Iron in the Hallervorden-Spatz syndrome.

The dark discoloration of globus pallidus and substantia nigra pars reticularis in the Hallervorden-Spatz syndrome is due to the accumulation of iron. Routine iron stains detect the metal mostly in microglia and macrophages, but scattered neurons are also reactive. Axonal spheroids are characteristic of the disease, and many of these expansions give a positive iron reaction. Globus pallidus and substantia nigra are normally rich in iron, and additional "storage" of the metal has often been considered the essential factor in the pathogenesis of Hallervorden-Spatz syndrome. However, other equally iron-rich structures, such as the red nucleus and the dentate nucleus, remain unaffected. In normal globus pallidus and substantia nigra pars reticularis, double-label immunofluorescence microscopy of ferritin, as an indirect marker of cellular iron localization, and phosphorylated neurofilament protein reveal close proximity of ferritin-reactive microglial and oligodendroglial processes to tightly packed axons. It is proposed that a primary axonal disorder allows the seepage of iron into the axoplasm. Iron may contribute to the axonal disease, but accumulation of the metal probably should be viewed as an epiphenomenon. Pallidal and nigral iron excess is not unique to Hallervorden-Spatz syndrome, and some previously reported postmortem examinations may actually represent pallidonigroluysian atrophy.

Genetic localization of an autosomal dominant leukodystrophy mimicking chronic progressive multiple sclerosis to chromosome 5q31.

The hereditary leukodystrophies represent a group of neurological disorders, in which complete or partial dysmyelination occurs in either the central nervous system (CNS) and/or the peripheral nervous system. Adult-onset autosomal dominant leukodystrophy (ADLD) is a slowly progressive, neurological disorder characterized by symmetrical widespread myelin loss in the CNS, and the phenotype is similar to that of chronic progressive multiple sclerosis. We report clinical, neuroradiological and neuropathological data from the originally reported ADLD family. Furthermore, we have localized the gene that causes ADLD to a 4 cM region on chromosome 5q31. Linkage analysis of this family yielded a LOD score of 5.72 at theta = 0.0 with the microsatellite marker D5S804. Genetic localization will lead to cloning and characterization of the ADLD gene and may yield new insights into myelin biology and demyelinating diseases.

Oculoleptomeningeal amyloidosis associated with a new transthyretin variant Ser64.

BACKGROUND: A Canadian family with oculoleptomeningeal amyloidosis with both central and peripheral nervous system disorders was described in 1988. Death of affected family members resulted from recurrent cerebral hemorrhage. OBJECTIVE: To determine if oculoleptomeningeal amyloidosis is caused by a mutation in transthyretin (prealbumin). METHODS: DNA isolated from peripheral blood and archival tissues of affected members of the kindred was studied by direct DNA sequencing and restriction fragment length polymorphism analysis. RESULTS: Direct DNA sequencing identified a thymine-to-cytosine transition at the second base of codon 64, which resulted in a replacement of serine for phenylalanine. This mutation, which creates an additional HinfI site was detected by restriction fragment length polymorphism analysis in each affected individual. CONCLUSION: In this kindred, oculoleptomeningeal amyloidosis is related to a mutation in transthyretin (Phe64Ser).

Synapses in the hereditary ataxias.

