Biomedical subjects

B Ghetti

Publications and source records attributed to B Ghetti.

At least 91 records · Page 5Linked to original sources

PubMed1996

Linkage mapping of microdissected clones from distal mouse chromosome 16.

A total of 38 unique segments generated by microdissection of mouse chromosome 16 (MMU16), sequence independent amplification (SIA) and cloning were sequentially mapped on the distal portion of the chromosome with two mouse backcross panels. Some reference markers from other sources were retyped in the panels and results integrated with those for our microdissected DNA segments. The clone map is most highly refined in its distal portion, which stretches from reference marker D16Mit71 to D16Mit5, and the highest density of clones is in the region defined by markers D16Mit5 and D16Mit141. This map on distal mouse chromosome 16 should be a useful tool for the mouse genome project and for studies of genes in the region.

Animals↗

PubMed1996

Proteinase-K-resistant prion protein isoforms in Gerstmann-Sträussler-Scheinker disease (Indiana kindred).

Gerstmann-Sträussler-Scheinker (GSS) disease is a cerebral prion protein (PrP) amyloidosis associated with mutations in the PrP gene (PRNP). A GSS disease variant with mutation at codon 198 (F198S) has been studied in a large Indiana kindred. Biochemical investigations showed that the amyloid protein consists of 11 and 7 kDa fragments of PrP. Immunohistochemical studies showed that in addition to amyloid, these patients accumulate PrP deposits which are neither fluorescent nor birefringent when stained with thioflavin S and Congo red. In the present paper, we analyzed proteinase-K (PK)-resistant PrP in 7 patients with GSS F198S disease. Immunoblots of PK-treated brain extracts show prominent bands of ca. 27-29, 18-19, and 8 kDa. Immunohistochemistry and thioflavin-S-fluorescence show that the amyloid deposits are conspicuous in the cerebellum but sparse in the caudate nucleus. However, immunoblot analysis reveals PK-resistant PrP bands of similar intensity in both regions. Treatment with PK and PNGase F generates a pattern similar to that of PK alone. Our findings suggest that brain extracts from GSS F198S disease contain 3 prominent nonglycosylated PK-resistant PrP fragments forming a pattern not previously described in other prion diseases, which may in part explain the pathology of this GSS disease variant.

Adult↗

PubMed1996

Direct cDNA selection with DNA microdissected from mouse chromosome 16: isolation of novel clones and construction of a partial transcription map of the C3-C4 region.

A group of cDNA segments was selected by direct hybridization of mouse cerebellar cDNAs against genomic DNA pools generated by microdissection of the mouse chromosome 16 (MMU16) C3-C4 region. After elimination of repetitive sequences and adjustment for redundancy among clones, 34 novel cDNA fragments were isolated. The MMU16 origin of clones was confirmed by genetic linkage mapping. Reverse transcription PCR indicated that approximately 68% of the cDNAs represent transcripts that are expressed in adult mouse cerebellum. Northern blotting showed that some of these are predominantly or solely expressed in brain. This work demonstrates that DNA microdissected from banded MMU16 can be used for direct cDNA selection, thus enabling construction of a new, region-specific partial transcription map. This selected cDNA library should be a useful reagent for further molecular neurobiological studies.

Animals↗

PubMed1996

Prion protein amyloidosis.

The prion protein (PrP) plays an essential role in the pathogenesis of a group of sporadic, genetically determined and infectious fatal degenerative diseases, referred to as "prion diseases", affecting the central nervous system of humans and other mammals. The cellular PrP is encoded by a single copy gene, highly conserved across mammalian species. In prion diseases, PrP undergoes conformational changes involving a shift from alpha-helix to beta-sheet structure. This conversion is important for PrP amyloidogenesis, which occurs to the highest degree in the genetically determined Gerstmann-Sträussler-Scheinker disease (GSS) and prion protein cerebral amyloid angiopathy (PrP-CAA), while it is less frequently seen in other prion diseases. GSS and PrP-CAA are associated with point mutations of the prion protein gene (PRNP); these conditions show a broad spectrum of clinical presentation, the main signs being ataxia, spastic paraparesis, extrapyramidal signs and dementia. In GSS, parenchymal amyloid may be associated with spongiform changes or neurofibrillary lesions; in PrP-CAA, vascular amyloid is associated with neurofibrillary lesions. A major component of the amyloid fibrils in the two diseases is a 7 kDa peptide, spanning residues 81-150 of PrP.

