Loss of Perineuronal Net in ME7 Prion Disease

Microglial activation and behavioral abnormalities occur before neuronal loss in experimental murine prion disease; the behavioral changes coincide with a reduction in synaptic plasticity. Because synaptic plasticity depends on an intact perineuronal net (PN), a specialized extracellular matrix that surrounds parvalbumin (PV)-positive GABAergic (gamma-aminobutyric acid [GABA]) inhibitory interneurons, we investigated the temporal relationships between microglial activation and loss of PN and PV-positive neurons in ME7 murine prion disease. Anesthetized C57Bl/6J mice received bilateral intracerebral microinjections of ME7-infected or normal brain homogenate into the dorsal hippocampus. Microglial activation, PrP accumulation, the number of PV-positive interneurons, and Wisteria floribunda agglutinin-positive neurons (i.e. those with an intact PN) were assessed in the ventral CA1 and subiculum at 4, 8, 12, 16, and 20 weeks postinjection. Hippocampal areas and total neuron numbers in the ventral CA1 and subiculum were also determined. Loss of PN coincided with early microglial activation and with a reduction in synaptic plasticity. No significant loss of PV-positive interneurons was observed. Our findings suggest that the substrate of the earliest synaptic and behavioral abnormalities in murine prion disease may be inflammatory microglia-mediated degradation of the PN.     

Inflammatory response and retinal ganglion cell degeneration following intraocular injection of ME7

Scrapie is a prion disease which occurs naturally in sheep and which can be transmitted experimentally to rodents. After intracerebral injection of ME7 into mouse, an atypical inflammatory response, characterized by T-lymphocytes and activated microglia is present early in the course of the disease. In the present work, we have investigated the relationship between this inflammatory response, astrocytosis and neuronal loss along the visual pathway after intraocular injection (intraocular) of ME7 in C57BL/6J mice. We have demonstrated that microglia activation and T-lymphocyte recruitment accompanies the spread of prion pathology along the visual pathway and in the early stages of the disease is restricted to the subcortical visual pathway. Inflammation was also present in non-visual areas in association with PrPsc deposition at late stages of the disease, possibily indicating that diffusion of the scrapie agent also contributes to the spread of the disease. After intraocular injection of the prion agent, the disease is believed to be transported into the brain via axons of retinal ganglion cells (RGCs). Despite the high levels of infectivity reported to be present in the retina early in the disease after intraocular injection of ME7, retinal pathology has not been extensively investigated. We have studied the RGCs response in whole mount retinas after intraocular injection of ME7. We have shown that RGCs degenerate after intraocular injection of ME7 whereas amacrine cells, retinal interneurones, are more resistant. Our results suggest that two distinct population of neurones, exposed in vivo at the same time to the same agent scrapie strain, show different susceptibility to the toxic effects of PrPsc.     

The acute inflammatory response in CNS following injection of prion brain homogenate or normal brain homogenate

The neuropathological hallmarks of end-stage prion disease are vacuolation, neuronal loss, astrocytosis and deposition of PrPSc amyloid. We have also shown that there is an inflammatory response in the brains of scrapie-affected mice from 8 weeks post-injection. In this study we have investigated the acute CNS response to the intracerebral injection of scrapie-affected brain homogenate. The ME7 strain of scrapie (Neuropathogenesis Unit, Edinburgh) was used, and control mice were injected with brain homogenate derived from normal C57BL/6 J mice. One microlitre of 10% w/v ME7 (n = 33) and normal brain homogenate (n = 28) was injected stereotaxically into the right dorsal hippocampus. Cryostat sections of brains taken at 1, 2, 5, 7, 14 and 28 days post-injection were examined histologically for neuronal loss, and immunocytochemically to study the inflammatory response. This study shows that ME7 is not acutely neurotoxic in vivo. There is also no difference (ANOVA) in the inflammatory response, which peaked between 2 and 5 days and resolved by 4 weeks after intracerebral injection of either ME7 or normal brain homogenate. The well circumscribed inflammatory response seen previously at 8 weeks is therefore a consequence of a disease process rather than a surgical artefact. This disease process may be related to a localized accumulation of PrPSc sufficient to stimulate an inflammatory response which in turn may contribute to neuronal loss. The role of the inflammatory response in chronic neurodegeneration can be usefully studied using this mouse model of prion disease, and this will undoubtedly shed light on the pathogenic mechanisms underlying other chronic neurodegenerative diseases.     