The goal of this investigation was the systematic assessment of synapses in the hereditary ataxias by the immunocytochemical and immunofluorescent visualization of SNAP-25, a protein of the presynaptic membrane. Sections were prepared from the cerebellar cortex, dentate nucleus, basis pontis, inferior olivary nuclei, and the spinal cord in 57 cases of autosomal dominant and recessive ataxia. The neuropathological phenotype included 18 cases of olivopontocerebellar atrophy (OPCA), 14 cases of familial cortical cerebellar atrophy (FCCA), 4 cases of Machado-Joseph disease (MJD), and 21 cases of Friedreich's ataxia (FA). Among the autosomal dominant ataxias, spinocerebellar ataxia type 1 (SCA-1), SCA-2, MJD/SCA-3, and SCA-6 were represented. Expanded guanine-adenine-adenine trinucleotide repeats were confirmed in 7 patients with FA. The abundance of SNAP-25 was estimated by comparing the fluorescence of the regions of interest to that of the frontal cortex, which was considered unaffected by the disease process. Despite severe Purkinje cell loss, abundant SNAP-25 reaction product remained in the molecular layer of FCCA and OPCA. Among the cases of OPCA, those identified as SCA-2 showed the most severe overall synaptic destruction in cerebellum and brain stem. In SCA-1, which caused either OPCA or FCCA, significant synaptic loss was restricted to the inferior olivary nuclei. Sparing of cerebellar cortex and inferior olivary nuclei was the rule for MJD/SCA-3 and FA, though the dentate nucleus showed reduced SNAP-25 immunoreactivity in both ataxias. In FA, preservation of SNAP-25 in the dentate nucleus was characteristic of long survival. Severe cases with short survival revealed synaptic depletion of the dentate nucleus. At the level of the spinal cord, synaptic loss in the dorsal nuclei of Clarke characterized FA and MJD/SCA-3. The inexorable clinical progression of the hereditary ataxias could not be attributed to synaptic loss in a single anatomic structure of cerebellum, brain stem, or spinal cord. Nevertheless, synaptic loss in dentate and inferior olivary nuclei correlated more precisely with the severity of the ataxia than the changes in the cerebellar cortex.

The hereditary ataxias.

Efforts to classify the hereditary ataxias by their clinical and neuropathological phenotypes are troubled by excessive heterogeneity. Linkage analysis opened the door to a new approach with the methods of molecular biology. The classic form of autosomal recessive ataxia, Friedreich's ataxia (FA), is now known to be due to an intronic expansion of a guanine-adenine-adenine (GAA)-trinucleotide repeat. The autosomal dominant ataxias such as olivopontocerebellar atrophy (OPCA), familial cortical cerebellar atrophy (FCCA), and Machado-Joseph disease (MJD) have been renamed the spinocerebellar ataxias (SCA). Specific gene loci are indicated as SCA-1, SCA-2, SCA-3, SCA-4, SCA-5, SCA-6, and SCA-7. In 5 of them (SCA-1, SCA-2, SCA-3, SCA-6, and SCA-7), expanded cytosine-adenine-guanine (CAG)-trinucleotide repeats and their abnormal gene products cause the ataxic condition. The most common underlying loci for olivopontocerebellar atrophy (OPCA) are SCA-1 and SCA-2, although other genotypes may be added in the future. A major recent advance was the identification of the gene for SCA-3 and MJD, and the high prevalence of this form of autosomal dominant ataxia. In FA and the SCA with expanded CAG-trinucleotide repeats, clinical and neuropathological severity are inversely correlated with the lengths of the repeats. Anticipation in the dominant ataxias can now be explained by lengthening of the repeats in successive generations. Progress is being made in the understanding of the pathogenesis of FA and SCA as the absent or mutated gene products are studied by immunocytochemistry in human and transgenic murine brain tissue. In FA, frataxin is diminished or absent, and an excess of mitochondrial iron may cause the illness of the nervous system and the heart. In SCA-3, abnormal ataxin-3 is aggregated in neuronal nuclei, and in SCA-6, a mutated alpha1A-calcium channel protein is the likely cause of abnormal calcium channel function in Purkinje cells and in the death of these neurons.

Somatic mosaicism in the central nervous system in spinocerebellar ataxia type 1 and Machado-Joseph disease.

Spinocerebellar ataxia type 1 and Machado-Joseph disease are two autosomal dominant cerebellar ataxias caused by expansions of unstable CAG repeats in the coding region of the causative genes. The selectivity of cell death and the resulting characteristic neuropathological features in each of these diseases are not explained by the gene expression patterns. Since the repeat size correlates with age at onset and severity of these diseases, somatic mosaicism, the result of mitotic instability of the CAG repeat, could be the basis for specificity of neurodegeneration; brain structures with larger expanded repeats would be more severely affected. To study the association between neuropathological changes and somatic mosaicism of the CAG repeat size in the central nervous system of patients with these two ataxias, we determined the size of the (CAG)n expansion in 20 different regions of the brain, brainstem, cerebellum, and spinal cord from 3 patients with spinocerebellar ataxia type 1 and 3 with Machado-Joseph disease; these regions were selected for their differential neuropathological involvement in the two disorders. We observed a considerable homogeneity of repeat size ranges in all but 1 of the 20 regions examined: The cerebellar cortex showed slightly smaller (CAG)n tracts in all specimens from both groups of patients. Our results suggest that the pattern of repeat size mosaicism, similar in spinocerebellar ataxia type 1 and Machado-Joseph disease, reflects the developmental pathways and cell composition of different central nervous system regions and is not the cause of selective cell death in these disorders.

