Oral aluminum administration during pregnancy and lactation produces gastric and renal lesions in rat mothers and delay in CNS development of their pups.

The expression of the neurofilament protein of the highest molecular weight (NF-H) is developmentally and spatially regulated. For example, the MAb RMO24.9, directed against a phosphorylated epitope in the tail domain of NF-H, immunohistochemically labels specific tracts within the rat brainstem prenatally, but does not label diencephalic tracts until after postnatal day 10 (P10). A diet providing 300 mg/kg/d Al (as Al lactate) to rat dams throughout gestation causes behavioral deficits in their offspring (Bernuzzi et al., 1989). We repeated this regimen by substituting 120 mM Al lactate (pH 6.5) for drinking water during gestation and lactation, and examined the distribution of immunolabeling by RMO 24.9 after exposure to Al. Tracts within the diencephalon that bind RMO 24.9 on P11 in control pups did not bind the MAb until P14 in Al-treated pups. In these preliminary experiments, Al seemed to have caused a developmental delay in the expression of phosphorylated NF-H in the pups of mothers that received Al during gestation. However, subsequent experiments showed that the neuropathology observed--and that reported by other investigators using similar Al levels--may not be the result of the direct effects of Al on the pups. Throughout lactation, treated dams appeared progressively more cachexic. Unlike the normal viscera of pair-watered controls, the stomachs of treated dams were ulcerated, and their kidneys had decreased cortical thickness and contained stones. Lesions such as these compromise a rat's ability to absorb nutrients, to excrete toxins, and to regulate water and electrolytes. In a lactating dam, these alterations could compromise the dam's ability to nourish her pups. Our experiments point out that the mechanisms of Al toxicity-- already complex in the adult--are further complicated in a system in which the pup is dependent on the mother for delivery of both nutrients and toxins. It is therefore impossible to determine the cause of any neuropathology in the pup in a system where Al delivery overlies a background of multisystem defect and altered maternal homeostasis.

Expression of the neurofilament protein NF-H in L cells.

We have inserted a Not1-Sal1 fragment of the mouse gene coding for the neurofilament protein NF-H behind the dexamethasone-inducible transcription promoter of MMTV in a vector derived from pMAMneo (Clonetech Labs). This construct, which includes all four exons of the NF-H gene, was amplified and incorporated into liposomes for transfection of L cells. Transfectants were selected in G418-containing medium and cloned. Clones were grown in serum-containing medium and screened for expression of the NF-H mRNA by extraction of total RNA, generation of cDNAs by reverse transcription, and amplification of a 900-base portion of the NF-H cDNA by PCR. Positive clones were detected by the presence of a band of the correct size on agarose gels. This was confirmed by Southern blotting of the gels probed with a 185-base segment of the amplified region. Immunofluorescent analysis of two positive clones, C33 and C34, showed that C33 cells grown in serum-containing medium or in serum-free medium in the presence of dexamethasone have a network of SMI32 (Sternberger/Meyer Inc.--monoclonal antibody against a nonphosphorylated epitope on NF-H)-positive filaments with the same distribution as filaments stained with antibodies to vimentin, while C34 cells do not react with antibodies against neurofilament proteins. Neither clone reacted with antibodies against highly phosphorylated NF-H (SMI31).

cDNA coding for the tail region of the high molecular weight rabbit neurofilament protein NF-H.

A cDNA library was prepared from rabbit brain mRNA, in the expression vector, lambda gt11. The library was screened with polyclonal antibodies raised against the neurofilament protein NF-H, and a cloned cDNA (KMRH-1) was selected and characterized. The fusion protein coded for by KMRH-1 includes epitopes for two monoclonal antibodies which react with nonphosphorylated sites in the tail region of NF-H. The selected cDNA includes 891 base pairs. It hybridizes to human genomic DNA, as well as to rabbit genomic DNA, and to a rabbit brain mRNA with a size of 4.7 kilobases (kb). The sequence of KMRH-1 includes extensive repeating regions, including one duplicated 60-base segment. Within the first 196 bases, one 13-base segment is repeated 9 times. The cDNA codes for the carboxy-terminal 184 amino acid residues of NF-H, including a series of 9 serines, each surrounded by a similar group of amino acids: ..Ala.Lys.Ser.Pro.(Glu./Val.).Lys.. Comparison of the derived amino acid sequence for KMRH-1 indicates considerable divergence from the sequence information available for rodent NF-H (Robinson et al.: FEBS Lett 209:203-205, 1986). This diversity in amino acid sequence may account for the failure to induce tangles of neurofilaments in animals, such as rats, following treatment with doses of aluminum which are sufficient to induce such tangles in rabbits and to bring on seizures and behavioral pathology in both species.

