Prion replication-once again blaming the dendritic cell.

The lymphoid system is known to be involved in the propagation and spread of scrapie. However, the identity of the cell type responsible for scrapie replication remains controversial. A new study provides evidence that the follicular dendritic cells in the spleen are the targets of this infectious form of prion (pages 1308-1312).

Allelic origin of the abnormal prion protein isoform in familial prion diseases.

The hallmark of prion diseases is the presence of an aberrant isoform of the prion protein (PrP(res)) that is insoluble in nondenaturing detergents and resistant to proteases. We investigated the allelic origin of PrP(res) in brains of subjects heterozygous for the D178N mutation linked to fatal familial insomnia (FFI) and a subtype of Creutzfeldt-Jakob disease (CJD178), as well as for insertional mutations associated with another CJD subtype. We found that in FFI and CJD178 subjects, only mutant PrP was detergent-insoluble and protease-resistant. Therefore, PrP(res) derives exclusively from the mutant allele carrying the D178N mutation. In contrast, in the CJD subtype harboring insertional mutations, wild-type PrP was also detergent-insoluble and likely to be protease-resistant. Our findings indicate that the participation of the wild-type PrP in the formation of PrP(res) depends on the type of mutations, providing an insight into the molecular mechanisms underlying the phenotypic heterogeneity in familial prion diseases.

Presence of soluble amyloid beta-peptide precedes amyloid plaque formation in Down's syndrome.

Abnormal and excessive accumulation of the amyloid beta-peptide (A beta) in the brain is a major and common characteristic of all Alzheimer's disease (AD) forms irrespective of their genetic background. Insoluble aggregates of A beta are identified as amyloid plaques. These deposits are thought to form when the amount of A beta is increased in the brain parenchyma as a result of either overexpression or altered processing of the amyloid precursor protein (APP). Soluble A beta ending at carboxyl-terminal residue 40 (A beta 40) and, in lesser amount, the form ending at residue 42 (A beta 42), are normal products of the APP metabolism in cell cultures. Increased secretion of soluble A beta 42 has been observed in cells transfected with constructs modeling APP gene mutations of familial forms of AD (refs 4, 5). On the basis of these in vitro data it has been hypothesized that the presence of soluble A beta 42 plays a role in the formation of amyloid plaques. Subjects affected by Down's syndrome (DS) have an increased APP gene dosage and overexpress APP. Apparently because of this overexpression, they almost invariably develop amyloid deposits after the age of 30 years, although they are free of them at earlier ages. Moreover, it has been observed that A beta 42 precedes A beta 40 in the course of amyloid deposition in DS brain. Thus, DS subjects provide the opportunity to investigate in the human brain the metabolic conditions that precede the formation of the amyloid deposits. Here we report that soluble A beta 42 is present in the brains of DS-affected subjects aged from 21 gestational weeks to 61 years but it is undetectable in age-matched controls. It is argued that overexpression of APP leads specifically to A beta 42 increase and that the presence of the soluble A beta 42 is causally related to plaque formation in DS and, likely, in AD brains.

Tau gene mutation in familial progressive subcortical gliosis.

Familial forms of frontotemporal dementias are associated with mutations in the tau gene. A kindred affected by progressive subcortical gliosis (PSG), a rare form of presenile dementia, has genetic linkage to chromosome 17q21-22. This kindred (PSG-1) is included in the 'frontotemporal dementias and Parkinsonism linked to chromosome 17' group along with kindreds affected by apparently different forms of atypical dementias. Some of these kindreds have mutations in the tau gene. We report here that PSG-1 has a tau mutation at position +16 of the intron after exon 10. The mutation destabilizes a predicted stem-loop structure and leads to an over-representation of the soluble four-repeat tau isoforms, which assemble into wide, twisted, ribbon-like filaments and ultimately result in abundant neuronal and glial tau pathology. The mutations associated with PSG and other atypical dementias can be subdivided into three groups according to their tau gene locations and effects on tau. The existence of tau mutations with distinct pathogenetic mechanisms may explain the phenotypic heterogeneity of atypical dementias that previously led to their classification into separate disease entities.

The fine structure of puromycin-induced changes in mouse entorhinal cortex.

