L. Autilio-Gambetti

Pathogenesis of experimental giant neurofilamentous axonopathies: A unified hypothesis based on chemical modification of neurofilaments

This review summarizes current evidence suggesting that the pathogenetic basis of giant axonal neuropathies induced by neurotoxic chemicals involves a direct chemical modification of neurofilaments (NF) and/or related cytoskeletal proteins. Chemical modification of NF is believed to disrupt the normal cytoskeletal organization, which results in an alteration in NF transport rate and accumulation of NF at prenodal sites along the axon. The exact location at which axonal enlargements occur appears to be a continuous function, dependent on both the structure and dosage schedule of the chemical toxin. A unified hypothesis for the neuropathologic effect of the diverse spectrum of toxic chemicals known to induce giant axonopathies is presented, based on recently published data on the structure of NF protein. Neurotoxic chemicals are believed to alter the charge balance of highly ionic domains of NF proteins which are thought to enter into intermolecular coulombic interactions in forming the supramolecular cytoskeletal framework.

Neuronal and astrocytic differentiation in human neuroepithelial neoplasms: An immunohistochemical study

Neuroepithelial neoplasms of childhood were examined immunohistochemically using antibodies against a neurofilament polypcptide and glial fibrillary acidic protein. Ninety-one cases, including 11 controls, were examined. Positively reacting cells, indicating neuronal and glial differentiation, were found in 59 of the 80 tumors. The study supports a neuroepithelial origin for medulloblastomas, central neuroblastomas, and primitive neuroectoder-mal tumors of childhood. The results also indicate that only a small number of the tumor cells differentiate along either neuronal or glial cell lines.

Function and evolution of neurofilament proteins.

The intermediate filament (IF) proteins have evolved by repeated duplications of a single ancestral gene (FIGURE 1). At least five subfamilies of IF proteins have been identified in mammals, and each subfamily is expressed preferentially in a particular type of differentiated cell: cytokeratins in epithelia, neurofilament (NF) proteins in neurons, glial fibrillary acidic protein (GFAP) in glia, desmin in myogenic cells, and vimentin in early embryonic cells and in mesenchymal cells.’-’ All of the IF proteins have a conserved 40 kD rod-shaped domain, which has probably been derived from the ancestral IF gene?-” This 40 kD domain is distinguished by its large content of alpha helical structure and by the presence of a highly conserved epitope that is recognized by a mouse monoclonal antibody that reacts specifically with all IF proteins.” It appears that the 40 kD domain has been highly conserved because of its essential role in IF IF proteins also contain two hypervariable regions that flank the conserved 40 kD d ~ m a i n . ~ These regions form the amino-terminal (head) and the carboxy-terminal (tail) portions of the IF proteins. Evolutionary variation in both of these regions has led to substantial differences between the subfamilies of IF proteins? In particular, the tail segment of the NF proteins has varied extensively and now contains a number of features not found in other IF proteins. One of the most evident differences between the tail segment of the NF proteins and other IF proteins is size. The tail segment of many NF proteins is larger than 100 kD, whereas the typical tail segment of other IF proteins is less than 5 kD.5,’ The tail region is heavily phosphorylated in higher molecular weight NF subunits16 and amino acid sequencing of the 68 kD subunit has identified a unique domain, the b domain, at the carboxy-terminal end of the protein.’ The b domain is characterized by an unusually high content of acidic residues (44% glutamic acid), and also contains the binding site for the Bodian

Analysis of the prion protein gene in thalamic dementia

Thalamic degenerations or dementias are poorly understood conditions. The familial forms are (1) selective thalamic degenerations and (2) thalamic degenerations associated with multiple system atrophy. Selective thalamic degenerations share clinical and pathologic features with fatal familial insomnia, an autosomal dominant disease linked to a mutation at codon 178 of the prion protein (PrP) gene that causes the substitution of asparagine for aspartic acid (178Asn mutation). We amplified the carboxyl terminal coding region of the PrP gene from subjects with selective thalamic dementia or thalamic dementia associated with multiple system atrophy. Three of the four kindreds with selective thalamic dementia and none of the three kindreds with thalamic dementia associated with multiple system atrophy had the PrP 178Asn mutation. Thus, analysis of the PrP gene may be useful in diagnosing the subtypes of thalamic dementia. Moreover, since selective thalamic dementia with the PrP 178Asn mutation and fatal familial insomnia share clinical and histopathologic features, we propose that they are the same disease.

MOLECULAR PATHOLOGY OF FATAL FAMILIAL INSOMNIA

Fatal familial insomnia (FFI) is linked to a mutation at codon 178 of the prion protein gene, coupled with the methionine codon at position 129, the site of a methionine/valine polymorphism. The D178N mutation coupled with the 129 valine codon is linked to a subtype of Creutzfeldt‐Jakob disease (CJD178) with a different phenotype. Two protease resistant fragments of the pathogenic PrP (PrPres), which differ in molecular mass, are associated with FFI and CJD178, respectively, suggesting that the two PrPres have different conformations and hence they produce different disease phenotypes. FFI transmission experiments, which show that the endogenous PrPres recovered in affected syngenic mice specifically replicates the molecular mass of the FFI PrPres inoculated and is associated with a phenotype distinct from that of the CJD178 inoculated mice, support this idea. The second distinctive feature of the FFI PrPres is the underrepresentation of the unglycosylated PrPresform. Cell models indicate that the underrepresentation of this PrPres form results from the PrP dys‐metabolism caused by the D178N mutation and not from the preferential conversion of the glycosylated forms. Codon 129 on the normal allele further modifies the FFI phenotype determining patient subpopulations of 129 homozygotes and heterozygotes: disease duration is generally shorter, insomnia more severe and histopathology more restricted to the thalamus in the homozygotes than in the heterozygotes The allelic origin of PrPres fails to explain this finding since in both cases FFI PrPres is expressed only by the mutant allele. Despite remarkable advances, many issues remain unsolved precluding full understanding of the FFI pathogenesis.