Amino Acid Sequence↗

PubMed1996

Familial and sporadic Alzheimer's disease: neuropathology cannot exclude a final common pathway.

Whether all etiologic forms of Alzheimer's disease (AD) share a final common pathway is a major issue. We determined the severity and regional distribution of neuronal loss, amyloid plaques, neuritic plaques (NPs), and neurofibrillary tangles (NFTs), and calculated the ratio of neuronal loss to NPs and NFTs in brains of 19 familial AD (FAD) patients with linkage to chromosome 14, six AD patients with mutations of chromosome 21 (codon 717 of the beta-amyloid percursor protein gene), and 11 sporadic AD (SAD) patients. There was no difference in the pattern of distribution of the various pathologic features or in the ratio of neuronal loss to NPs or NFTs in any AD group. However, FAD groups could be distinguished from SAD by the greater severity and the lack of influence of apolipoprotein E genotype on pathology. These differences may reflect differences in age at onset rather than different etiopathologic mechanisms. The similarity of pathologic findings in the different AD groups provides evidence for a final common pathophysiologic pathway in AD.

Age of Onset↗

PubMed1995

Detection of apoptosis in weaver cerebellum by electron microscopic in situ end-labeling of fragmented DNA.

Massive degeneration of granule cell precursors occurs perinatally in the cerebellum of weaver mutant mice. We have studied the electron microscopic (EM) features of granule cell death in weaver and control mice, using an in situ end-labeling (ISEL) technique for detecting DNA fragmentation, a hallmark of apoptosis. In all animals, EM-ISEL revealed the pattern of apoptosis, with an enhanced expression in weaver mice. The weaver gene appears to accelerate the death program, most likely through potassium channel-mediated signals.

Aging↗

PubMed1995

Selective vulnerability of late-generated dopaminergic neurons of the substantia nigra in weaver mutant mice.

In homozygous weaver (wv/wv) mutant mice, nearly 50% of the dopaminergic substantia nigra neurons degenerate by postnatal day 20. We have now determined that the total number of dopaminergic neurons in the ventral midbrains of a litter of obligatory homozygous weaver pups and a litter of normal wild-type control pups indicates that no significant differences are present between groups at birth. To test the hypothesis that the subsequent degeneration of these neurons is linked to their time of origin, [3H]thymidine autoradiography was combined with tyrosine hydroxylase immunocytochemistry to construct neurogenetic timetables on postnatal day 20 in wild-type mice and weaver homozygotes. Both groups have the same span of neurogenesis but have statistically different proportions of neurons generated on specific days. In wild-type mice, more than half of the dopaminergic neurons originate on or after embryonic day 12. In contrast, over two-thirds of the surviving dopaminergic neurons in homozygous weaver mice originate on or before embryonic day 11. Our data suggest that the weaver gene does not interfere with the generation of dopaminergic neurons, but it preferentially kills late-generated dopaminergic neurons between birth and postnatal day 20.

Aging↗

PubMed1995

Time of neuron origin and gradients of neurogenesis in midbrain dopaminergic neurons in the mouse.

Previous [3H]thymidine studies in Nissl-stained sections in rats established that the substantia nigra pars compacta and the ventral tegmental area originate sequentially according to an anterolateral to posteromedial neurogenetic gradient. We investigated whether that same pattern is found in mice in the dopaminergic neurons in each of these structures. Using tyrosine hydroxylase immunostaining combined with [3H]thymidine autoradiography, the time of origin of dopaminergic midbrain neurons in the retrorubral field, the substantia nigra pars compacta, the ventral tegmental area, and the interfascicular nucleus was determined in postnatal day 20 mice. The dams of the experimental animals were injected with [3H]thymidine on embryonic days (E) 11-E12, E12-E13, E13-E14, and E14-E15. The time of origin profiles for each group indicated significant differences between populations. The retrorubral field and the substantia nigra pars compacta arose nearly simultaneously and contained the highest proportion of neurons, 49 to 37%, generated on or before E11. Progressively fewer early-generated neurons were found in the ventral tegmental area (20%), and the interfascicular nucleus (8.5%). In addition, anterior dorsolateral neurons in the substantia nigra and ventral tegmental area were more likely to be generated early than the posterior ventromedial neurons. These findings indicate that mouse and rat brains have nearly identical developmental patterns in the midbrain, and neurogenetic gradients in dopaminergic neurons are similar to those found in Nissl studies in rats.