The doublet microtubules of rods of the rabbit retina.

The connecting cilium of the rabbit photoreceptor rod is composed of nine outer doublets, lacking dynein side arms. The central singlet microtubules are absent. In cross section, there is an inner dense ring situated between the doublets and the center core of the cilium. As the doublet microtubules progress from the connecting region into outer segments, the cylindrical array of the nine pairs of doublets spreads out as a brush-like arrangement into the incisure cavity of the outer segment. The microtubules continue as doublets for much of the length of the outer segment. The B-tubules terminate first; the A-tubules extend as single tubules into the apical region of the photoreceptor. Before the B-tubules end, they open up, forming hook-shaped projections from the A-tubules. The gradual reduction in length of these hook-shaped structures suggests that near their distal ends each B-tubule opens because of the separation of protofilament 1 of the B-tubule from protofilament 1 of the adjacent A-tubule. Subsequently, the B-tubule protofilaments terminate individually.

Reduction of neuronal specific protein and some neurotransmitters in the infantile neuroaxonal dystrophy (INAD).

INAD with classical clinicopathological features was seen in three children of one family. They presented with a history of regression after the age of 1.5 years and died in mental institutions at the ages of 6, 7 and 9. Two of them had postmortem neuropathological studies, one had brain biopsy and one biochemical study. The following observations have been made; I. Histological--(1) some axonal loss with almost total absence of neurofilaments in dystrophic neurites and (2) marked degenerative changes and loss of synaptic vesicles; II. Biochemical--(1) the decrease of the neurofilament polypeptides by up to 75% and (2) a reduction of some neurotransmitter enzymes. The presence of intermitochondrial septate junctions. The neurofilament protein is specific to neurons and makes up a large percentage of their protein content. Neurofilaments have been implicated in several cellular functions such as intracellular transport and in the maintenance of cell structure. The decrease of neurofilament protein in our case is compatible with our morphological findings where we found decreased numbers of axons and an almost total absence of neurofilaments in the affected neurites.

Similarity of neurofilament proteins from different parts of the rabbit nervous system.

In the nervous system, the various populations of neurons perform a large spectrum of functions. Although neurofilaments are a major constituent of the different neurons, the neurofilament protein composition and the expression of the genes specifying these proteins may not be the same throughout the entire nervous system. To investigate these two aspects of the biology of neurofilaments, we have prepared neurofilament-rich fractions from different regions of the nervous system of strains of rabbits known to present a genetically determined polymorphism involving one of the neurofilament polypeptides (P200). Filaments were isolated from brain, spinal cord, sciatic, optic and trigeminal nerves, and lumbar ventral and dorsal roots by a procedure not involving axonal flotation and yielding material suitable for comparative analysis within a single animal. The filaments were compared for their variability as a function of the region from which they were prepared. For any given animal, the neurofilament peptides migrate to identical positions on SDS-gel electropherograms. Whatever allele of P200 is expressed in filaments from one region, the same allele is also expressed in all of the other filament preparations from that animal. On two-dimensional analysis isomorphs of the P68 neurofilament protein are not present in the same amounts in different regions of the nervous system. These results indicate that, although it seems that the gene for the P200 neurofilament protein is expressed uniformly throughout the nervous system, there may be some topographic specificity in the distribution of the other constituent proteins of neurofilaments.

