PrP fragment 106-126 is toxic to cerebral endothelial cells expressing PrP(C)

A hydrophobic, fibrillogenic peptide fragment of human prion protein (PrP106-126) had in vitro toxicity to neurons expressing cellular prion protein (PrP(C)). In this study, we proved that primary cultures of mouse cerebral endothelial cells (MCEC) express PrP(C). Incubation of MCEC with PrP106-126 (25-200 microM) caused a dose-dependent toxicity assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase release, bis-benzimide staining for nuclear morphology, and trypan blue exclusion test. Pentosan polysulphate (50-100 microg/ml), a drug effective in scrapie prophylaxis, dose-dependently attenuated the injury. MCEC cultures from mice homogenous for the disrupted PrP gene were resistant to the toxicity of PrP106-126. In conclusion, cerebral endothelium expressing PrP(C) may be directly damaged during spongiform encephalopathies.     

The N-terminal, polybasic region of PrP(C) dictates the efficiency of prion propagation by binding to PrP(Sc)

Prion propagation involves a templating reaction in which the infectious form of the prion protein (PrP(Sc)) binds to the cellular form (PrP(C)), generating additional molecules of PrP(Sc). While several regions of the PrP(C) molecule have been suggested to play a role in PrP(Sc) formation based on in vitro studies, the contribution of these regions in vivo is unclear. Here, we report that mice expressing PrP deleted for a short, polybasic region at the N terminus (residues 23-31) display a dramatically reduced susceptibility to prion infection and accumulate greatly reduced levels of PrP(Sc). These results, in combination with biochemical data, demonstrate that residues 23-31 represent a critical site on PrP(C) that binds to PrP(Sc) and is essential for efficient prion propagation. It may be possible to specifically target this region for treatment of prion diseases as well as other neurodegenerative disorders due to β-sheet-rich oligomers that bind to PrP(C).     

The neurotoxicity of prion protein (PrP) peptide 106-126 is independent of the expression level of PrP and is not mediated by abnormal PrP species

A synthetic peptide homologous to region 106-126 of the prion protein (PrP) is toxic to cells expressing PrP, but not to PrP knockout neurons, arguing for a specific role of PrP in mediating the peptide's activity. Whether this is related to a gain of toxicity or a loss of function of PrP is not clear. We explored the possibility that PrP106-126 triggered formation of PrP(Sc) or other neurotoxic PrP species. We found that PrP106-126 did not induce detergent-insoluble and protease-resistant PrP, nor did it alter its membrane topology or cellular distribution. We also found that neurons expressing endogenous or higher level of either wild-type PrP or a nine-octapeptide insertional mutant were equally susceptible to PrP106-126, and that sub-physiological PrP expression was sufficient to restore vulnerability to the peptide. These results indicate that PrP106-126 interferes with a PrP function that requires only low protein levels, and is not impaired by a pathogenic insertion in the octapeptide region.     

Cobalamin (vitamin B(12)) regulation of PrP(C), PrP(C)-mRNA and copper levels in rat central nervous system

The pathogenesis of cobalamin (Cbl)-deficient (Cbl-D) neuropathy is not clear, nor is the role of prions (PrP(C)) in myelin maintenance. However, as it is known that Cbl deficiency damages myelin by increasing tumor necrosis factor (TNF)-α and decreasing epidermal growth factor (EGF) levels in rat spinal cord (SC), and that TNF-α and EGF regulate PrP(C) expression in vitro, we investigated whether Cbl deficiency modifies SC PrP(C) and PrP(C)-mRNA levels in Cbl-D rats. PrP(C) levels had increased by the time myelin lesions appeared. This increase was mediated by excess myelinotoxic TNF-α and prevented by EGF, which proved to be as effective as Cbl in preventing Cbl deficiency-induced lesions. There were no significant changes in hepatic PrP(C) levels of Cbl-D rats. Anti-octapeptide repeat (OR) region antibodies normalized SC myelin morphology. Cbl deficiency greatly reduced SC PrP(C)-mRNA levels, which were subsequently increased by Cbl and EGF. Cbl deficiency-induced excess OR is myelin-damaging, but new PrP(C) synthesis is a common effect of different myelinotrophic agents.     