Bitemporal intracerebral injections of puromycin in mice suppress indefinitely expression of memory of avoidance-discrimination learning. Ultrastructural studies of the entorhinal cortex of puromycin-treated mice revealed the following: (a) Abnormalities were not observed in presynaptic terminals and synaptic clefts; many postsynaptic dendrites or somas contained swollen mitochondria. (b) Dispersion of polyribosomes into single units or condensation of ribosomes into irregular aggregates with loss of "distinctiveness" was noted in a few neurons 7-27 hr after puromycin treatment. (c) Cytoplasmic aggregates of granular or amorphous material were frequently noted within otherwise normal neuronal perikarya. (d) Mitochondria in many neuronal perikarya and dendrites were swollen. Mitochondria in axons, presynaptic terminals, and glial cells were unaltered. The relationships between these lesions and the effect of puromycin on protein synthesis and memory are examined. It is suggested that the disaggregation of polysomes is too limited to explain the effect of puromycin on memory. Special emphasis is given to the swelling of mitochondria. The possible mechanisms and the significance of this lesion are discussed.

Proteins transported in slow components a and b of axonal transport are distributed differently in the transverse plane of the axon.

The distribution of the proteins migrating with the slow components a (SCa) and b (SCb) of axonal transport were studied in cross-sections of axons with electron microscope autoradiography. Radiolabeled amino acids were injected into the hypoglossal nucleus of rabbits and after 15 d, the animals were killed. Hypoglossal nerves were processed either for SDS-polyacrylamide gel electrophoresis fluorography to identify and locate the two components of slow transport, or for quantitative electron microscope autoradiography. Proteins transported in SCa were found to be uniformly distributed within the cross-section of the axon. Labeled SCb proteins were also found throughout the axonal cross-section, but the subaxolemmal region of the axon contained 2.5 times more SCb radioactivity than any comparable area in the remainder of the axon.

Reorganization of axoplasmic organelles following beta, beta'-iminodipropionitrile administration.

beta, beta'-Iminodipropionitrile (IDPN), a synthetic compound that selectively impairs slow axonal transport, produced a rearrangement of the axonal cytoskeleton, smooth endoplasmic reticulum, and mitochondria. Immunoperoxidase staining using an antiserum to the 68,000-dalton neurofilament subunit demonstrated a displacement of neurofilaments toward the periphery of the axons of IDPN-treated rats. This change occurred simultaneously along the entire length of the sciatic nerve. Ultrastructural morphometry of the axonal organelles confirmed the peripheral relocation of neurofilaments and also showed a displacement of microtubules, smooth endoplasmic reticulum, and mitochondria to the center of the axons. The overall density of axonal mitochondria was increased, whereas those of other organelles were not significantly changed. Axons were reduced in size by 10--24%, the large axons being more affected than the small ones. The observed rearrangement of axonal organelles may be due to an effect of IDPN on microtubule-neurofilament interactions, which could in turn explain the impairment of the slow transport. Axons in IDPN intoxication are a useful model to study the organization of the axoplasm and the mechanism of axonal transport.

Axonal growth during regeneration: a quantitative autoradiographic study.

The intraaxonal distribution of labeled glycoproteins in the regenerating hypoglossal nerve of the rabbit was studied by use of quantitative electron microscope autoradiography. 9 d after nerve crush, glycoproteins were labeled by the administration of [3H]fucose to the medulla. The distribution of transported 3H-labeled glycoproteins was determined 18 h later in segments of the regenerating nerve and in the contralateral, intact nerve. At the regenerating tip, the distribution was determined both in growth cones and in non-growth cone axons, 6 and 18 h after labeling. The distribution within the non-growth cone axons of the tips was quite different at 6 and 18 h. At 6 h, the axolemma region contained < 10% of the radioactivity; at 18 h, it contained virtually all the radioactivity. In contrast, the distribution within the growth cones was similar at both time intervals, with 30% of the radioactivity over the axolemmal region. Additional segments of the regenerating nerve also showed a preferential labeling of the axolemmal region. In the intact nerve, 3H-labeled glycoproteins were uniformly distributed. These results suggest that: (a) in this system the labeled glycoproteins reaching the tip of the regenerating axons are inserted into the axolemma between 6 and 18 h after leaving the neuronal perikaryon; (b) at the times studied, there is a fairly constant ratio between glycoproteins reaching the growth cone through axoplasmic transport and glycoproteins inserted into the growth cone axolemma; (c) the axolemma elongates by continuous insertion of membrane precursors at the growth cone; the growth cone then advances, leaving behind an immature axon with a newly formed axolemma; and (d) glycoproteins are preferentially inserted into the axolemma along the entire regenerating axon.