The loss of neuron-specific proteins during the course of Wallerian degeneration of optic and sciatic nerve.

To identify axonal proteins which are unique constituents of neurons, the spectrum of detectable proteins of degenerating nerves has been compared with that of intact control nerves from the same animals. Wallerian degeneration was induced in rabbits by unilateral transection of the optic and sciatic nerves. Proteins of nerve homogenates were compared by sodium dodecyl sulfate-gel electrophoresis and by two-dimensional electrophoresis. Four non-myelin proteins disappear from degenerating nerve. These include the three neurofilament proteins (P68, P150, and P200) and a polypeptide with a molecular weight of about 65,000 daltons (P65) which is not associated with filaments.

Association of newly synthesized tubulin with brain microsomal membranes.

Tubulin has been found to be synthesized on both membrane-bound and free polyribosomes prepared from brain. Cell-free studies indicate that tubulin made on rough microsomes is incorporated into the endoplasmic reticulum membrane as it is synthesized. This tubulin remains associated with the membrane after sedimentation and washing. The tubulin is not removed from the membrane after stripping ribosomes from the membranes in KCl-puromycin, followed by repeated washing by either sedimentation or flotation in 0.05 M-KCl. The membrane tubulin is partially susceptible to proteolysis by trypsin and chymotrypsin: beta-tubulin is more accessible to the proteases than in alpha-tubulin. Nonionic detergents extract mostly beta-tubulin from the microsomal membrane. Newly synthesized tubulin which has been extracted from microsomal membranes in 0.5% Nonidet P-40, coassembles and disassembles with carrier microtubule protein. The insertion of newly synthesized tubulin into endoplasmic reticulum membrane may be the first step in the incorporation of tubulin into the plasma membrane.

Heterogeneity of intermediate filament proteins from rabbit spinal cord.

Large amounts of a nerofilament-enriched fraction may be prepared from spinal cord homogenates by a simple, three-step procedure. This involves flotation of filament-containing axon fragments, extraction with Triton X-100, and washing by sedimentation through a sucrose density gradient. The material obtained by this procedure includes both large mats of individual 10-nm filaments and tightly packed bundles of filaments. SDS-gel electrophoresis of these fractions indicates that the fractions are formed of four polypeptides: the three which are generally considered to form neurofilaments (P200, P150, and P68) and another, with a molecular weight of about 50,000 daltons (P50), which is thought to be derived from fibrous astrocytes. Analysis of these filament fractions on two-dimensional gels indicate heterogeneity among each of the different molecular weight classes. The largest polypeptide of neurofilaments, P200, focuses at several spots in the pH gradient. P68 and P150 are more acidic: each appears as a pair of overlapping spots. P50 resolves into a complex of spots of about the same molecular weight but with different isoelectric points. Heterogeneity is not unique to these filament polypeptides but appears to be a characteristic of all fibrous proteins of the nervous system.

Studies on the biosynthesis of neurofilament proteins.

To determine whether the triplet polypeptides of neurofilaments arise by degradation of precursor, we studied the biosynthesis of neurofilament polypeptides both in vivo and in cell-free systems. Neurofilament-enriched fractions and polyribosomes were prepared from the same rabbit spinal cord homogenates. At 1 h after intracisternal administration of [34S]methionine, radiolabeled neurofilament proteins were detected in spinal cord homogenates as well as in isolated filaments. When polyribosomes from rabbit spinal cord were allowed to incorporate [35S]methionine into protein, triplet polypeptides were among the proteins labeled. Addition of spinal cord polyribosomes to rabbit reticulocyte lysates led to several cycles of translation of the spinal cord mRNA; the three neurofilament polypeptides were among the proteins synthesized in this system. The results demonstrate that the triplet polypeptides of neurofilaments are synthesized as such in the course of individual translational events and do not arise from degradation of P200 or a larger precursor.

Poly(A)- and nonpoly(A)-RNA associated with rat brain microsomal fractions: in vivo labelling studies.