The octapeptide repeat PrP(C) region and cobalamin-deficient polyneuropathy of the rat

Introduction: Cobalamin (Cbl) deficiency affects the peripheral nervous system (PNS) morphologically and functionally. We investigated whether the octapeptide repeat (OR) region of prion protein (PrP^C) (which is claimed to have myelinotrophic properties) is involved in the pathogenesis of rat Cbl‐deficient (Cbl‐D) polyneuropathy. Methods: We intracerebroventricularly administered antibodies (Abs) against the OR region (OR‐Abs) to Cbl‐D rats to prevent myelin damage and maximum nerve conduction velocity (MNCV) abnormalities, and PrP^Cs to normal rats to reproduce PNS Cbl‐D–like lesions. We measured nerve PrP^C levels and MNCV. Results: The OR‐Abs normalized myelin ultrastructure, MNCV values, and tumor necrosis factor (TNF)‐α levels in the sciatic and tibial nerves of Cbl‐D rats. PrP^C levels increased in Cbl‐D nerves. The nerves of the PrP^C‐treated rats showed typical Cbl‐D lesions, significantly decreased MNCV values, and significantly increased TNF‐α levels. Conclusions: OR‐Abs prevent the myelin damage caused by increased OR regions, and excess TNF‐α is involved in the pathogenesis of Cbl‐D polyneuropathy. Muscle Nerve 2011     

Expression of PrP(C) in the rat brain and characterization of a subset of cortical neurons

The cellular prion protein (PrP(C)) is a membrane-bound glycoprotein mainly present in the CNS. The scrapie prion protein (PrP(Sc)) is an isoform of PrP(C), and it is responsible for transmissible spongiform encephalopathies (TSEs), a group of neurodegenerative diseases affecting both humans and animals. The presence of the cellular form is necessary for the establishment and further evolution of prion diseases. Here, we map the regional distribution of PrP(C) in the rat brain and study the chemical nature of these immunopositive neurons. Our observations are congruent with retrograde transport of prions, as shown by the ubiquitous distribution of PrP(C) throughout the rat brain, but especially in the damaged areas that send projections to primarily affected nuclei in fatal familial insomnia. On the other hand, the presence of the cellular isoform in a subset of GABAergic neurons containing calcium-binding proteins suggests that PrP(C) plays a role in the metabolism of calcium. The lack of immunostaining in neurons ensheathed by perineuronal nets indicates that prions do not directly interact with components of these nets. The destruction of these nets is more likely to be the consequence of a factor needed for prions during the early stages of TSEs. This would cause destruction of these nets and death of the surrounded neurons. Our results support the view that destruction of this extracellular matrix is caused by the pathogenic effect of prions and not a primary event in TSEs.     

Small is not beautiful: antagonizing functions for the prion protein PrP(C) and its homologue Dpl

A conformational variant of the normal prion protein PrP(C) is believed to be identical to PrP(Sc), the agent that causes prion diseases. Recently, a novel protein, named Doppel (Dpl), was identified that shares significant biochemical and structural homology with PrP(C). In specific strains of PrP(C)-deficient mouse lines, Dpl is overexpressed and causes a neurological disease. Dpl neurotoxicity is counteracted and prevented by PrP(C), but the mechanism of antagonistic PrP(C)-Dpl interaction remains elusive. In contrast to its homologue PrP(C), initial studies suggest that Dpl is dispensable for prion disease progression and for the generation of PrP(Sc). Although we are only beginning to understand its function, the discovery of Dpl has already provided some answers to long-standing questions and is transforming our understanding of prion biology.     