Quantitative autoradiographic study of labeled RNA in rabbit optic nerve after intraocular injection of (3H)uridine.

The distribution of labeled RNA in the optic nerve of the rabbit was studied by quantitative ultrastructural autoradiography after the intraocular injection of [(3)H]uridine. The highest density of silver grains related to [(3)H]RNA (27-40 grains/100 microm(2)) was found in glial cell perikarya; a slightly lower density was present in the glial nuclei (19-20 grains/100 microm(2)). Axons (4-5 grains/100 microm(2)) and myelin (2-3 grains/100 microm(2)) had the lowest grain densities. 74-83% of all counted grains were located outside the axons. By comparing the grain density distribution over the axon with that expected in the case of an exclusive labeling of the surrounding myelin and glial cell processes, it was concluded that the axons contained a number of grains representing [(3)H]RNA significantly higher than that expected to scatter from myelin and glial processes. Most of these grains were concentrated at the periphery of the axon and were not related to axonal mitochondria.

Morphological and biochemical changes in rat synaptosome fractions during neonatal development.

A biochemical and quantitative morphologic study of presynaptic endings during postnatal development was carried out in subcellular fractions from cerebral cortex of 1, 4, 8, 12, and 18 day old and adult rats. Crude mitochondrial fractions were subfractionated in Ficoll gradients and all resulting fractions were examined in the electron microscope. Presynaptic terminals and other intact processes were counted. Protein content and enzyme activities were assayed in the fractions and in total brain homogenate. In the first and fourth day of life, most of the presynaptic terminals were found in two "light" fractions, between supernatant and 7.5% Ficoll, where they accounted, respectively, for 6 and 22% of all the processes. Progressively with age, more presynaptic terminals were found in the traditional "synaptosomal" fractions between 7.5 and 13% Ficoll. In that region of the gradient, 40, 54, 75, and 89% of the processes were presynaptic endings at 8, 12, and 18 postnatal days and in the adult animal, respectively. A similar shift from the lighter to the heavier fractions was observed in the distribution of choline acetyltransferase and acetylcholinesterase between days 8 and 12. The rate of increase of the specific activity of these two enzymes paralleled that of the percentage of the presynaptic endings after day 8. This study indicates that subcellular fractions can be used to study formation and maturation of synapses during postnatal development.

Calcium-containing structures in vertebrate glial cells. Ultrastructural and microprobe analysis.

Electron probe microanalysis has revealed that vesicular or cisternal structures containing electron-dense material in frog ependymal glial cells contain deposits of calcium and phosphorus. The so-called "osmiophilic particles" in human astrocytes also contain calcium. It is suggested that these organelles are storage sites of calcium.

Redistribution of proteins of fast axonal transport following administration of beta,beta'-iminodipropionitrile: a quantitative autoradiographic study.

Beta,beta'-iminodipropionitrile (IDPN) produces a rearrangement of axoplasmic organelles with displacement of microtubules, smooth endoplasmic reticulum, and mitochondria toward the center and of neurofilaments toward the periphery of the axon, whereas the rate of the fast component of axonal transport is unchanged. Separation of microtubules and neurofilaments makes the IDPN axons an excellent model for study of the role of these two organelles in axonal transport. The cross-sectional distribution of [3H]-labeled proteins moving with the front of the fast transport was analyzed by quantitative electron microscopic autoradiography in sciatic nerves of IDPN-treated and control rats, 6 h after injection of a 1:1 mixture of [3H]-proline and [3H]-lysine into lumbar ventral horns. In IDPN axons most of the transported [3H] proteins were located in the central region with microtubules, smooth endoplasmic reticulum and mitochondria, whereas few or none were in the periphery with neurofilaments. In control axons the [3H]-labeled proteins were uniformly distributed within the axoplasm. It is concluded that in fast axonal transport: (a) neurofilaments play no primary role; (b) the normal architecture of the axonal cytoskeleton and the normal cross-sectional distribution of transported materials are not indispensable for the maintenance of a normal rate of transport. The present findings are consistent with the models of fast transport that envision microtubules as the key organelles in providing directionality and propulsive force to the fast component of axonal transport.

Protein synthesis in synaptosomal fractions. Ultrastructural radioautographic study.

