Lesions akin to transmissible spongiform encephalopathy in the brains of rats inoculated with immature cerebellum

Summary Fourteen BD IX rats were inoculated intracerebrally with a homogenate prepared from the immature cerebellar cortex of 10-day-old rats, when synaptogenesis is at its peak in this species. Eight controls were inoculated with mature cerebellar cortex. Transient ultrastructural changes were observed between 2 and 23 weeks' incubation in those animals which had received an inoculum of immature cerebellum. These changes pointed to a re-activation of embryonic or neo-natal growth mechanisms and were identical to those occurring in kuru-inoculated spider monkeys. With longer incubation histopathological lesions such as intracytoplasmic vacuolation, chromatolysis and neuronophagia appeared in neurons of the brain stem reticular formation. Such features are common in all the spongiform encephalopathies. All controls were negative. It is suggested that the transmissible agent in these diseases might be the factor which influences the various stages of normal neuronal maturation. A hypothesis is developed which would reconcile the infectious character of these diseases with a genetic factor and explain the unconventional behaviour of the agent as well as the mode of its transmission.Preliminary results of this work were included in a paper read at the 28th meeting of the Deutsche Gesellschaft für Neuropathologie und Neuroanatomie in October 1983 [3]. The work is dedicated to the memory of Herbert Butler (James) Parry 1912–1980     

A morphometric and immunohistochemical study of the vestibular nuclear complex in bovine spongiform encephalopathy

Summary A morphometric and immunohistochemical study of the vestibular nuclear complex was performed on five bovine spongiform encephalopathy (BSE) and five control cow brains. Neurons of the lateral and superior vestibular nuclei were counted at 500-m intervals in 10-m-thick sections, using an image analysis system comprosing a projection microscope and digitising pad linked to a computer. A bimodal distribution of neuron diameters was recognised in the brains of normal cattle. One population of neurons had a mean diameter of 30 m and the other had a mean diameter of 60 m. The vestibular nuclei from BSE cattle had an approximately 50% reduction in total numbers of neurons when compared with controls (P<0.01). Cattle which were clinically diseased longer had the fewest number of neurons preserved. Diminshed numbers of neurons were detected throughout the area studied and affected neurons of all diameters. Immunohistochemical staining for synaptophysin a protein present in synapses throughout the CNS, showed no significant reduction in axon terminals synapsing with vestibular neurons, including vacuolated neurons of BSE brains, when controls and BSE brains were compared. This suggests that de-afferentation of neurons is not the cause of neuronal loss. Prion protein was detected in the neuropil of the vestibular nuclear complex of BSE brains but not control brains. These studies show that previously unsuspected neuronal loss is a significant feature of BSE.     

Spontaneous mutations in the prion protein gene causing transmissible spongiform encephalopathy

We analyzed the prion protein gene (PRNP) region in patients with transmissible spongiform encephalopathy associated with the PRNP D178N mutation. The results suggest that the D178N chromosomes had independent origins in each affected pedigree or apparently sporadic case. A de novo spontaneous PRNP mutation was observed. We provide evidence that hereditary and apparently sporadic transmissible spongiform encephalopathy cases associated with the D178N mutation result from multiple recurrent mutational events.     

Abnormal prion protein is associated with changes of plasma membranes and endocytosis in bovine spongiform encephalopathy (BSE)-affected cattle brains