Animals↗

PubMed1995

Systematic differences in time of dopaminergic neuron origin between normal mice and homozygous weaver mutants.

Immunocytochemical labeling for tyrosine hydroxylase and [3H]thymidine autoradiography were combined in wild-type mice and in mice homozygous for the weaver mutant gene (wv) to see whether the neurogenetic patterns of midbrain dopaminergic neurons was normal in the mutants and whether the degeneration of dopaminergic neurons was linked to their time of origin. Dams of wild-type and homozygous weaver mice were injected with [3H]thymidine on embryonic days (E) 11-E12, E12-E13, E13-E14, and E14-E15 to label neurons in the retrorubral field, the substantia nigra pars compacta, the ventral tegmental area, and the interfascicular nucleus as they were being generated. The quantitatively determined time of origin profiles indicated that wv/wv mice have the same time span of neurogenesis as +/+ mice (E10 to E14), but have significant deficits in the proportion of late-generated neurons in each dopaminergic population. In the retrorubral field and substantia nigra, weaver homozygotes had substantial losses of dopaminergic neurons and had a greater deficit in the proportion of neurons generated late while, in the ventral tegmental area and interfascicular nucleus, there were slight losses of dopaminergic neurons and only slight deficits in the proportion of late-generated neurons. These findings lead to the conclusion that the weaver gene is specifically targeting dopaminergic neurons that are generated late, mainly on E13 and E14.

Animals↗

PubMed1995

Neurofibrillary tangles in Niemann-Pick disease type C.

Niemann-Pick disease type C (NPC) is an autosomal recessive disease, belonging to a clinically heterogeneous group of lipid storage diseases, distinguished by a unique error in cellular trafficking of exogenous cholesterol, associated with lysosomal accumulation of unesterified cholesterol. Unlike Niemann-Pick disease types A and B, there is no primary genetic defect in sphingomyelinase in NPC. During the routine neuropathological study of NPC patients, we found neurofibrillary tangles (NFT) in a series of cases with a slowly progressive chronic course. These were not associated with beta-amyloid deposits. The NFT were most frequent in the orbital gyrus, cingulate gyrus and entorhinal region of the cerebral cortex, but were also frequently found in the basal ganglia, thalamus and hypothalamus. In one of the most severely affected case, the NFT were even found in the neurons in the inferior olivary nucleus and in the spinal cord. The NFT were immunostained with Alz 50, and consisted of paired helical filaments. The distribution of the neurons bearing the NFT was generally similar to that of the swollen storage neurons, and storage neurons often contained NFT in their perikarya and/or in the meganeurites. However, neurons with NFT could be noted without swollen perikarya. The coexistence of neuronal storage and NFT in NPC without amyloid deposits suggests that perturbed cholesterol metabolism and/or lysosomal membrane trafficking may play a role in the formation of NFT, and that amyloid deposits are not necessarily the prerequisite for NFT formation. The results of our study also suggest that NFT formation may be a rather nonspecific cellular reaction of neurons to certain slowly progressive metabolic perturbations of an as yet undefined nature.

Adolescent↗

PubMed1995

Endogenous serotonin release from the dopamine-deficient striatum of the weaver mutant mouse.

In addition to an altered dopaminergic input, the striatum of the weaver mutant mouse (wv/wv) has increased serotonin tissue content and uptake compared to the wild-type mouse (+/+). To gain information regarding the functional status of serotonergic inputs to the wv/wv striatum, endogenous serotonin release from wv/wv and +/+ striatum was measured under basal conditions as well as in the presence of fenfluramine or elevated concentrations of potassium (K+). Fractional basal release of serotonin from the +/+ striatum was significantly greater than that from the wv/wv striatum. In the presence of K+, evoked release (stimulated release minus basal release) was greater from the +/+ striatum than from the wv/wv striatum. In the presence of fenfluramine, evoked serotonin release was greater from the wv/wv striatum compared to the +/+ striatum. These data are consistent with the involvement of an additional transmitter(s) in modulating serotonin release to a greater extent in the wv/wv than the +/+ striatum. The data on fenfluramine-stimulated serotonin release suggest that the additional serotonin content found in the wv/wv striatum is in a releasable pool but that striatal serotonin release might be attenuated more in wv/wv than in +/+ mice.