The history of iron in the brain.

Brain iron research began in the late nineteenth century when Zaleski (1886) made a quantitative analysis of one human brain and correlated iron levels with observations on stained slices and some microscopic sections. Gradually, the realization grew that the central nervous system (CNS) contained iron which was different from hemoglobin-iron. This non-heme iron was found in highest concentrations in globus pallidus, substantia nigra, red nucleus, and dentate nucleus. The enhancement of the traditional histochemical stain, potassium ferrocyanide in hydrochloric acid, by incubating the reacted sections in a solution of diaminobenzidine and hydrogen peroxide, revealed iron in many cell types of the CNS, including neurons, microglia, oligodendroglia, and some astrocytes. A large proportion of the soluble brain iron was shown to be present in ferritin. Brain ferritin was found to be very similar to the protein from other organs in that it contained heavy and light subunits. Several investigators reported the presence of other iron-related proteins in the central nervous system, including transferrin, transferrin receptor, and the ferritin repressor protein. Brain was shown to respond to the extravasation of blood by converting the iron in heme to hemosiderin by a sequence of steps which was quite similar to the process in extracerebral organs. The methods of molecular biology have contributed greatly to our understanding of brain iron but many questions remain about its unique anatomical distribution and its role in degenerative diseases such as Parkinson's disease and Alzheimer's dementia.

The cellular reactions to experimental intracerebral hemorrhage.

The resolution of an intracerebral hemorrhage can be measured by the occurrence of hemosiderin. Extravasation of blood elicits a cellular reaction in the adjacent surviving tissue where the lesion activates resident microglia and attracts many more phagocytes from the blood stream. The signals for this migration into the perifocal reactive zone are not fully understood but it is likely that proteins in the coagulated blood contribute to cellular activation. In order to study the role of plasma proteins in the pathogenesis of the perifocal reactive zone, intracerebral injections of either autologous whole blood (0.1 ml) or an equal volume of washed autologous red blood cells (RBC) in lactated Ringer's solution were made in adult rabbits. The amount of total iron was the same (30 micrograms). The cellular responses to the injections were studied by iron histochemistry and immunocytochemistry for ferritin, the ferritin repressor protein (FRP), the glial fibrillary acidic protein (GFAP), and the complement receptor CR3. Experimental hematomas resolved much more slowly after the injection of whole blood than after the injection of RBC. Qualitative microglial and astrocytic responses were quite similar. However, at 48 h, iron- and ferritin-reactive microglia were more numerous following the injection of whole blood. After injections of either type, ferritin-immunoreactive cells were more abundant than iron-positive cells. This observation implied that the biosynthesis of holoferritin protein and iron incorporation proceeded independently. Expression of CR3 on the surface of microglia was much more prominent after whole blood, suggesting a role of inactivated complement 3b in the attraction of additional phagocytes. Conversion to hemosiderin began at 5 days after the injection of either blood or RBC. The lesions caused initial destruction of astrocytes in the perifocal zone as judged by GFAP- and FRP-immunoreactivity. However, at 5 days, astrocytic processes reentered the perifocal zone and intermingled with microglia and macrophages. It is proposed that this contact between astrocytes and microglia reversed the uncoupling of ferritin biosynthesis and iron incorporation and initiated the storage of iron and formation of hemosiderin.

The heterogeneous distribution of brain transferrin.