Aims: Transmissible spongiform encephalopathies (TSEs) or prion diseases are fatal neurodegenerative diseases of man and animals characterized by vacuolation and gliosis of neuropil and the accumulation of abnormal isoforms of a host protein known as prion protein (PrP). It is widely assumed that the abnormal isoforms of PrP (PrP^d, disease-specific form of PrP) are the proximate cause of neurodegeneration. Methods: To determine the nature of subcellular changes and their association with PrP^d we perfusion-fixed brains of eight bovine spongiform encephalopathy (BSE)-affected cows and three control cattle for immunogold electron microscopy at two different neuroanatomical sites. Results: All affected cattle presented plasma membrane alterations of dendrites and astrocytes that were labelled for PrP^d. PrP^d on membranes of dendrites and occasionally of neuronal perikarya was associated with abnormal endocytotic events, including bizarre coated pits and invagination of the plasma membrane. BSE-affected cattle also presented excess and abnormal multivesicular bodies, sometimes associated to the plasma membrane perturbations. In contrast, two TSE-specific lesions, vacuolation and rare tubulovesicular bodies, were not labelled for PrP^d as were a number of other nonspecific lesions, such as autophagy and dystrophic neurites. At least two different morphological pathways to vacuoles were recognized. Conclusions: When compared with other TSEs, these changes are common to those of sheep and rodent scrapie and shows that there are consistent membrane toxicity properties of PrP^d. This toxicity involves an aberration of endocytosis. However, it is by no means clear that the lesions are of sufficient severity to result in clinical deficits.     

Oxidative stress and the prion protein in transmissible spongiform encephalopathies

Transmissible spongiform encephalopathies form a group of fatal neurodegenerative disorders that have the unique property of being infectious, sporadic or genetic in origin. These diseases are believed to be the consequence of the conformational conversion of the prion protein into an abnormal isoform. Their exact pathogenic mechanism remains uncertain, but it is believed that oxidative stress plays a central role. In this article, we will first review in detail the data supporting the latter hypothesis. Subsequently, we will discuss the relationship between the prion protein and the cellular response to oxidative stress, attempting ultimately to link PrP function and neurodegeneration in these disorders.     

Prion encephalopathy with insertion of octapeptide repeats: the number of repeats determines the type of cerebellar deposits

C. Vital, F. Gray, A. Vital, P. Parchi, S. Capellari, R. B. Petersen, X. Ferrer, D. Jarnier, J. Julien and P. Gambetti (1998) Neuropathology and Applied Neurobiology 24, 125–130
Prion encephalopathy with insertion of octapeptide repeats: the number of repeats determines the type of cerebellar deposits
We studied modifications of the molecular layer of the cerebellum in three patients with octapeptide repeat insertion (OPRI). Two brothers carrying a six-OPRI showed only spongiosis in haematoxylin & eosin preparations (H&E), whereas immunocytochemical examination (ICC) with an antiprion protein (PrP) antibody revealed numerous elongated PrP deposits. The third patient from a family with an eight-OPRI had numerous plaques visible in H&E preparations and had been diagnosed as Gerstmann-Straüssler–Scheinker syndrome. So far, 15 other cases from seven families and three individual cases with OPRI have undergone neuropathological examination. Characteristic PrP deposits were seen in six other cases, two isolated cases with a four- and a seven-OPRI, whereas four cases with a six-OPRI came from three different families. Such deposits have never been reported in other cases of prion encephalopathy, without OPRI. Genuine plaques were observed in five out of the 15 other patients. Interestingly, four had an eight-OPRI and one a nine-OPRI. Cases with OPRI are prone to develop different PrP deposits: those only visible on ICC are not to be confused with genuine plaques visible in H&E preparations. Elongated PrP deposits are present in cases with a four- to seven-OPRI, whereas plaques are present when there is an eight- or a nine-OPRI. All these cases should be termed prion encephalopathy with OPRI.     

Review: Membrane‐associated misfolded protein propagation in natural transmissible spongiform encephalopathies (TSEs), synthetic prion diseases and Alzheimer's disease

