Abnormal activation of glial cells in the brains of prion protein-deficient mice ectopically expressing prion protein-like protein, PrPLP/Dpl.

BACKGROUND: Some lines of mice homozygous for a disrupted prion protein gene (Prnp), including Ngsk Prnp(0/0) mice, exhibit Purkinje cell degeneration as a consequence of the ectopic overexpression of the downstream gene for prion protein-like protein (PrPLP/Dpl) in the brain, but others, such as Zrch I Prnp(0/0) mice, show neither the neurodegeneration nor the expression of PrPLP/Dpl. In the present study, we found that Ngsk Prnp(0/0), but not Zrch I Prnp(0/0) mice, developed gliosis involving both astrocytes and microglia in the brain. MATERIALS AND METHODS: The brains from wild-type (Prnp(+/+)), Ngsk Prnp(0/0), Zrch I Prnp(0/0), and reconstituted Ngsk Prnp(0/0) mice carrying a mouse PrP transgene, designated Tg(P) Ngsk Prnp(0/0) mice, were subjected into Northern blotting and in situ hybridization using probes of glial fibrillary acidic protein (GFAP) and lysozyme M (LM) specific for astrocytes and microglia, respectively. Immunohistochemistry was also performed on the brain sections using anti-GFAP and anti-F4/80 antibodies. RESULTS: Northern blotting demonstrated upregulated expression of the genes for GFAP and LM in the brains of Ngsk Prnp(0/0), but not in Zrch I Prnp(0/0) mice. A transgene for normal mouse PrP(C) successfully rescued Ngsk Prnp(0/0) mice from the glial activation. In situ hybridization and immunohistochemistry revealed activated astrocytes and microglia mainly in the white matter of both the forebrains and cerebella. In contrast, there was no evidence of neuronal injury except for the Purkinje cell degeneration. Moreover, the glial cell activation was notable well before the onset of the Purkinje cell degeneration. CONCLUSIONS: These findings strongly suggest that ectopic PrPLP/Dpl in the absence of PrP(C) is actively involved in the glial-cell activation in the brain.

Early appearance but lagged accumulation of detergent-insoluble prion protein in the brains of mice inoculated with a mouse-adapted Creutzfeldt-Jakob disease agent.

1. To elucidate mechanisms for the generation of the detergent-insoluble, proteinase K-resistant prion protein (PrP(Sc)) from the detergent-soluble, proteinase K-sensitive PrP (PrP(C)) and the replication of the infectious agent in prion diseases, we followed the kinetics of detergent-insoluble PrP and PrP(Sc) levels, infectious titers, and associated pathological changes in the brains of mice inoculated with a mouse-adapted Creutzfeldt Jakob disease agent. 2. PrP(Sc) in brain homogenate and detergent-insoluble PrP enriched by two-cycle ultracentrifugation were detected by immunoblotting and their relative amounts were estimated according to a standard curve plotted between the amount of PrP and signal intensity on immunoblotting. The titer of infectivity was determined by the incubation periods of mice inoculated with the unfractionated homogenate on the basis of a standard curve plotted between the titer and incubation period. 3. Detergent-insoluble PrP became detectable 4 weeks postinoculation (p.i.) well before the detection of PrP(Sc). The low level of detergent-insoluble PrP continued until dramatic accumulation occurred at 14 weeks p.i., correlating well with the accumulation of PrP(Sc) and development of pathological changes. The infectious titer was undetectable at 4 weeks p.i. and its logarithmic increase occurred 10 weeks p.i. preceding the logarithmic accumulation of PrPs. 4. The lag time of detergent-insoluble PrP accumulation and the discrepancy between infectious titers and PrPs observed during the early period after inoculation suggest a slow and rate-limiting step for the detergent-insoluble PrP to become the infectious agent-associated PrP(Sc).

Physiological expression of the gene for PrP-like protein, PrPLP/Dpl, by brain endothelial cells and its ectopic expression in neurons of PrP-deficient mice ataxic due to Purkinje cell degeneration.

Recently, a novel gene encoding a prion protein (PrP)-like glycoprotein, PrPLP/Dpl, was identified as being expressed ectopically by neurons of the ataxic PrP-deficient (PRNP(-/-)) mouse lines exhibiting Purkinje cell degeneration. In adult wild-type mice, PrPLP/Dpl mRNA was physiologically expressed at a high level by testis and heart, but was barely detectable in brain. However, transient expression of PrPLP/Dpl mRNA was detectable by Northern blotting in the brain of neonatal wild-type mice, showing maximal expression around 1 week after birth. In situ hybridization paired with immunohistochemistry using anti-factor VIII serum identified brain endothelial cells as expressing the transcripts. Moreover, in the neonatal wild-type mice, the PrPLP/Dpl mRNA colocalized with factor VIII immunoreactivities in spleen and was detectable on capillaries in lamina propria mucosa of gut. These findings suggested a role of PrPLP/Dpl in angiogenesis, in particular blood-brain barrier maturation in the central nervous system. Even in the ataxic Ngsk PRNP(-/-) mice, the physiological regulation of PrPLP/Dpl mRNA expression in brain endothelial cells was still preserved. This strongly supports the argument that the ectopic expression of PrPLP/Dpl in neurons, but not deregulation of its physiological expression in endothelial cells, is involved in the neuronal degeneration in ataxic PRNP(-/-) mice.