Animals↗

PubMed1995

Dopaminergic parameters in the striatum and substantia nigra of seven strains of mice: higher density in striatum of CAST compared to BALB mice.

Tyrosine hydroxylase activity was assayed in microdissected substantia nigra and striata from seven strains of mice (BALB, CBA, YBR, WB, IS, MOLG, and CAST). In the substantia nigra where tyrosine hydroxylase activity is thought to be proportional to dopaminergic neuron number, only CBA had a different (lower) enzyme activity compared with BALB. However in the striatum, tyrosine hydroxylase activity was larger for IS, MOLG and CAST compared with BALB. Further investigation of the CAST striatum showed that dopamine content and dopamine uptake activity were also higher in comparison with BALB. All three dopaminergic parameters were larger because of lower protein levels in the CAST striatum. A lower absolute amount of glutamic acid decarboxylase activity in CAST versus BALB striatum was consistent with the possibility of a smaller CAST striatum. In contrast to dopamine, the serotonin content in CAST striatum was reduced in proportion to the decrease in protein content. We suggest that the CAST striatum is smaller than BALB striatum and is innervated by proportionally fewer serotoninergic terminals, but the amount of dopaminergic innervation of the CAST striatum is not altered by the size of the target.

Animals↗

PubMed1995

Altered IGFBP5 gene expression in the cerebellar external germinal layer of weaver mutant mice.

The IGF system components play important roles in cerebellar development as demonstrated by their specific spatial-temporal expression. IGF-I, type I IGF receptor (IGFR-I), IGFBP2 and IGFBP5 mRNA are localized in distinct cell populations, and all are expressed at the highest levels at the peak of Purkinje cell growth, active synaptogenesis and dendritic formation. To understand IGF-I's action at the cellular level, in situ hybridization was employed to investigate the distribution of IGF system gene transcripts in the cerebellum of weaver mutant mice (wv/wv). Although located ectopically, the surviving Purkinje cells express IGF-I mRNA at the same level in wv/wv as in +/+. No alteration in the cellular distribution or mRNA levels was observed with IGFBP2, or IGFR-I mRNAs. However, the pattern of IGFBP5 expression is altered in the external germinal layer of wv/wv mice. Not only is IGFBP5 expressed by more granule cell precursors of wv/wv cerebellum, but its mRNA level is 2.3 fold that of +/+. The altered IGFBP5 gene expression in granule cell precursors may modulate the interaction of IGF-I with IGFR-I in ways that contribute to their massive death occurring in the development of wv/wv cerebellum.

Animals↗

PubMed1995

Dopamine transporter-dependent and -independent endogenous dopamine release from weaver mouse striatum in vitro.

The weaver mutant mouse (wv/wv) has an approximately 70% loss of nigrostriatal dopamine (DA) neurons, but the fractional DA release evoked by amphetamine (but not a high potassium level) has been shown to be greater from striatal slices of the weaver compared with +/+ mice. In the present work we tested the hypothesis that fractional DA release from weaver striatum would be greater when release was mediated by the DA transporter. Serotonin (5-HT)-stimulated fractional DA release was greater from weaver than from +/+ striatum. The release evoked by 5-HT in the presence of 10 microM nomifensine (an antagonist of the DA transporter) was less than in its absence, but the difference between weaver and +/+ striatum remained. In the presence of nomifensine, 1-(m-chlorophenyl)biguanide, classified as a 5-HT3 agonist, also induced a greater fractional release from weaver compared with +/+ striatum. When veratridine was used at a low concentration (1 microM), the fractional evoked release of DA was higher from the weaver in the presence and absence of nomifensine. These findings suggest that the reason for the difference in the responsiveness of the two genotypes to these release-inducing agents is not related to DA transporter function.

Animals↗

PubMed1995

The tyrosine kinase inhibitor genistein increases endogenous dopamine release from normal and weaver mutant mouse striatal slices.

