development. At 10-20 weeks of gestation, EGF-like immunoreactivity appeared in proliferating and migrating cells in the cerebrum, disappeared thereafter, and appeared again in cortical neurons after 27 weeks of gestation. Astrocytes also showed EGF-like immunoreactivity from 27 weeks of gestation. These results suggest developmental regulation of EGF expression in the human brain, suggesting Its physiological role in both neuronal and glial cells. © 1995 Wiley-Liss, Inc.     

Immunochemical detection of sodium channel in human tissue and cell lines

Mouse monoclonal antibodies have been raised to the C-terminal region of rat type II voltage-dependent sodium channel. One of these, designated 13A4-G4, recognizes a 260 Kd putative sodium channel protein in human fetal brain, heart and skeletal muscle, at 14-18 weeks of gestation. Faint immunoreactivity is also present in human fetal kidney but none has been detected in human fetal liver, lung or spleen. The antibody reacts in both western blots and immunocytochemical preparations with the human neuroblastoma cell lines SK-N-SH and SK-N-MC, the rhabdomyoblastoma cell line TE671, Y79 retinoblastoma cells and IPSB-18 astrocytoma cells. No 13A4-G4 immunoreactivity has been detected in several human cell lines derived from tissues that do not normally express sodium channels.     

Clinical and neuropathological picture of familial encephalopathy with bifunctional protein deficiency

Peroxisomal diseases are a heterogeneous group of genetic metabolic disorders which are caused by incorrect biogenesis of peroxisomes or a defect in activity of particular enzymes located in those organelles.D-bifunctional protein (D-BP) deficiency belongs to the second group of peroxisomal diseases characterised by dysfunction of a single peroxisomal enzyme. Bifunctional protein is a catalyst in the second and third stage of the beta-oxidation of fatty acids. Gene locus of bifunctional protein deficiency comprises chromosomes 5q2 and 3p23-p22. The authors present two siblings with progressing family encephalopathy. In the younger brother the diagnosis of a bifunctional protein deficiency was made. The girl died before a diagnosis was made;however, due to the presence of a very similar clinical condition a suspicion arises that the girl had a peroxisomal disease. In the siblings were ascertained characteristic dysmorphic features, delayed psychomotor development, polymorphic epileptic seizures and generalized muscular hypotonia with areflexia. The neuropathological findings were consistent in general with MRI findings showing features of hypomyelination. Also neuron heterotopias that were found in autopsy are a form of pathology typical for D-BP.     

Effects of exposure to chronic placental insufficiency on the postnatal brain and retina in sheep

Chronic placental insufficiency (CPI) has the potential to affect fetal brain development and to cause brain injury. Our aim was to determine the effects of exposure to CPI during late gestation on brain and retinal structure and brain neurotrophin expression 8 weeks after birth. Six fetal sheep were exposed to CPI, induced by umbilico-placental embolization, from 120 days of gestation until term (approximately 147 days) such that fetal arterial oxygen saturation (SaO2) was reduced by approximately 50%. Nine untreated animals served as controls. During CPI, fetal arterial PO2, SaO2, pH, and growth were reduced (p < 0.05); these animals remained small at 8 weeks after birth. Structural abnormalities were present in the brains and retinae of all CPI-exposed lambs. There was a reduction in retinal width and in the number of retinal tyrosine hydroxylase-immunoreactive dopaminergic amacrine cells (p < 0.05). In the dorsal hippocampus the combined width of strata oriens and pyramidale was significantly reduced (p < 0.05). In the cerebellum there was a significant reduction (p = 0.05) in cerebellar cross-sectional area, most notably in the inner granule cell layer, and a reduction (p < 0.05) in immunoreactivity for the cytoskeletal protein neurofilament-200 in the white matter. Gliosis was present in either the cerebral white matter or cerebellum in all animals and degeneration was seen around blood vessels in 4/6 umbilico-placental embolization animals. There were reductions in brain-derived neurotrophic factor immunoreactivity in the hippocampus (p < 0.05) and tyrosine kinase B immunoreactivity in the cerebellum (p < 0.05). This study shows that late gestational CPI affects morphology and neurotrophin expression of the postnatal brain. These alterations in the brain can apparently persist from fetal life or become established after birth; some changes that were present in the fetus at term did not persist into postnatal life.     