Identification of a novel gene encoding a PrP-like protein expressed as chimeric transcripts fused to PrP exon 1/2 in ataxic mouse line with a disrupted PrP gene.

1. Mouse lines lacking prion protein (PrP(C)) have a puzzling phenotypic discrepancy. Some, but not all, developed late-onset ataxia due to Purkinje cell degeneration. 2. Here, we identified aberrant mRNA species in the brain of Ngsk Prnp0/0 ataxic, but not in nonataxic Zrch Prnp0/0 mouse line. These mRNAs were chimeric between the noncoding exons 1 and 2 of the PrP gene (Prnp) and the novel sequence encoding PrP-like protein (PrPLP), a putative membrane glycoprotein with 23% identity to PrP(C) in the primary amino acid structure. The chimeric mRNAs were generated from the disrupted Prnp locus of Ngsk Prnp0/0 mice lacking a part of the Prnp intron 2 and its splice acceptor signal. 3. In the brain of wild-type and Zrch Prnp0/0 mice, PrPLP mRNA was barely detectable. In contrast, in the brain of Ngsk Prnp0/0 mice, PrP/PrPLP chimeric mRNAs were expressed in neurons, at a particularly high level in hippocampus pyramidal cells and Purkinje cells under the control of the Prnp promoter. 4. In addition to the functional loss of PrP(C), ectopic PrPLP expression from the chimeric mRNAs could also be involved in the Purkinje cell degeneration in Ngsk Prnp0/0 mice.

Upregulation of the genes encoding lysosomal hydrolases, a perforin-like protein, and peroxidases in the brains of mice affected with an experimental prion disease.

In an attempt to identify the molecules involved in the pathogenesis of prion diseases, we performed cDNA subtraction on the brain tissues of mice affected with an experimental prion disease and the unaffected control. The genes identified as being upregulated in the prion-affected brain tissue included those encoding a series of lysosomal hydrolases (lysozyme M and both isoforms of beta-N-acetylhexosaminidase), a perforin-like protein (macrophage proliferation-specific gene-1 [MPS-1]), and an oxygen radical scavenger (peroxiredoxin). Dramatic increases in the expression level occurred at between 12 and 16 weeks after intracerebral inoculation of the prion, coinciding with the onset of spongiform degeneration. The proteinase K-resistant prion protein (PrP(Sc)) became detectable by immunoblotting well before 12 weeks, suggesting a causal relationship between this and the gene activation. Immunohistochemistry paired with in situ hybridization on sections of the affected brain tissue revealed that expression of the peroxiredoxin gene was detectable only in astrocytes and was noted throughout the affected brain tissue. On the other hand, the genes for the lysosomal hydrolases and MPS-1 were overexpressed exclusively by microglia, which colocalized with the spongiform morphological changes. A crucial role for microglia in the spongiform degeneration by their production of neurotoxic substances, and possibly via the aberrant activation of the lysosomal system, would have to be considered.

A mouse prion protein transgene rescues mice deficient for the prion protein gene from purkinje cell degeneration and demyelination.

Disruption of both alleles of the prion protein gene, Prnp, renders mice resistant to prions; in a Prnp o/o line reported by some of us, mice progressively developed ataxia and Purkinje cell loss. Here we report torpedo-like axonal swellings associated with residual Purkinje cells in Prnp o/o mice, and we demonstrate abnormal myelination in the spinal cord and peripheral nerves in mice from two independently established Prnp o/o lines. Mice were successfully rescued from both demyelination and Purkinje cell degeneration by introduction of a transgene encoding wild-type mouse cellular prion protein. These findings suggest that cellular prion protein expression may be necessary to maintain the integrity of the nervous system.

Prion protein is necessary for latent learning and long-term memory retention.

1. The cellular prion protein, designated PrPc, is a key molecule in the prion diseases but its physiological function remains unknown. To elucidate whether PrPc plays some role in the central nervous system, we established a line of mice in which the PrP gene had been disrupted and subsequently conducted long-term observations. 2. Performance in latent learning and passive avoidance was evaluated using water-finding and step-through tests, respectively. 3. PrP-/- mice showed impaired performance in the water-finding test, indicating a disturbance in latent learning, at 23 weeks of age. In the step-through test, although the PrP-/- mice showed normal learning ability and short-term memory retention, they evidenced a significant disturbance in long-term memory retention. 4. These results indicate that PrPc is needed for certain types of learning and memory and that the loss of function of this protein may contribute to the pathogenesis of prion diseases.