Immunocytochemistry with antisera to transferrin has often been used to identify oligodendroglia in tissue sections and cultures, but reaction product also occurs in blood vessel walls and nerve cells. There is considerable species variation. Serum transferrin is largely biosynthesized in the liver, and its established physiological role is the transport of iron to tissue sites and delivery of the metal to the interior of cells that have transferrin receptors on their surfaces. In sections of the central nervous system, the visualization of iron and transferrin generally does not coincide, and transferrin may have importance to normal brain function beyond iron transport. For a comparative analysis of transferrin in rabbit and rat brain, polyclonal antisera were raised against purified serum transferrins of these species. The antisera were used for transferrin immunocytochemistry on vibratome sections and for immunochemical detection on electroblots. Transferrin immunocytochemistry and iron histochemistry were compared. The electrophoretic separation of brain extracts and transfer to nitrocellulose membranes permitted the quantitation of the protein and the study of the carbohydrate chains of tissue-bound transferrins by biotinylated lectins. An unexpected result in the rabbit was the dense immunocytochemical reaction product in Bergmann glia and Golgi epithelial cells. Reaction in the cytoplasm of oligodendrocytes was relatively faint in this species except for some selected white matter tracts, e.g. the inferior cerebellar peduncles. In sections of rat brain, oligodendrocytes and vessel walls reacted vigorously in all locations. Transferrin levels in rat brain were substantially higher than in rabbit brain. In the rabbit, maximum transferrin levels occurred in the cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)

Striatal dihydroxyphenylalanine decarboxylase and tyrosine hydroxylase protein in idiopathic Parkinson's disease and dominantly inherited olivopontocerebellar atrophy.

We measured the levels of dopamine, tyrosine hydroxylase (TH) protein, and dihydroxyphenylalanine (DOPA) decarboxylase (DDC) protein in the striatum of 10 patients with idiopathic Parkinson's disease (PD) and 23 patients with dominantly inherited olivopontocerebellar atrophy (OPCA). The levels of dopamine were markedly reduced (2% of control) in the striatum of the patients with PD, whereas striatal dopamine in the patients with OPCA ranged from normal (> 60% of control) to moderately reduced (20-60% of control) to severely depleted (< 20% of control). Both TH and DDC protein levels were significantly lower than those of the controls in the striatum of all of the patients with PD and in the subgroup of patients with OPCA having severely depleted dopamine. In contradistinction, TH but not DDC protein levels were reduced in those patients with OPCA having moderately reduced dopamine levels. This suggests that in the early stage of nigrostriatal dopamine neurone degeneration, DDC levels may be less susceptible to neurodegenerative influences than is TH synthesis or, alternatively, DDC synthesis may be more aggressively upregulated. Unexpectedly, from the blot immunolabeling analysis an additional DDC-immunoreactive band of slightly lower apparent molecular mass was detected in two of the patients with PD and in 12 of the patients with OPCA. This additional DDC band, which was not present in any of the control subjects, may reflect posttranslational modification(s) of DDC related to the neurodegenerative process.

The cellular localization of malonyl-coenzyme A decarboxylase in rat brain.

Malonyl-coenzyme A (CoA) decarboxylase (E.C.4.1.1.9) activity in brain is low but steadily increases after birth. The main physiological role of this mitochondrial enzyme is thought to be the stabilization of malonyl-CoA levels which change very little with brain growth. In an effort to visualize malonyl-CoA decarboxylase by immunocytochemistry, and to determine its developmental changes, the enzyme was purified by an efficient small-scale procedure involving isolation of mitochondria, extraction at high ionic strength, isoelectric focusing, column chromatography, and preparative polyacrylamide gel electrophoresis. The enzyme from brain showed the same apparent molecular weight (160 kDa) and was immunoreactive with antisera raised against malonyl-CoA decarboxylase from liver. Immunocytochemistry revealed early and extensive labeling of hepatocytes in rat liver but only delayed visualization in the brain. Most nerve cells of the cerebral cortex and many microglia were stained but the neurons of the cerebellar cortex did not become reactive. Golgi epithelial cells and their processes, the Bergmann glia, also showed reaction product.

The pathogenesis of superficial siderosis of the central nervous system.