Development of GABAergic neurons and their transporter in human temporal cortex

The γ-aminobutyric acid (GABA) system plays an important role in the early development of the hippocampal formation. The immunohistochemical expression of γ-aminobutyric acid transporter-1, GAT-1, in the human developing temporal cortex was examined, and the distribution of GAT-1 was compared with that of the 67-kDa isoform of glutamic acid decarboxylase as a marker of GABAergic neurons. Four postmortem tissue specimens from young patients with hippocampal sclerosis were also examined. GAT-1 immunoreactivity was present, with a few puncta, in the neuropil of the stratum oriens and in the molecular layer of the dentate gyrus from 21-22 weeks of gestation, and in the stratum lacunosum-moleculare from 26 weeks of gestation. The peak expression of GAT-1 was seen in early infancy and that of glutamic acid decarboxylase in the perinatal period. These findings may reflect the development of GABAergic inhibitory systems, and may be related to the seizure susceptibility in infancy and early childhood. In the temporal lobes with hippocampal sclerosis, GAT-1 immunoreactivity of the neuropil was preserved in the vicinity of the neuronal loss of the hippocampus. This finding may result from the neurotrophic function of GAT-1 and may be related to its ability of neuronal repair and plasticity in childhood.     

Circadian rhythmicity after neural transplant to hamster third ventricle: specificity of suprachiasmatic nuclei

Neural transplants into the third ventricle were utilized to quantitatively assess the effectiveness of fetal tissue from selected brain sites in restoring circadian locomotor rhythmicity of adult hamsters rendered arrhythmic by lesions of the suprachiasmatic nuclei (SCN). Circadian function was continuously monitored in recording wheel cages under controlled environmental conditions. Animals which remained arrhythmic for 3–4 weeks after SCN lesions received transplants of neural tissue from 13–14-day-old fetuses: either SCN tissue or non-SCN tissue (cerebral cortex or hypothalamus excluding SCN). Quantitative evaluation of the data indicated partial restoration of circadian rhythmicity in 37% of 19 animals with SCN transplants, but in 0% of the 9 animals with non-SCN neural transplants. The mean time for reappearance of rhythmicity was 20 days after SCN transplantation. Animals were sacrificed 8–10 weeks after transplantation for histological analysis in order to visualize lesion placement and to characterize transplants. The cytoarchitecture and neuropeptide organization of the transplants were consistent with the donor brain region. Only SCN transplants were characterized by aggregates of small neurons with codistributed immunoreactivity for SCN-characteristic neuropeptides.     

Benign transient downward gaze in preterm infants

Five preterm infants who had episodes of conjugate downward gaze 2–3 months after birth were evaluated. Three were extremely preterm, born at 22–25 weeks gestation, and 2 were twins, born at 28 weeks gestation. In all patients, multiple, brief episodes of downward gaze occurred daily, with each lasting for several seconds. In 3 infants, downward gaze occurred more often with stimulation, while in another it occurred more frequently prior to feeding. There was never an associated change in vital signs, level of consciousness, muscle tone, or focal neurologic deficit. All infants had bronchopulmonary dysplasia. Neuroimaging studies were normal in 3 patients and demonstrated nonspecific abnormalities in 2. At follow-up (6 –34 months corrected age), intermittent downward gaze had completely resolved in 3 infants and was decreased in frequency and severity in the youngest 2. Four infants had normal development and neurologic examinations, while 1 child demonstrated mildly increased asymmetric muscle tone with normal development. Intermittent tonic downward gaze can be seen in extremely preterm infants who subsequently have normal development, and may represent a maturational delay in upward gaze reflex systems.     

Developmental immunohistochemistry of catalase in the human brain

The immunohistochemical studies on a peroxisomal enzyme, catalase, were done on brains from human fetuses to adults. The catalase-positive neurons appeared in the basal ganglia, thalamus and cerebellum at 27-28 weeks of gestation, and in the frontal cortex at 35 weeks. They then increased in number with gestational age. The extent of immunopositive staining increased with enlargement of perikaryonal size. However, the extent gradually decreased with postnatal age. On the other hand, catalase-positive glia appeared in the deep white matter at 31-32 weeks of gestation, their appearance shifting from the deep to the superficial white matter with increasing age. These results suggest that peroxisomes are closely related to neuronal growth and myelinogenesis in the human brain during development.     