A quantitative ultrastructural radioautographic study of in vitro protein synthesis has been carried out in rat synaptosomal fractions incubated with tritiated leucine or a tritiated amino acid mixture. Analysis of grain density distribution demonstrated that presynaptic endings are labeled. 30-50% of the developed grains, representing tritiated amino acids incorporated into proteins, were related to presynaptic endings which accounted for 75-77% of the total processes. 34-45% of the grains were related to processes containing ribosomes which accounted for only 4-7% of the total processes. The relative specific activity of these ribosome-containing processes, some of which could be identified as postsynaptic elements, was up to ten times higher than that of the presynaptic ending. These findings indicate that protein synthesis takes place in vitro in presynaptic terminals although to a significantly lesser degree than that occurring in ribosome-containing processes, which, with other nonpresynaptic processes, are at the present time unavoidable contaminants of synaptosomal fractions. Presynaptic endings that in radioautographs contained no mitochondria were labeled. Also, presynaptic endings were labeled after incubation in the presence of chloramphenical which inhibited 20% of the protein synthesis of the synaptosomal fraction. It is concluded that besides mitochondrial protein synthesis, another protein synthesizing system operates in presynaptic endings in vitro.

Puromycin: action on neuronal mitochondria.

Puromycin, in dosages that inhibit cerebral protein synthesis and expression of memory in mice, produces swelling of neuronal mitochondria. Acetoxycycloheximide, which inhibits cerebral protein synthesis to the same extent as puromycin, fails to produce swelling of neuronal mitochondria. Puromycin and heximide mixtures produce severe inhibition of protein synthesis, but result in a minimal swelling of neuronal mitochondria and in a decrease of peptidylpuromycin complexes to a level of 30 percent of that following the injection of puromycin alone. It is concluded that swelling of neuronal mitochondria in the presence of puromycin is not due to inhibition of cerebral protein synthesis per se, but is related to a specific action of puromycin on ribosomal protein synthesis. The findings are consistent with the hypothesis that peptidyl-puromycin complexes are responsible for mitochondrial swelling.

Evidence for the conformation of the pathologic isoform of the prion protein enciphering and propagating prion diversity.

The fundamental event in prion diseases seems to be a conformational change in cellular prion protein (PrPC) whereby it is converted into the pathologic isoform PrPSc. In fatal familial insomnia (FFI), the protease-resistant fragment of PrPSc after deglycosylation has a size of 19 kilodaltons, whereas that from other inherited and sporadic prion diseases is 21 kilodaltons. Extracts from the brains of FFI patients transmitted disease to transgenic mice expressing a chimeric human-mouse PrP gene about 200 days after inoculation and induced formation of the 19-kilodalton PrPSc fragment, whereas extracts from the brains of familial and sporadic Creutzfeldt-Jakob disease patients produced the 21-kilodalton PrPSc fragment in these mice. The results presented indicate that the conformation of PrPSc functions as a template in directing the formation of nascent PrPSc and suggest a mechanism to explain strains of prions where diversity is encrypted in the conformation of PrPSc.

Fatal insomnia and agrypnia excitata: sleep and the limbic system.

Fatal familial insomnia, a human prion disease, Morvan's chorea, an autoimmune limbic encephalopathy, and delirium tremens, the well-known alcohol (or benzodiazepine [BDZ]) withdrawal syndrome, share a clinical phenotype largely consisting in an inability to sleep associated with motor and autonomic activation. Agrypnia excitata is the term which aptly defines this clinical condition, whose pathogenetic mechanism consists in an intralimbic disconnection releasing the hypothalamus and brainstem reticular formation from corticolimbic inhibitory control. Severance of cortical-subcortical limbic structures is due to visceral thalamus degeneration in fatal familial insomnia, and may depend on autoantibodies blocking voltage-gated potassium channels within the limbic system in Morvan's chorea, and the sudden changes in gabaergic synapses down-regulated by chronic alcohol abuse within the limbic system in delirium tremens. On the basis of these findings, we suggest that a neuronal network, extending from the medulla to the limbic cortex, controls the sleep-wake cycle, operating in an integrated fashion following a caudorostral organization.

Identification of an epitope in the C terminus of normal prion protein whose expression is modulated by binding events in the N terminus.