In advanced cases of superficial siderosis of the human central nervous system, the clinical triad of hearing loss, cerebellar ataxia, and myelopathy permits the diagnosis at the bedside, and magnetic resonance imaging readily confirms the hemosiderin deposits in brainstem, cerebellum, and spinal cord. To study the pathogenesis of this condition and explain the selective vulnerability of the cerebellum, experimental siderosis was induced in rabbits by the repeated intracisternal injection of autologous red blood cells. The earliest cellular response in the cerebellar molecular layer was hyperplasia and hypertrophy of microglia as displayed by immunocytochemistry for ferritin. Microglia also contained iron, but ferritin biosynthesis appeared to proceed without commensurate iron accumulation. This early apoferritin response probably occurred due to the presence of heme, rather than iron, in the cerebrospinal fluid and subpial tissue. Ferritin biosynthesis is accelerated when the ferritin repressor protein is dissociated from ferritin messenger ribonucleic acid. A specific antiserum localized ferritin repressor protein predominantly to astrocytes including Bergmann glia. It is proposed that abundance and proximity of ferritin repressor protein--immunoreactive Bergmann glia and ferritin-containing microglia in the cerebellar molecular layer permit prompt cellular interaction in the conversion of heme to ferritin and ultimately hemosiderin.

Immunocytochemical studies on the vimentin distribution and cell proliferation of fibroblasts in patients with Friedreich's ataxia.

Fibroblasts obtained from patients with Friedreich's ataxia and normal control subjects were studied by immunocytochemistry for intermediate filament vimentin and also for in vitro proliferation. Trypsinized cells were seeded on coverslips and incubated for 1.5 h and 24 h. The expression of vimentin in cells was investigated by immunofluorescence microscopy. Cell proliferation was studied with BrdU antibody technique. Cells from patients with Friedreich's ataxia showed a slower outgrowth of vimentin filaments in comparison to cells from normal controls. These cells also incorporated less 5-bromo-2'-deoxyuridine (BrdU) into their DNA. The observations may be relevant to the clinical manifestations of the disease which involves many organs in addition to brain and spinal cord.

Experimental superficial siderosis of the central nervous system: biochemical correlates.

The pathogenesis of superficial siderosis of the central nervous system (CNS) may be examined by the repeated intracisternal injection of washed autologous red blood cells (RBC). In rabbits, the injections cause the accumulation of iron in the cytoplasm of microglial cells and astrocytes of cerebellar and cerebral cortices. Immunocytochemistry for ferritin reveals enhanced reaction product mainly in microglia but hemosiderin occurs only after extending the injections to 6 months. In an effort to determine the biochemical correlates of these morphological changes, iron, ferritin, ferritin subunits and the ferritin repressor protein (FRP) were quantitated. There was no increase of total iron or ferritin in the exposed cortical areas. However, the injections of RBC caused dramatic shifts of the relative contributions by heavy (H-) and light (L-) ferritin subunits. The initial response was a prompt increase of the H/L ratio to over 4.0 from the normal ratio near 1.0. Extended injections caused the ratio to drop to below unity, and the predominance of L-ferritin at 6 months coincided with the appearance of granular hemosiderin. This investigation also confirmed the presence of FRP in rabbit brain cytosols but the induction of experimental superficial siderosis did not change its levels or in vitro affinity for the iron-responsive element in ferritin messenger ribonucleic acid. It is proposed that the incrustation by hemosiderin which characterizes superficial siderosis of the CNS in humans occurs when prolonged exposure to hemoglobin produces persistent shifts of the H/L-ratios by accumulation of L-ferritin.

Myelin deficiency in female rats due to a mutation in the PLP gene.

Myelin deficiency (md) in female rats due to a mutation in the X-linked proteolipid protein (PLP) gene is caused by X-chromosome monosomy. Cytogenetic analysis revealed a single X karyotype [41,X(md/0)]. An immunocytochemical, electron microscopic, and biochemical study was performed on male and female md rats. The central nervous system (CNS) of the female md rat [41,X(md/0)] revealed the same total lack of PLP as the CNS of the affected male littermate [42,XY(md/Y)]. Immunocytochemistry for myelin basic protein (MBP), myelin-associated glycoprotein (MAG), and 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNP) revealed "islands" of myelin sheath-like reaction product in both. Electron microscopy showed great paucity of compact myelin sheaths in 41,X(md/0) and 42,XY(md/Y). Reduced levels of MPB, MAG, and CNP were confirmed for both sexes but MAG and CNP were substantially higher in 41,X(md/0). Sexual differentiation of the brain may account for the observed differences since normal female reproductive organs are present in the md female rat.