Involvement of the 5-lipoxygenase pathway in the neurotoxicity of the prion peptide PrP106-126

Transmissible spongiform encephalopathies are characterised by the transformation of the normal cellular prion protein (PrP(C)) into an abnormal isoform (PrP(TSE)). Previous studies have shown that N-methyl-D-aspartate (NMDA) receptor antagonists can inhibit glutathione depletion and neurotoxicity induced by PrP(TSE) and a toxic prion protein peptide, PrP106-126, in vitro. NMDA receptor activation is known to increase intracellular accumulation of Ca(2+), resulting in up-regulation of arachidonic acid (AA) metabolism. This can stimulate the lipoxygenase pathways that may generate a number of potentially neurotoxic metabolites. Because of the putative relationship between AA breakdown and PrP106-126 neurotoxicity, we investigated AA metabolism in primary cerebellar granule neuron cultures treated with PrP106-126. Our studies revealed that PrP106-126 exposure for 30 min significantly up-regulated AA release from cerebellar granule neurons. PrP106-126 neurotoxicity was mediated through the 5-lipoxygenase (5-LOX) pathway, as shown by abrogation of neuronal death with the 5-LOX inhibitors quinacrine, nordihydroguaiaretic acid, and caffeic acid. These inhibitors also prevented PrP106-126-induced caspase 3 activation and annexin V binding, indicating a central role for the 5-LOX pathway in PrP106-126-mediated proapoptosis. Interestingly, inhibitors of the 12-lipoxygenase pathway had no effect on PrP106-126 neurotoxicity or proapoptosis. These studies clearly demonstrate that AA metabolism through the 5-LOX pathway is an important early event in PrP106-126 neurotoxicity and consequently may have a critical role in PrP(TSE)-mediated cell loss in vivo. If this is so, therapeutic intervention with 5-LOX inhibitors may prove beneficial in the treatment of prion disorders.     

Immunohistochemical localization of three different prepro-GnRHs in the brain and pituitary of the European sea bass (Dicentrarchus labrax) using antibodies to the corresponding GnRH-associated peptides

The distribution of the cells expressing three prepro-gonadotrophin-releasing hormones (GnRH), corresponding to salmon GnRH (sGnRH), seabream GnRH (sbGnRH), and chicken GnRH-II (cGnRH-II) forms, was studied in the brain and pituitary of the sea bass (Dicentrarchus labrax) by using immunohistochemistry. To circumvent the cross-reactivity problems of antibodies raised to GnRH decapeptides, we used specific antibodies generated against the different sea bass GnRH-associated peptides (GAP): salmon GAP (sGAP), seabream GAP (sbGAP), and chicken-II GAP (cIIGAP). The salmon GAP immunostaining was mostly detected in terminal nerve neurons but also in ventral telencephalic and preoptic perikarya. Salmon GAP-immunoreactive (ir) fibers were observed mainly in the forebrain, although sGAP-ir projections were also evident in the optic tectum, mesencephalic tegmentum, and ventral rhombencephalon. The pituitary only receives a few sGAP-ir fibers. The seabream GAP-ir cells were mainly detected in the preoptic area. Nevertheless, sbGAP-ir neurons were also found in olfactory bulbs, ventral telencephalon, and ventrolateral hypothalamus. The sbGAP-ir fibers were only observed in the ventral forebrain, innervating strongly the pituitary gland. Finally, chicken-II GAP immunoreactivity was only detected in large synencephalic cells, which are the origin of a profuse innervation reaching the telencephalon, preoptic area, hypothalamus, thalamus, pretectum, posterior tuberculum, mesencephalic tectum and tegmentum, cerebellum, and rhombencephalon. However, no cIIGAP-ir fibers were detected in the hypophysis. These results corroborate the overlapping of sGAP- and sbGAP-expressing cells in the forebrain of the sea bass, and provide, for the first time, unambiguous information on the distribution of projections of the three different GnRH forms expressed in the brain of a single species.     

Polymorphisms within the prion (PrP) and prion-like protein (Doppel) genes in AD

The authors present a study on the association of PRNP and PRND gene polymorphisms with the occurrence and age at onset of Alzheimer's disease (AD). DNA from 79 Polish patients with probable AD and 107 healthy control subjects was studied. The PRNP codon 129 homozygosity seemed to be associated with the occurrence of AD: In AD patients, the percentage of Val/Val and Met/Met genotypes was higher than in the control subjects. A significant difference appeared also between early-onset (<70 years) and late-onset (> or = 70 years) AD patients in the PRND genotypes.     