Zn-binding globulin in human fetal brain and liver: a marker for passive blood/CSF transfer of protein

The presence of Zn-binding globulin (ZnbG) during human fetal development was studied in cerebrospinal fluid (CSF) and plasma with immunodiffusion methods and in brain, CSF, plasma and liver using immunocytochemical methods. At the earliest stages examined with immuno-cytochemistry (5–6 weeks gestation) no staining for ZnbG was visible in liver, plasma, CSF or brain. However, the primitive mesenchyme exhibited a prominent staining reaction. In late embryonic and early fetal stages, staining for the protein was most prominent in the spinal cord, brain stem and diencephalon and in the choroid plexuses and marginal and subplate zones in the telencephalon. At the cellular level, synaptic strata and territories were most strongly stained. The distribution of ZnbG in the early developing central nervous system suggests that this protein may be involved in the initial establishment of CNS circuitry. Embryonic brain was positive for ZnbG well before the protein could be detected in CSF, plasma or liver. The early occurrence of ZnbG in brain tissue prior to its presence in liver or plasma also suggests that the protein is synthesized in early fetal brain. At the time when CSF first became positive (17 weeks gestation), the brain staining had largely disappeared. ZnbG in plasma increased throughout gestation to reach 2.6 ± 0.4 mg/100 ml at term and subsequently increased to an adult value of 6.8 ± 1.5 mg/100 ml. The CSF to plasma ratio did not change significantly during development and was low (1–2%) at all fetal ages examined when compared with other plasma proteins; this is consistent with passive transfer between the two compartments both in the fetus and in the adult.     

Distribution and characteristics of a 90 kDa protein, KG-CAM, in the rat CNS

The distribution of a 90 kDa protein, termed KG-CAM, was examined in the developing and adult rat central nervous system (CNS) using the monoclonal antibody 11–59. The amino acid sequence of this protein revealed a sequence homology with a group of chick cell adhesion molecules from the immunoglobulin superfamily: DM-GRASP; SC1; and BEN. Immunolabeling of cells cultured from the embryonic and neonatal rat brain demonstrates that the protein recognized by 11–59 is on the external surface of a subpopulation of neurons and a limited population of glial cells. When the 11–59 antibody was used to stain sections of the adult brain and spinal cord, a number of different structures were labeled. The most intense immunoreactivity was found in the somatosensory system, the basal ganglia, the cortex, the olfactory system, and the circumventricular organs. One of the more interesting aspects of KG-CAM is the spatially and temporally regulated patterns of expression observed during the development of the CNS. For example, the dendrites of layer II pyramidal cells in the granular retrosplenial cortex are immunopositive for 11–59 while the dendrites are in the process of bundling in layer I, but not before bundling begins or after the process is completed. These findings reveal the varied roles of this adhesion molecule in the developing brain and spinal cord, as well as its potential role in the maintenance of the structural integrity of the adult CNS.     

Developmental expression of myotonic dystrophy protein kinase in brain and its relevance to clinical phenotype

To investigate the pathophysiologic role of myotonic dystrophy protein kinase (DMPK) in the brain in myotonic dystrophy (MD), the developmental characteristics of DMPK immunoreactivity in the central nervous system and its alteration with disease were studied. Eleven patients’ brain with MD (5 congenital form, 6 adult form) were examined by immunohistochemistry using a specific antibody against synthetic DMPK peptides, anti-peptide DM1, and compared with 30 control brains, including 16 age-matched controls. In controls, DM1-immunoreactive neurons appeared in the early fetal frontal cortex and cerebellar granule cell layer, persisting through 29 weeks of gestation and then disappearing. In contrast, immunoreactive neurons continued to persist in the cerebral cortex and cerebellar granule cell layer of MD patients. When we counted DM1-immunoreactive neurons, the increase over controls was greater in the congenital form of MD than in the adult form, and was greater in the cerebrum than in the cerebellum in both forms of MD. DM1 immunostaining was predominantly nuclear, mirroring Western blotting of subcellular fractions. Differences in DM1 expression related to development and to the two forms of MD may be closely related to the pathogenesis of mental retardation in this disease. Received: 30 July 1999 / Revised: 21 January 2000 / Accepted: 1 February 2000     

Ontogenic changes in cerebral concentration of homovanillic acid in response to haloperidol treatment

Pregnant rabbits of 24–29 days gestation, and young rabbits from birth to 4 weeks of age, were treated with haloperidol (0.2 mg/kg), dopamine-receptor blocking agent. Control animals received glucose. The animals were killed 4 h after haloperidol, and the brains of the young animals (and also of the foetuses, delivered by caesarian section) were analyzed for homovanillic acid (HVA), 5-hyroxyindoleacetic acid (5-HIAA) or dopamine.

The levels of 5-HIAA and dopamine were unchanged after haloperidol, but from the 29th day of gestation the drug induced an increase in the brain levels of HVA. The magnitude of increase, however, was much smaller in the young animals (29 day foetuses and newborn) than in the older animals (3–4 weeks of age.) This change with age was interpreted as an ontogenic development of dopamine receptors, or of a link in the feed-back control of transmitter release, or as an alternative possibility, development of the enzymes involved in the transformation of dopamine to HVA.     