We have characterized the epitopes of a panel of 12 monoclonal antibodies (Mabs) directed to normal human cellular prion protein (PrP(C)) using ELISA and Western blotting of recombinant PrP or synthetic peptide fragments of PrP. The first group of antibodies, which is represented by Mabs 5B2 and 8B4, reacts with PrP(23-145), indicating that the epitopes for these Mabs are located in the 23 to 145 N-terminal region of human PrP. The second group includes Mabs 1A1, 6H3, 7A9, 8C6, 8H4, 9H7 and 2G8. These antibodies bind to epitopes localized within N-terminally truncated recombinant PrP(90-231). Finally, Mabs 5C3, 2C9 and 7A12 recognize both PrP(23-145) and PrP(90-231), suggesting that the epitopes for this group are located in the region encompassing residues 90 to 145. By Western blotting with PepSpot(TM), only three of Mabs studied (5B2, 8B4 and 2G8) bind to linear epitopes that are present in 13-residue long synthetic peptides corresponding to human PrP fragments. The remaining nine Mabs appear to recognize conformational epitopes. Two N terminus-specific Mabs were found to prevent the binding of the C terminus-specific Mab 6H3. This observation suggests that the unstructured N-terminal region may influence the local conformation within the folded C-terminal domain of prion protein.

Hirano body filaments contain actin and actin-associated proteins.

Hirano bodies are eosinophilic, rod-shaped intraneuronal inclusions whose frequency increases with age and with Alzheimer's disease. To investigate their composition and possible relationship to the neuronal cytoskeleton, we employed immunocytochemistry and immunoelectronmicroscopy by using antisera to cytoskeletal proteins. The presence of actin, alpha-actinin, vinculin and tropomyosin was demonstrated diffusely throughout the Hirano body. The presence of these proteins supports the contention that Hirano bodies are derived from an abnormal organization of the neuronal cytoskeleton. The staining of Hirano bodies with fluorescent labelled phalloidin, a probe with a unique affinity for F-actin, indicates that the actin in Hirano bodies is in the F-state. Results of high voltage electron microscopy on 1.0 and 0.5 micron sections confirm the purely filamentous nature of Hirano bodies. These findings suggest that the mechanism of formation of Hirano bodies is different from that of the neurofibrillary tangle, another characteristic intraneuronal inclusion seen in Alzheimer patients.

The presence of glial fibrillary acidic protein in the human pituitary gland.

The presence of glial fibrillary acidic protein (GFAP) was studied in human pituitary glands with the peroxidase-antiperoxidase (PAP) method. Positive reaction was observed in cells and processes of the neurohypophysis, in occasional cells lining the Rathke's cysts of the pars intermedia, and in scattered star-shaped cells and small follicles of the pars distalis. GFAP immunoreactivity was sparse and variable in amount from case to case. An increase in GFAP-immunoreactivity was observed as a reaction to injury. GFAP-positive cells were seen within and around pituitary adenomas regardless of their secretory cell type. Evidence is presented to indicate that these cells do not contain conventional pituitary hormones. It is postulated that the GFAP-positive cells of the pars distalis are nonsecretory elements, identical to the folliculostellate cells. They may become visible by immunostaining following increased synthesis of GFAP. The latter may be a response to cell injury or metabolic changes in adjacent secretory elements. A similar reaction in pituicytes may explain the appearance of immunoreactive GFAP in the neural lobe. The presence of GFAP in the adenohypophysis suggests that some of their cells are neuroectodermal in origin.

Neurofibrillary changes in human brain. An immunocytochemical study with a neurofilament antiserum.

Brain samples from cases of Alzheimer's disease, postencephalitic Parkinson's disease, progressive supranuclear palsy, amyotrophic lateral sclerosis, and Pick's disease, as well as from a case of Alzheimer's disease with a large number of Hirano bodies, were stained with the peroxidase-anti-peroxidase method using an antiserum previously shown to immunoreact with normal neurofilaments and neurofilament polypeptides. The specificity of this serum was confirmed by absorption an purified neurofilament proteins. Neurofibrillary tangles of Alzheimer's disease, postencephalitic Parkinson's disease, and progressive supranuclear palsy, Pick's bodies, and the fibrillary inclusions of amyotrophic lateral sclerosis were all immunostained. Hirano bodies showed no immunostaining. Thus, with the exception of the Hirano bodies, all the neuronal fibrillary inclusions examined appeared to share common antigenic characteristics. The orgin of all these structures from normal neurofilaments is postulated.