Protein misfolding has long been recognized as a primary cause of systemic amyloidosis and, increasingly, template‐mediated misfolding of native host proteins is now also considered to be central pathogenetic events in some neurodegenerative diseases. Alzheimer's disease, naturally occurring transmissible spongiform encephalopathies (TSEs) and experimental disorders caused by misfolded prion protein (PrP) generated in vitro all share an imbalance of protein synthesis, aggregation and clearance that leads to protein aggregation, prompting some to suggest that Alzheimer's disease is caused by a prion‐like mechanism. In TSEs, the host‐coded, glycosyl‐phosphoinositol (GPI) membrane‐anchored prion protein (PrP^c) is misfolded into disease‐associated, putatively infectious aggregates known as prions. In Alzheimer's disease the membrane‐spanning Alzheimer's precursor protein (APP) is progressively cleaved within the plasmalemma to form Aβ peptide fragments that can form pathogenic extracellular aggregates while microtubule‐associated tau proteins may also aggregate within neurones. Oligomeric Aβ peptides and full‐length misfolded PrP show a common potential to convert native protein and aggregate on plasma membranes before subsequent release to form amyloid fibrils in the extracellular space. However, the nature, membrane topography and processing of the precursor and propagated proteins in prion and Alzheimer's disease all differ, and each group of diseases has distinctive spectra of additional pathological changes and clinical signs suggesting that fundamentally different disease mechanisms are involved.     

Advances in the detection of prion protein in peripheral tissues of variant Creutzfeldt-Jakob disease patients using paraffin-embedded tissue blotting

The accumulation of PrP(Sc), an abnormal and disease-associated form of the normal prion protein (PrP(c)), within the central nervous system (CNS) is a key pathological feature of Creutzfeldt-Jakob disease (CJD). Following limited proteolytic digestion of PrP(Sc), the detection of PrP(res) within lymphoid tissues is a unique characteristic of variant CJD in comparison with other human prion diseases, raising fears of an increased risk of iatrogenic spread. Because levels of PrP(res) in lymphoid tissues are lower than those found in CNS tissue, there is concern that other peripheral tissues may harbour infectivity at levels that current detection systems cannot demonstrate PrP(res). We have modified the paraffin-embedded tissue blot (PET blot), a technique combining immunohistochemistry (IHC), histoblot and Western blotting, for the detection of PrP(res) in paraffin sections in peripheral tissues in variant CJD. Five cases of variant CJD were examined, using a panel of anti-PrP antibodies. In each of these five cases, spleen, tonsil, lymph nodes and dorsal root ganglia showed an increase in the sensitivity and specificity of labelling using the PET blot when compared with optimized PrP(res) IHC methods. Control cases showed no evidence of PrP accumulation in either peripheral or CNS tissues. Autopsy and biopsy brain material from sporadic CJD cases also showed an increased sensitivity of PrP(res) detection with the PET blot, confirming its value as an important diagnostic and research tool in human prion diseases.     

Brain histamine metabolism in transmissible spongiform encephalopathy (Scrapie)

Summary The histamine (Hi) content and the activity of L-histidine decarboxylase (HD) in brains of scrapie infected hamsters were measured. No significant changes in Hi levels in particular brain areas were found when compared to controls. Decreased activity of HD was found in hypothalamus (p<0.02). Increased activity of the enzyme was observed in rest of brain, which consisted mainly of thalamus and striatum (p<0.05).     

Prion disease with a 144 base pair insertion: unusual cerebellar prion protein immunoreactivity

Sporadic, acquired, and genetic human prion diseases are characterized neuropathologically by distinct deposition patterns of the abnormal, disease-associated form of the prion protein (PrP^sc). In addition to mutations in the prion protein gene (PRNP), PrP^sc immunostaining patterns correlate with molecular phenotypes of prion diseases defined by the PRNP polymorphism at codon 129 and with protease-resistant PrP classified by Western blotting. Some point or insertional PRNP mutations share similar clinical and neuropathological phenotypes, whereas others show great variability even within the same family. Here we report a patient who presented clinically as sporadic Creutzfeldt-Jakob disease (CJD). Histologically moderate spongiform change was seen in cerebral and cerebellar cortical areas. Neuronal loss was restricted mainly to the occipital cortex and the basal ganglia. Surprisingly, numerous eosinophilic globular structures were noted in the molecular layer and the parahippocampal gyrus. These globules showed intense PrP immunopositivity using anti-PrP antibodies against different epitopes. They were stained with PAS but lacked congophilia and birefringence in polarized light. Ultrastructurally, globules were composed of 21-nm-thick intermingled filaments without dense core. Genetic analysis revealed a PRNP 144 base pair insertion. Our case reinforces the importance of molecular genetic diagnosis, especially in those patients who lack a family history of prion disease and show unusual neuropathological changes. It also widens the phenotypic spectrum of prion diseases. The phenotypic variability within the same mutation suggests further, yet uncharacterized, genetic or epigenetic influence on phenotype in these diseases.     