Neonatal seizures and severe hypotonia in a male infant suffering from a defect in peroxisomal beta-oxidation.

In this paper, we describe a baby male born to healthy non-consanguineous parents presenting at birth with hypotonia and seizures. Additional salient clinical features included the development of glaucoma, the absence of significant facial dysmorphism and the absence of liver enlargement or renal cysts. The patient died at the age of 3 months. At autopsy, liver fibrosis and kidney glomerulosclerosis were noted. Neuropathological findings included pachygyria of the olivary nuclei and cerebellar neuronal heterotopias. There was no evidence for a demyelinating process. Biochemically, the patient was found to have elevated plasma levels of very-long-chain fatty acids (VLCFA) and abnormal bile acid intermediates, whereas other indicators of peroxisomal function (plasmalogen biosynthesis and plasma pipecolic acid) were normal. Catalase staining of a liver biopsy specimen revealed peroxisomes to be present in normal numbers, although some were abnormally large. Trilamellar inclusions typical of a peroxisomal fatty acid oxidation defect were present in macrophages. Indeed, beta-oxidation of the very-long-chain fatty acid hexacosanoic acid (C26:0) was found to be strongly deficient. Fatty acyl-CoA oxidase activity in the patient's liver was normal, however. Furthermore immunocytochemical studies using antibodies against acyl-CoA oxidase, bifunctional protein and peroxisomal thiolase, revealed the normal localization of all three enzyme proteins within the peroxisomes. We suggest that our patient has a selective peroxisomal beta-oxidation defect, a recently identified heterogeneous group of early-onset peroxisomal disorders distinct from the Zellweger syndrome and other generalized peroxisomal disorders.     

Immunohistochemical study of the early human fetal brain

Summary To assess the cytogenesis of the central nervous system we studied the spinal cord and the cerebrum in 11 human embryos and fetuses of gestation age 7–25 weeks immunohistochemically using anti-vimentin, anti-neurofilament protein (NFP), anti-neuron-specific enolase (NSE), anti-glial fibrillary acidic protein (GFAP), anti-S-100 protein, anti-Leu 7 and anti-myelin basic protein (MBP) antibodies. Vimentin was demonstrated in ventricular cells at 7 weeks and older. NFP-68-kDa and-160-kDa components were observed in neuroblastic cells of the neural tube at 7 weeks. NFP (68 and 160 kDa) was mainly located in the marginal zone of the spinal cord and the cerebrum at 8–9 weeks. NSE was not found in the neural tube at 7 weeks, although NSE was demonstrable at 9 weeks both in the spinal cord and in the cerebrum. GFAP-positive cells started to appear at 9 weeks in the spinal cord and at 15 weeks in the cerebrum, respectively. S-100 immunoreactivity was almost coincident with GFAP. S-100, however, was observed in more numerous glioblastic cells. Leu 7 was detected at 7 weeks and located in the neuropil of the central nervous tissue. MBP was not demonstrable in this study. Our study indicates that neuronal differentiation occurs much earlier than glial differentiation in the human brain and that neuronal and glial cell classes do not coexist in the ventricular zone of the early human fetal brain.     

A case of pseudo-Zellweger syndrome with a possible bifunctional enzyme deficiency but detectable enzyme protein: Comparison of two cases of Zellweger syndrome

Three infants with peroxisomal disorders were investigated clinicobiochemically and neuroradiologically. Two had classical Zellweger syndrome, and cranial CT scans showed typical disproportionate enlargement of the occipital horns of the lateral ventricles (colpocephaly) with marked hypodensity of the white matter. In one female infant, although the clinical findings were similar to those in Zellweger syndrome, some findings, such as elevated transaminase levels, liver fibrosis, the absence of renal cortical cysts and colpocephaly, were negative or milder. Biochemical analyses revealed increased very long-chain fatty acids, dicar☐ylic aciduria and impaired β-oxidation of lignoceric acid. However, peroxisomes were abundantly present in hepatocytes and cultured fibroblasts, and all peroxisomal β-oxidation enzyme proteins were detected on immunoblot analysis. A cell fusion study suggested that the enzyme responsible for this case of ‘pseudo-Zellweger syndrome’ is bifunctional.     