The time course of incorporation of radiolabelled precursor into RNA associated with rat brain free polyribosomes, rough membranes, and smooth membranes was measured following a single intracranial injection of [3H] orotic acid. Polyadenylated RNAs were separated from nonpolyadenylated RNAs by affinity chromatography on oligo (dT)-cellulose columns. Poly(A)-RNA associated with each of the microsomal fractions became more rapidly labelled than did the nonpoly(A)-RNA of the same fractions. While the labelling profiles of the nonpoly(A)-RNA isolated from the polyribosomes and rough membranes are similar from one fraction to another, the specific radioactivity of the poly(A)-RNA isolated from free polyribosomes increased much more drastically than that of the poly(A)-RNA associated with rough membranes. The labelling profiles of RNA species isolated from smooth membranes were very different in this respect from the two ribosomal fractions. There was a lag of more than four hours before significant label appeared in the RNA associated with the smooth membrane fraction. These studies demonstrate that the different populations of brain microsomal RNA are labelled at different rates, perhaps reflecting differences in the turnover of these RNAs and differences in their function.

Isolation and characterization of free and membrane-bound polyribosomes from rabbit spinal cord.

A procedure is described for the preparation of free and membrane-bound polyribosomes from rabbit spinal cord. First, myelin and filaments are floated away from other subcellular components. The latter are then fractionated by differential centrifugation followed by sedimentation through a discontinuous sucrose gradient. The ribosomal fractions are characterized by their electron microscopic appearance, RNA/protein ratios and sedimentation profile in a linear sucrose gradient. Both membrane-bound and free polyribosomes are active in incorporating amino acids in a cell-free system, whether heterologous or homologous pH 5 enzyme fractions are employed. Autoradiographic analysis of translation products separated by electrophoresis on SDS-polyacrylamide gels shows that both ribosome fractions are active in the synthesis of a variety of proteins including polypeptides which comigrate with alpha- and beta-tubulins and actin. The utility of these polyribosome preparations for the cell-free study of protein biosynthesis is indicated by the high molecular weight of many of the translation products, and by the similarity of the electrophoretic pattern of translation products from the cell-free systems to the pattern of radioactive protein synthesized by rabbit spinal cord during the hour prior to sacrifice.

Neurofibrillary pathology: current status and research perspectives.

The neurofibrils seen in the light microscope are shown, by electron microscopy, to be heterogeneous structures, formed of neurotubules and neurofilaments. A variety of pathological conditions (especially, presenile and senile dementia (are characterized by the presence of neurofibrillary tangles, formed either of paired helical filaments or of single filaments. The morphology, distribution and biochemistry of these various fibrillary structures is reviewed. Particular attention is devoted to the assembly of neurotubules, to the mechanism of action of drugs which prevent assembly, and to possible implications for the experimental induction of neurofibrillary pathology. Of central importance in the arguments is the emphasis on the normal neurofibrillary structures being single forms of pleomorphic proteins. The healthy neuron assembles these proteins into the required form as is necessary for the needs of that neuron. Interference with this process in neuronal cell biology may lead to the deposition of neurofibrillary tangles. On the basis of the morphological and biochemical evidence, several approaches to the experimental study of neurofibrillary pathology are proposed.

Isolation and characterization of an enriched Golgi fraction from rat brain.

A fraction rich in membranes of the Golgi apparatus was isolated from rat brain by discontinuous density gradient centrifugation. The fraction sedimented at the characteristic Golgi density of 1.11--1.15 (g/cm3, 5 degrees C) and had specific activities of Golgi-marker enzymes (N-acetyllactosaminyl synthetase, glycoprotein (Fetuin) galactosyltransferase, thiamine pyrophosphatase), 6--7 times over those of the original homogenates. The recovery of the enzyme activities in this fraction ranged from 17 to 31 %. The incorporation [3H]fucose into glycoproteins was 3-fold higher than in homogenate. Recovery and relative specific activities of marker enzymes for other subcellular organelles were low. Electron microscopic analysis of the fraction revealed in the presence of Golgi structures, namely, large sacs or plates with attached tubules and "blebbing" of the tubules into the vesicles.