Enhanced CD9 expression in the mouse and human brains infected with transmissible spongiform encephalopathies

A tetraspan protein CD9, normally expressed in the myelin sheath of the central and peripheral nervous system, was identified to be up-regulated in mouse brains infected with transmissible spongiform encephalopathy (TSE), by mRNA differential display screening. To elucidate its role in the neurodegeneration process observed in TSE, CD9 expression was examined in the murine disease model and in the human disease materials. Up-regulation of CD9 gene expression in the TSE-infected mouse brains was detected as early as a preclinical stage, when abnormal prion protein deposition and vacuolation were obviously manifested in the internal capsule and thalamus. In contrast, other myelin protein genes showed a reverse pattern of CD9 gene expression. Enhanced CD9 expression was immunohistochemically detected in the astrocytes of such pathological regions. In human specimens of TSE, enhanced CD9 immunoreactivity was observed in the astrocytes and some oligodendrocytes in the brains, but no relevant alteration in CD9 immunoreactivity was observed in the other organs or tissues. Positive CD9 immunoreactivity in astrocytes was also manifest in other neurological disorders in a less prominent manner. The findings indicate that up-regulated CD9 plays a role in glial cells in pathological conditions, especially in such a devastating condition as TSE.     

Neuronal and astrocytic responses involving the serotonergic system in human spongiform encephalopathies

The relationships between the degree of cortical prion protein (PrP) deposition, tissue vacuolation and astrocytosis were studied in the frontal cortex of 27 cases of human spongiform encephalopathy, encompassing 13 cases of sporadic Creutzfeldt-Jakob disease (sCJD), four cases of familial CJD (fCJD) (one owing to E200K mutation, one owing to 144 bp insertion, one owing to P102L mutation and one owing to A117V mutation), five cases of iatrogenic CJD (iCJD) owing to growth hormone therapy and five cases of variant CJD (vCJD). The size and number of tryptophan hydroxylase (TPH) positive cells in the dorsal raphe were determined as an index of the function of the brain's serotonergic system. The amount of PrP deposited in frontal cortex in vCJD was significantly greater than that in both sCJD and iCJD, which did not differ significantly from each other. The extent of grey matter deposition of PrP correlated with that of white matter deposition. Deposition of PrP as plaques was greater in cases of sCJD bearing valine at codon 129 of PrP gene, especially when homozygous. However, all cases of vCJD displayed florid plaque formation yet these were homozygous for methionine at codon 129. Prion protein deposition as plaques was greater in cases of sCJD with 2A PrP isotype than those with 1 PrP isotype, similar to that seen in cases of vCJD all of which are 2B PrP isotype. There were no significant differences in the extent of astrocytosis between the different aetiological groups, in either grey or white matter, as visualized with glial fibrillary acidic protein (GFAP) or 5HT-2A receptor (5HT-2AR) immunostaining, although there was a strong correlation between the severity of 5HT-2AR and GFAP reactions within both grey and white matter. The extent of PrP deposition within the grey, but not white, matter correlated with the degree of astrocytosis for both GFAP and 5HT-2AR and the extent of tissue vacuolation in grey and white matter, although the latter did not correlate with degree of astrocytosis for either GFAP or 5HT-2AR. Astrocytes may be responding directly to the presence of PrP within the tissue, rather than the vacuolar damage to neurones. Although S100beta immunoreactivity was present in astrocytes in control cases, no S100beta staining was seen in astrocytes in either grey or white matter in most CJD cases. There were no differences in the number of TPH-positive cells between CJD and control cases, although the mean TPH-positive cell size was significantly greater, and cells were more intensely stained, in CJD compared to controls, suggesting a pathological overactivity of the brain's serotonergic system in CJD. This may result in excessive release of 5HT within the brain triggering increased 5HT-2AR expression within activated astrocytes leading to release and depletion of S100beta protein from such cells. The clinical symptoms of fluctuating attention and arousal could be mediated, at least in part, by such alterations in function of the serotonergic system.     