Familial progressive subcortical gliosis Presence of prions and linkage to chromosome 17

Article abstract—Progressive subcortical gliosis (PSG) is a sporadic and familial dementing disease characterized pathologically by astrogliosis at the cortex-white matter junction, a feature present in some prion diseases. With im-munocytochemical and Western blot analyses, we investigated the presence of deposits of the prion protein (PrP) and of the protease-resistant PrP isoform, the hallmarks of prion diseases, in six affected members of two large kindreds with PSG. The coding region of the PrP gene was sequenced and chromosomal linkage determined. We demonstrated “diffuse” PrP plaques in the cerebral cortex of two subjects from one kindred and protease-resistant PrP fragments in four of the five subjects examined. We found no mutation in the coding region of the PrP gene. Moreover, the disease was linked to chromosome 17 and not to chromosome 20, where the PrP gene resides. The familial form of PSG is the first human genetic disease characterized by the presence of protease-resistant PrP that lacks a mutation in the coding region of the PrP gene. The linkage to chromosome 17 suggests that other genes are involved in the PrP metabolism. Whether the protease-resistant PrP plays a primary or secondary role in the pathogenesis of this form of PSG remains to be determined.

Differential APP gene expression in rat cerebral cortex, meninges, and primary astroglial, microglial and neuronal cultures

Differential amyloid precursor protein (APP) gene expression was investigated in primary cultures of astrocytes, neurons and microglia from neonatal rat cerebral cortex as well as in meninges, and young and adult cerebral cortex tissues in order to define the possible contribution of individual CNS cell types in βAP deposition. Meninges and neurons contained higher levels of total APP mRNA than glial cells and APP695 mRNA was abundant in neurons while glial cells and meninges contained higher levels of KPI‐containing mRNAs. These results demonstrate cell‐specific transcriptional and post‐transcriptional regulation of APP gene expression in CNS cell types. In addition, the steady‐state level of APPs in each cell type did not reflect mRNA levels indicating translational or post‐translational regulation.

Ubiquitin in Motor Neuron Disease: Study at the Light and Electron Microscope

Several neurodegenerative diseases, including motor neuron disease (MND), are characterized by formation of abnormal cytoskeleton-derived inclusions which contain ubiquitin (Ubq). We have studied the distribution of Ubq in 26 cases of MND with light and electron microscopic immunocytochemistry. Ubiquitin-positive inclusions were found in neurons of anterior horns in most cases of amyotrophic lateral sclerosis (ALS) but were not present in other forms of MND. Ubiquitin immunoreactivity was observed in 10–15 nm intraneuronal filaments, which were not stained by antibodies to neurofilaments, and on dense bodies of dystrophic neurites throughout the neuropil of anterior horns and pyramidal tracts. Data analysis showed a trend toward lower percentage of Ubq-positive neurons in cases with longer duration of illness or lower number of neurons. A high percentage of Ubq-positive inclusions occurred in cases with an aggressive clinical course, suggesting that ubiquitination takes place at early stages of the disease.

Bodian's silver method reveals molecular variation in the evolution of neurofilament proteins

The recent demonstration that Bodians silver method specifically stains mammalian neurofilament subunits (NFs), but not other intermediate filament proteins (IFs), provides a specific marker for the identification of neurofilament polypeptides. We have applied the Bodian stain to SDS-PAGE separated polypeptides in nervous tissues from 9 species, representing neuronal evolution in 4 major phyla: chordata, mollusca, arthropoda and annelida. Every species tested except the arthropod showed intense silver staining of a set of polypeptides, each subsequently identified as NFs by immunomethods. These results demonstrate that the affinity of NFs for Bodians silver stain is conserved during the evolution of nervous systems in a diverse spectrum of animals. Further, considerable variation in the molecular weight of NF subunits was found among the 6 vertebrates studied. This variation suggests that the molecular weight of NFs has not been conserved during evolution, a quality which appears to be unusual for a structural protein.

Experimental increase of neurofilament transport rate: Decreases in neurofilament number and in axon diameter

In 2,5-hexanedione (2,5-HD)-induced axonal neuropathy, the rate of neurofilament (NF) transport increases in optic axons. To test the prediction that increases in the rate of polymer transport in any one locality of the axon lead directly to a decrease in the number of NF in that locality, NF and microtubules (MT) were quantitatively analyzed in axonal cross sections. In 2,5-HD axons the number of NF was 38% of that in control axons while the number of MT was not significantly changed; it appears that the drug treatment decreases NF number in the proximal axon regions, most directly through an increase in rate of NF transport. In those regions, the cross-sectional areas of the 2,5-HD-treated axons were 45% smaller than those of control axons; although the axons had shrunk in diameter, they retained their normal cylindrical shapes as measured by the index of circularity. Reduced internal expansive forces in the axon, working in conjunction with the normal external compressive forces, appear to reduce the radius of the axon. Quantitative analyses demonstrated that the average and the maximum lateral spacings between NF-NF, NF-MT, and MT-MT were all 30% larger in 2,5-HD-treated axons than in control axons. This suggests that polymers are relatively free to move laterally away from one another and to fill the available, space within the axon. These observations are not consistent with models wherein 2,5-HD acts to crosslink the NF into an immobile network that can no longer advance within the axon. Instead, it appears more likely that 2,5-HD acts selectively on the interaction between some NF and the slow transport mechanism to increase the rate of NF transport.