Experimental superficial siderosis of the central nervous system. I. Morphological observations.

Autologous washed red blood cells were injected weekly over a period of three to six months into the cisterna magna of adult New Zealand white rabbits. After three months, the surface of the brain stem, cerebellum, and piriform cortex showed a distinct brown color, and staining of the gross specimens for iron produced an intense blue color which extended for a distance of 1-2 mm into the brain parenchyma. Enhanced iron stains of vibratome sections revealed the accumulation of reaction product in microglia and Bergmann glia of the cerebellar cortex, and in microglia and astrocytes of the piriform cortex. Ferritin immunocytochemistry revealed reaction product in cerebellar microglia and Bergmann glia which strongly resembled that obtained by the enhanced iron stain. In the piriform cortex, only microglia were reactive with anti-ferritin. Electron microscopy confirmed the accumulation of electron-dense ferritin granules only in the cytoplasm of microglia. Bergmann glia in the cerebellum and astrocytic processes in the piriform cortex were replete with intermediate filaments and contained an excess of glycogen. After six months, small granules of hemosiderin began to appear in cerebellar and piriform cortices. The observations support that the sequence of conversion of hemoglobin to ferritin and hemosiderin occurs in brain as in other organs.

The Purkinje cell and its afferents in human hereditary ataxia.

The cerebellar cortex in patients with autosomal dominant and recessive ataxia was studied by Golgi impregnation and immunocytochemistry in order to gain further insight into the pathogenesis of neuronal atrophy which accompanies these disorders. Monoclonal antisera were used to visualize phosphorylated and non-phosphorylated neurofilament proteins, and a synapse-specific protein (P38; synaptophysin). Golgi stain and immunocytochemistry for non-phosphorylated neurofilament protein revealed partial or complete loss of distal Purkinje cell dendrities in the dominant cases and in one recessive case. Many preserved parallel fibres were shown by the monoclonal antibody to phosphorylated neurofilament protein. This antibody also gave strong reaction product in torpedoes. Axosomatic and axodendritic terminals on Purkinje cells were reduced in number, and loss of mossy fiber terminals was revealed by monoclonal anti-P38. The described methods provided additional morphological evidence of the heterogeneity of the hereditary ataxias. Purkinje cell atrophy progressed from loss and simplification of the dendritic tree to disappearance of the cell body. While these cells appeared to be especially vulnerable, other neurons of the molecular and granular layers were not exempt. There was evidence that at least some extracerebellar afferents, such as mossy fibers, were also affected by the disease process.

The origin of free brain malonate.

Rat brain contains substantial concentrations of free malonate (192 nmol/g wet weight) but origin and biological importance of the dicarboxylic acid are poorly understood. A dietary source has been excluded. A recently described malonyl-CoA decarboxylase deficiency is associated with malonic aciduria and clinical manifestations, including mental retardation. In an effort to study the metabolic origin of free malonate, several labeled acetyl-CoA precursors were administered by intracerebral injection. [2-14C]pyruvate or [1,5-14C]citrate produced radioactive glutamate but failed to label malonate. In contrast, [1-14C]acetate, [2-14C]acetate, and [1-14C]butyrate were converted to labeled glutamate and malonate after the same route of administration. The intracerebral injection of [1-14C]-beta-alanine as a precursor of malonic semialdehyde and possibly free malonate did not give rise to radioactivity in the dicarboxylate. The labeling pattern of malonic acid is compatible with the reaction sequence: acetyl-CoA----malonyl-CoA----malonate. The final step is thought to occur by transfer of the CoA-group from malonyl-CoA to succinate and/or acetoacetate. Labeling of malonate from acetate is most effective at the age of 7 days when the net concentration of the dicarboxylic acid in rat brain is still very low. At this age, butyrate was a better precursor of malonate than acetate. It is proposed that fatty acid oxidation provides the acetyl-CoA which functions as the precursor of free brain malonate. Compartmentation of malonate biosynthesis is likely because the acetyl-CoA precursors citrate and pyruvate are ineffective.