The phenotypic expression of different mutations in transmissible human spongiform encephalopathy.

Clinical, pathological, and experimental transmission characteristics are reviewed for each of the known mutations in the amyloid precursor gene (PRNP) associated with familial spongiform encephalopathies. All mutation groups show an earlier age at onset and longer duration of illness than sporadic disease, and more or less distinctive patterns of illness can be recognized for each mutation, although much variability may occur even among affected members of the same family. Experimental transmission of disease has been accomplished for most of the mutations, with shortened incubation periods in the inoculated animals that parallel the earlier age at onset of human illness in these cases, implying a shortened pre-clinical phase of disease rather than an earlier 'infecting event'. Mutations thus not only predispose to spongiform encephalopathy, but also accelerate its pathogenetic tempo and influence its phenotypic expression.     

Disease associated prion protein may deposit in the peripheral nervous system in human transmissible spongiform encephalopathies

There is increasing evidence indicating involvement of the peripheral nervous system (PNS) in the pathogenesis of transmissible spongiform encephalopathies (TSEs). Immunocytochemically detectable deposits of TSE-specific abnormal prion protein (PrP^sc) are considered as a surrogate marker for infectivity. We used anti-PrP immunocytochemistry to trace PrP^sc deposition in spinal and enteric ganglia, and peripheral nerve in Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker disease (GSS), and fatal familial insomnia. Discrete PrP^sc deposits were detectable only in a few posterior root nerve fibers in an adaxonal location in one of nine CJD and the one GSS patients examined. Follicular dendritic cells of the gut and enteric nervous system were not labeled. Thus, PrP^sc may spread to the PNS in different forms of human prion disease. In contrast to our observations in experimental scrapie (Groschup et al., Acta Neuropathol, this issue), the deposits were scant. Possible explanations for this discrepancy comprise strain difference, or centripetal (experimental scrapie) versus centrifugal (sporadic and genetic human prion diseases) spread of PrP^sc, resulting in different patterns and amounts of PrP^sc accumulation in the PNS.     

Prion protein immunocytochemistry: reliable protocols for the investigation of Creutzfeldt–Jakob disease

Current criteria for the histological diagnosis of Creutzfeldt–Jakob disease (CJD) include features such as spongiform change, neuronal loss and reactive gliosis which are shared to a varying extent with other neurodegenerative disorders. Reliable visualization of prion protein (PrP) has substantial potential value in diagnostic practice and as a research tool, since accumulation of the disease–associated isoform of this protein is apparently specific for spongiform encephalopathies. A number of antisera against PrP have previously been employed in conjunction with a range of pre–treatments designed to optimize the specificity of immunostaining; such varied usage makes the comparison and interpretation of results difficult. This study was undertaken to identify optimal combinations of each of three PrP antisera and five pre–treatments designed to specifically demonstrate disease–specific PrP in a series of seven CJD cases, six cases of Alzheimer–type dementia and six non–demented control cases. Specific staining of amyloid plaques, spongiform neuropil, neurons and, occasionally, astrocytes was achieved in CJD cases. Alzheimer and control cases were unstained. Use of formic acid with guanidine thiocyanate, and hydrolytic autoclaving with IB 3 and SP30 antisera proved most effective and can be recommended for future immunocytochemical studies. PrP immunocytochemistry revealed a greater extent of subcortical neural involvement than routine histological techniques in CJD; the relationship between classical neuropathology in CJD and PrP accumulation as revealed by immunocytochemistry is not clear cut and requires further investigation. These findings may help to broaden our understanding of human spongiform encephalopathies, and have implications for diagnostic practices in neuropathology.     