Protein tyrosine kinases that are known to have major roles in the control of cell growth and transformation are abundant and have numerous phosphoprotein substrates in the adult CNS. Although less well characterized than serine/threonine kinases, tyrosine kinases are also concentrated in the synapse. The effect of genistein, a selective inhibitor of tyrosine kinase activity, was examined on the in vitro release of endogenous dopamine (DA) from superfused mouse striatal slices. Fractional release of DA was significantly increased over basal release levels by genistein (100 and 200 microM). The effect was concentration dependent and rapidly reversible on washout of the kinase inhibitor. No significant change from basal release levels was observed with two structural analogues of genistein that do not inhibit tyrosine kinase activity at the same concentration. We have previously described alterations in basal and evoked DA release from the striatum of the weaver (wv/wv) mutant mouse, and genotypic differences in fractional release were also observed with genistein stimulation. The total evoked release was 25-50% greater from the wv/wv striatum. These results suggest a modulatory role for tyrosine kinase activity in neurotransmitter release and perhaps an alteration of kinase-regulated mechanisms in the DA-deficient wv/wv striatum.

Animals↗

PubMed1995

Gerstmann-Sträussler-Scheinker disease (PRNP P102L): amyloid deposits are best recognized by antibodies directed to epitopes in PrP region 90-165.

Gerstmann-Sträussler-Scheinker (GSS) disease is a familial neurological disorder pathologically characterized by accumulation of prion protein (PrP) in the form of fibrillary and non-fibrillary deposits within the cerebrum and cerebellum. We have studied two patients in whom the disease is caused by a leucine for proline amino acid substitution at residue 102 of PrP. In both patients, the neuropathologic findings are similar, consisting of spongiform changes, amyloid deposits, and gliosis. To investigate the antigenic profile of PrP deposits, we used antibodies raised against several peptides that correspond to segments of the N-terminus, repeat region, midregion, and C-terminus of PrP. By immunohistochemistry, PrP amyloid cores are best labeled by antibodies directed to epitopes spanning PrP residues 90-165. In GSS disease caused by a substitution of thymine to cytosine at PRNP codon 198 (Indiana kindred), the major amyloidogenic peptide spans residues 58-150; therefore, in these two genetic forms of GSS disease, amyloid may be composed of different peptides.

Adult↗

PubMed1995

Gerstmann-Sträussler-Scheinker disease and the Indiana kindred.

Gerstmann-Sträussler-Scheinker disease is an autosomal dominant disorder with a wide spectrum of clinical presentations including ataxia, spastic paraparesis, extrapyramidal signs, and dementia. The patients present with symptoms in the third to sixth decade of life and the mean duration of illness is five years. Mutations at codons 102, 105, 117, 145, 198 and 217 of the open reading frame of the prion protein gene have been associated with GSS disease. As a result of the mutations, a substitution at the corresponding residues of the prion protein occurs, or as in the case of the STOP mutation at codon 145, a truncated protein is produced. Neuropathologically, the common denominator is a cerebral prion protein amyloidosis; however, there is significant variability in the pattern of amyloid deposition in regions of the central nervous system among reported families. Amyloidosis coexists with severe spongiform degeneration in patients with the mutation at codon 102, and with neurofibrillary degeneration in the patients with mutation at codons 145, 198 and 217. The development of a transmissible spongiform encephalopathy in animals inoculated with brain tissue from affected subjects with mutation at codon 102 suggests that in some forms of genetically-determined Gerstmann-Sträussler-Scheinker disease, and particularly those characterized by severe spongiosis, amyloidogenesis and production of an infectious "agent" occur concomitantly via mechanisms that are only partially understood.

Amyloid↗

PubMed1995

Degeneration of Sertoli and spermatogenic cells in homozygous and heterozygous weaver mice.

In the neurological mutant mouse weaver (wv/wv), the majority of males are infertile due to hypospermatogenesis. Heterozygous weaver mice (wv/+) cease mating successfully when males reach an average age of 3.5 months. The contents of epididymal fluid were scored for the number of sperm and sperm motility in wv/wv, wv/+ and controls. Testes were examined in mice of the three genotypes at various ages using light and electron microscopy. In wv/+ males, sperm counts were significantly lower than in controls and were significantly higher than in wv/wv. The seminiferous epithelium in weaver mice appears depleted soon after puberty and a wide range of degenerative changes was identified in both germ cells and Sertoli cells. Analogous cellular aberrations were detected in heterozygous males, but they appeared at an older age and were not as severe as in wv/wv. We hypothesize that in weaver homo- and heterozygosity the damage of Sertoli cells may induce degeneration of germinal cells and particularly affect the most advanced spermatogenic cells.

Animals↗