Proper axonal distribution of PrP(C) depends on cholesterol-sphingomyelin-enriched membrane domains and is developmentally regulated in hippocampal neurons

Defects in cellular localization and trafficking seem to facilitate the conversion of PrP(C) into the disease-associated form, PrP(Sc). Still, it is not clear to which membrane compartments PrP(C) localizes in hippocampal neurons a population most affected in the prion disease. We here show that in developing hippocampal neurons in culture PrP(C) is equally distributed to all neurites yet enriched in growth cones. By contrast, in fully mature neurons PrP(C) is restricted to axons. The axonal distribution in mature stages is paralleled by the increased partitioning of PrP(C) into detergent-resistant cholesterol-sphingolipid-rich domains (DRMs). Consistent with a cause-effect mechanism, disruption of DRMs by sphingolipid or cholesterol depletion leads to the non-polarized distribution and impaired endocytosis of PrP(C). These results indicate that DRMs are essential for proper trafficking and distribution of PrP(C) at late stages of neuronal differentiation and that its function, at least in hippocampus, is restricted to the axonal domain.     

Caspase及细胞色素C在海人藻酸所致海马组织兴奋性毒性损伤中的作用

目的 探讨Ｃａｓｐａｓｅ及细胞色素Ｃ的蛋白质在兴奋性毒性损伤中的表达,并探讨其作用机制。方法 制备大鼠海马脑片,利用Ｗｅｓｔｅｒｎ印迹分析法,检测海人灌酸（ＫＡ）兴奋性损伤后大鼠海马组织Ｃａｓｐａｓｅ和细胞色素Ｃ的表达。结果 正常对照组Ｃａｓｐａｓｅ－３和Ｃａｓｐａｓｅ－９及细胞色素Ｃ有较低水平的表达,加用ＫＡ后可见Ｃａｓ－ｐａｓｅ－３和Ｃａｓｐａｓｅ－９及细胞色素Ｃ的表达增加。结论 （１）正常脑     

Paired helical filament tau (PHFtau) in Niemann-Pick type C disease is similar to PHFtau in Alzheimer's disease

Niemann-Pick Type C disease (NPC) is a cholesterol storage disease with defects in the intracellular trafficking of exogenous cholesterol derived from low density lipoproteins. In NPC cases with a chronic progressive course, neurofibrillary tangles (NFTs) that consist of paired helical filaments (PHFs) have been reported. To determine if NPC tangles contain abnormal tau proteins (known as PHFtau) similar to those found in Alzheimer's disease (AD) tangles, we examined the brains of five NPC cases by immunohistochemical and Western blot methods using a library of antibodies to defined epitopes of PHFtau. We show here that PHFtau in tangle-rich NPC brains is indistinguishable from PHFtau in AD brains. We speculate, that the generation of PHFtau in NPC may induce a cascade of pathological events that contribute to the widespread degeneration of neurons, and that these events may be similar in NPC and AD.     

Mental imagery of faces and places activates corresponding stiimulus-specific brain regions

What happens in the brain when you conjure up a mental image in your mind's eye? We tested whether the particular regions of extrastriate cortex activated during mental imagery depend on the content of the image. Using functional magnetic resonance imaging (fMRRI), we demonstrated selective activation within a region of cortex specialized for face perception during mental imagery of faces, and selective activation within a place-selective cortical region during imagery of places. In a further study, we compared the activation for imagery and perception in these regions, and found greater response magnitudes for perception than for imagery of the same items. Finally, we found that it is possible to determine the content of single cognitive events from an inspection of the fMRI data from individual imagery trials. These findings strengthen evidence that imagery and perception share common processing mechanisms, and demonstrate that the specific brain regions activated during mental imagery depend on the content of the visual image.     