朊蛋白基因多态性与老年性痴呆的相关性研究

目的探讨朊蛋白基因(PRNP)129密码子多态性与老年性痴呆(AD)发病的相关性。方法采用病例-对照研究方法,以PCR-RFLP方法检测驻京部队干休所60例晚发AD(LOAD)患者与92例健康老年对照的PRNP129密码子基因、ApoEε4等位基因多态性。结果驻京部队干休所汉族老年人群中PRNP129密码子甲硫氨酸纯合(MM)基因型频率为94.08%,甲硫氨酸缬氨酸杂合(MV)基因型频率为5.92%,未发现缬氨酸纯合(VV)型。ApoEε4等位基因分层前后,MM基因型发生LOAD的风险性差异无统计学意义。PRNP129密码子MM型不是LOAD的独立危险因素(OR=3.27,95%CI=0.402～26.605)。结论东亚人群与欧洲人群PRNP129密码子多态性分布不同。PRNP129密码子MM基因型携带者发生LOAD的风险无显著增高,提示MM基因型不参与LOAD发病。     

Brain biopsy in Creutzfeldt-Jakob disease: evolution of pathological changes by prion protein immunohistochemistry

The formation of protease-resistant prion protein (PrPsc) is considered to be an early event in the pathogenesis of Creutzfeldt-Jakob disease (CJD) and hence its demonstration in brain biopsies by immunohistochemistry is considered diagnostic. We analysed eight brain biopsies from the frontal cortex collected from different parts of India from cases diagnosed as CJD on clinical and pathological grounds for the expression of prion protein (PrP). The duration of illness in these cases varied from 2 months to 1 year. Immunohistochemistry was carried out on paraffin sections using two different clones (KG9 and 3F4) of monoclonal antibodies to PrP. Although all eight cases showed classical features of spongiform encephalopathy of varying severity, only five of the eight cases revealed PrP(sc) in the brain tissue. The immunolabelling was focal and all areas with spongiform change were not labelled. A temporal evolution in the staining pattern was evident - particulate diffuse labelling (synaptic type) in early stages (2 months), perivacuolar deposits in intermediate stages (5-6 months), and dense plaques in late stages (12 months).     

Pathogenetical significance of porencephalic lesions associated with intracerebral inoculation of sheep with the bovine spongiform encephalopathy (BSE) agent

Decreased rates of transmission of transmissible spongiform encephalopathies (TSEs) to sheep have been attributed to some polymorphisms of the prion protein (PrP) and to a 'species barrier' on interspecies experiments. In addition, the blood–brain barrier may be a further impediment to TSE neuroinvasion. The intracerebral (I/C) route is generally considered the most efficient for TSE transmission, as it may help to bypass those factors. Therefore, susceptibility of particular species to specific TSE agents is conducted by this route. Aims: This study characterizes the traumatic brain lesions associated with the I/C injection of the bovine spongiform encephalopathy agent in sheep, assesses the relevance of such lesions in the outcome of clinical disease and provides insight into the mechanisms of PrP^d conversion and amplification following I/C challenge. Methods: A total of 27 hemibrains have been macroscopically and immunohistochemically examined to investigate the presence of lesions compatible with the needle track and the PrP^d distribution, respectively. Results: No residual inoculum was found and the extension and severity of the traumatic brain lesions were unrelated to the clinical outcome. Sheep with PrP^d accumulation in the brain also showed conspicuous focal aggregates in the porencephalic lesions and in the circumventricular organs. In contrast, sheep without PrP^d deposits in the brain were also negative in the traumatic lesions. Conclusion: Overall, these findings suggest that the efficiency of the I/C route is due to effective absorption and blood recirculation of infection, rather than to primary amplification at the site of injection.