Characteristic neuropathology of leukomalacia in extremely low birth weight infants

Extremely low birth weight (ELBW) infants with periventricular leukomalacia (PVL) were examined by neuropathological and immunohistochemical methods. Thirteen ELBW infants of 85 infants with PVL, born at 23 to 27 weeks of gestation, showed a widespread type of distribution of PVL from the deep to intermediate white matter. Immunohistochemistry demonstrated glial fibrillary acidic protein (GFAP)-positive astrocytes to be increased in the deep white matter, often spreading to the intermediate white matter, in all cases of PVL. Tumor necrosis factor- (TNF-)-positive cells were found in the deep to intermediate white matter in 69% of PVL cases and appeared earlier, from 23 weeks of gestation, than in controls. β-Amyloid precursor protein (βAPP)-positive axons were found around PVL in the deep to intermediate white matter in 85% of the cases. In age-matched control ELBW infants, GFAP-, TNF--, or βAPP-positive cells were never found. Therefore, in ELBW infants, widespread axonal damage and glial activation with cytokine production occur in the progression of characteristic PVL lesions.     

Differential expression of peroxisomal matrix and membrane proteins during postnatal development of mouse brain

In peroxisomal biogenesis disorders, serious neurological abnormalities can be observed in the patients and the respective knockout mouse models. As a prerequisite for a better understanding of the relationship between the absence of peroxisomes and the observed neuropathology, knowledge of the regional and cell-type specific distribution of peroxisomal proteins in mouse brain is necessary. Therefore, we investigated the expression of distinct peroxins, peroxisomal membrane and matrix proteins (e.g. Pex5p, Pex14p, Pex13p, PMP70, catalase, peroxisomal thiolase, Acox1, "SKL"-PTS1 proteins) by indirect immunofluorescence 1) in primary cultures of the medial neocortex, hippocampus, and cerebellum of newborn mice and 2) in paraffin sections of mouse brain of different ages (newborn-adult). Quantitative analysis revealed a comparable abundance (number/microm(2)) of peroxisomes in cultured neurons and astrocytes of all three brain regions. In contrast, catalase immunoreactivity was higher in cultured astrocytes than in neurons. In mouse brain tissue, the abundance of peroxisomes decreased by half during postnatal development, also exhibiting prominent differences between distinct brain regions and cell types. Catalase protein levels in neuronal peroxisomes, however, decreased much more strongly in the neocortex, CA1-3 areas of the hippocampus, dentate gyrus, cerebellar nuclei, and cerebellar cortex but remained high in Bergmann glia and other astrocytes, epithelial cells of the choroid plexus, and ependyma. Similar age-dependent changes were found for thiolase and Acox1 protein levels. Developmental changes were confirmed by Western blot analysis using enriched peroxisomal and cytosolic fractions of the brain tissue as well as by measurement of catalase activity.     

Abnormal profiles of polyunsaturated fatty acids in the brain, liver, kidney and retina of patients with peroxisomal disorders.

The polyunsaturated fatty acid (PUFA) composition of the brain was studied in 8 patients with Zellweger's syndrome (ZS), 3 with neonatal adrenoleukodystrophy (NALD), one with bifunctional enzyme deficiency (BED), one with X-linked adrenoleukodystrophy (X-ALD), and one with adrenomyeloneuropathy (AMN). The PUFA composition of the liver, kidney and retina was studied in 8, 6 and 1 patients with ZS, respectively. An infant with NALD and a child with rhizomelic chondrodysplasia punctata (RCDP) were also studied for the PUFA composition of the liver. The liver and kidney of the patient with X-ALD and the liver of the patient with AMN were included in the study. The fatty acid values in the peroxisomal patients were compared with control data obtained in the normal developing brain (38 cases), liver (9 cases), kidney (7 cases) and retina (16 cases). The brain of a patient with metachromatic leukodystrophy (MLD) and the liver of a child with Krabbe's disease (KD) were also studied for comparison. The most constant and severe abnormality in all the peroxisomal patients was a drastic decrease in the total amount of docosahexaenoic acid (22:6 omega 3), especially in the brain. The other product of delta 4-desaturation, 22:5 omega 6, was generally decreased in the brain, liver and kidney of the ZS patients, but very much increased in the brain of two patients with NALD. The 22:6 omega 3/22:4 omega 6 ratio, which remains quite constant throughout normal brain development, was consistently decreased in the peroxisomal brain, in ZS as well as in NALD. This study confirms that, in classical Zellweger's syndrome, the two products of delta 4-desaturation are affected. In contrast, in neonatal adrenoleukodystrophy the deficiency is probably restricted to the omega 3 product of delta 4-desaturation, docosahexaenoic acid, especially in the brain, while the other product, 22:5 omega 6, is either normal or increased, perhaps in an attempt to compensate for the 22:6 omega 3 deficiency in brain membranes.