Neuroprotection by (R)-deprenyl and 7-nitroindazole in the MPTP C57BL/6 mouse model of neurotoxicity

The neurodegenerative properties of the parkinsonian inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are thought to result from inhibition of complex I of the mitochondrial respiratory chain by the monoamine oxidase-B (MAO-B) generated 1-methyl-4-phenylpyridinium metabolite MPP+. 7-Nitroindazole (7-NI) both a reversible MAO-B inhibitor and a neuronal nitric oxide synthase (nNOS) inhibitor, and (R)-deprenyl a potent MAO-B inactivator, provide neuroprotection in the C57BL/6 mouse model of MPTP neurotoxicity. The results reported here demonstrate the complexities of the effects of 7-NI in this model and examine the possibility of other mechanisms of neuroprotection by (R)-deprenyl.     

Deposition of the prion protein (PrP) during the evolution of experimental Creutzfeldt-Jakob disease

We studied the immunocytochemical distribution of the prion or proteinase-resistant protein (PrP) during the evolution of experimental Creutzfeldt-Jakob disease (CJD) in mice. Fifty-one brains were collected up to 22 weeks following intracerebral inoculation with the Fujisaki strain of the CJD agent. Slides were also immunostained for apolipoprotein E (apoE) and glial fibrillary acidic protein. Vacuolar changes with focal astrocytosis first occurred around the needle track at week 2 and later spread along white matter tracks. Until week 12, changes were asymmetrical, affecting more the side of inoculation. Spongiform change and astrogliosis spread subsequently to the gray matter. Time course and intensity of spongiform change and immunocytochemistry for PrP were discrepant: in most brain regions, severe vacuolation preceded immunocytochemically detectable PrP accumulation. PrP deposits in form of small dots were first detectable at week 6 in the area surrounding the needle track. After week 7, plaque-like amorphous PrP deposits were observed in white matter pathways. Finally, PrP was detectable also in basal ganglia and in the dorsal hippocampus (week 13) and in the neocortex (week 17), as the synaptic type of PrP immunopositivity. In the hippocampus, diffuse PrP deposits paralleled spongiform change, while in the cortex severe vacuolation was accompanied only by weak synaptic PrP deposits. Immunocytochemically detectable apoE was restricted to compact plaque-type PrP deposits after week 15. We conclude that disease-specific neuropathology spreads from the needle track along white matter pathways towards the gray matter; in this model, there is some discrepancy between development of tissue pathology and immunocytochemically detectable deposition of PrP. Immunocytochemically detectable apoE deposition follows PrP accumulation. Received: 22 December 1998 / Revised, accepted: 6 April 1999     

Development of the Behavioral Assessment and Research System (BARS) to detect and characterize neurotoxicity in humans

The Behavioral Assessment and Research System (BARS) is a computer-based testing system designed to assess neurobehavioral function in humans. It was developed to provide a series or battery of neurobehavioral tests optimized for the detection of neurotoxicity in non-mainstream human populations, specifically people with limited education or literacy. Key to meeting this goal were simply-stated instructions divided into an elemental series of steps, a 9BUTTON response unit to replace the computer keyboard for responding, and spoken instructions. Modifications all underwent serial testing in target populations to successively hone the changes to be more effective. A similar process was followed when developing adjustable parameters, test reliability assessments, and when implementing these tests with populations from different cultural groups and children. The principles and experiences that guided the development of BARS should inform the development of future testing systems to ensure that the new tests can be used with non-mainstream populations, which may be increasingly subject to neurotoxic exposures.     

Convulsant peptides related to corticotropin-releasing factor (CRF)

Corticotropin-releasing factor (CRF) and its related peptides have some conserved primary regions. We have taken an interest in the characteristic conserved sequence, Pro-Pro-Ile-Ser, which is located in the proximal N-terminal region of the CRF family. This sequence is not included in any other biologically active peptide and protein. Biological function of this N-terminal region has not become known yet. We synthesized seventeen peptide fragments of human CRF and sauvagine which involved either this entire N-terminal sequence or part of it and determined their behavioural effect after intracerebroventricular injections to mice. Several peptides which included the Pro-Pro-Ile region as the N-terminal segment and their caused a transient convulsion dose-dependently. H-Pro-Pro-Ile-OH (human CRF(4-6)) exhibited the strongest effect among these active peptides. However, these convulsant peptides did not affect the secretion of immunoreactive corticotropin in rats.