Dendritic and synaptic alterations of hippocampal pyramidal neurones in scrapie-infected mice

Neurone damage and eventual loss may underlie the clinical signs of disease in the transmissible spongiform encephalopathies (TSEs). Although neurone death appears to be through apoptosis, the trigger for this form of cell death in the TSEs is not known. Using two different murine scrapie models, hippocampal pyramidal cells were studied through microinjection of fluorescent dye, and synaptic integrity, using p38-immunoreactivity (p38-IR), both visualized using confocal laser scanning microscopy. Intradendritic distensions and dendritic spine loss were found to co-localize to areas of vacuolar and prion protein pathology in the hippocampus of mice infected with ME7 or 87 V scrapie. A significant reduction in p38-IR was found concomitantly in the hippocampus in ME7 scrapie mice. These results indicate that both pre- and post-synaptic sites are altered by scrapie infection; this would disrupt neuronal circuitry and may initiate apoptotic cell death, giving rise to the neurological disturbances manifested in clinical TSE cases.     

MCP-1 and murine prion disease: separation of early behavioural dysfunction from overt clinical disease

Prion diseases are chronic, fatal neurodegenerative conditions of the CNS. We have investigated the role of monocyte chemoattractant protein-1 (MCP-1) in the ME7 model of murine prion disease. MCP-1 expression increased in the CNS throughout disease progression and was positively correlated with microglial activation. We subsequently compared the inflammatory response, pathology and behavioural changes in wild-type (wt) mice and MCP-1 knockout mice (MCP-1-/-) inoculated with ME7. Late-stage clinical signs were delayed by 4 weeks in MCP-1-/- mice, and survival time increased by 2-3 weeks. By contrast, early changes in affective behaviours and locomotor activity were not delayed in onset. There was also no difference in microglial activation or neuronal death in the hippocampus and thalamus of wt mice and MCP-1-/- mice. These results highlight an important dissociation between prolonged survival, early behavioural dysfunction and hippocampal/thalamic pathology when considering therapeutic intervention for human prion diseases and other chronic neurodegenerative conditions.     

Neuropil and neuronal changes in hippocampal NADPH-diaphorase histochemistry in the ME7 model of murine prion disease

Nitric oxide (NO) has been implicated in neurotoxicity and cerebral blood flow changes in chronic neurodegeneration, but its activity in the mammalian prion diseases has not been studied in detail. Nicotine adenine dinucleotide phosphate (NADPH)-diaphorase (NADPH-d) histochemistry is a simple and robust histochemical procedure that allows localization of the tissue distribution of NO synthases. The aim of the present study is to assess whether NADPH-d histochemical activity is altered in the hippocampus in the ME7 model of prion disease in C57BL/6J mice. At early and late stages after the initiation of the disease we assessed features of the NADPH-d positive cells and the neuropil histochemical activity in CA1 and dentate gyrus using densitometric analysis. In C57BL/6J mice 13 weeks postinjection of the prion agent ME7, when behavioural changes first become apparent, neuropil NADPH-d histochemical staining increases, whereas at late stages it decreases dramatically. Both type I and type II NADPH-d positive cells were found to survive throughout the hippocampal formation into the late stages of the disease, but diaphorase activity was reduced in dendritic branches and abnormal varicosities were present in both dendritic and axonal processes of NADPH-d positive type I cells. The pathophysiological implications of the results remain to be investigated but both blood flow alteration and NO neurotoxicity may be features of the disease.     

Synaptic changes characterize early behavioural signs in the ME7 model of murine prion disease

Prion diseases are fatal, chronic neurodegenerative diseases of mammals, characterized by amyloid deposition, astrogliosis, microglial activation, tissue vacuolation and neuronal loss. In the ME7 model of prion disease in the C57BL/6 J mouse, we have shown previously that these animals display behavioural changes that indicate the onset of neuronal dysfunction. The current study examines the neuropathological correlates of these early behavioural changes. After injection of ME7-infected homogenate into the dorsal hippocampus, we found statistically significant impairment of burrowing, nesting and glucose consumption, and increased open field activity at 13 weeks. At this time, microglia activation and PrPSc deposition was visible selectively throughout the limbic system, including the hippocampus, entorhinal cortex, medial and lateral septum, mamillary bodies, dorsal thalamus and, to a lesser degree, in regions of the brainstem. No increase in apoptosis or neuronal cell loss was detectable at this time, while in animals at 19 weeks postinjection there was 40% neuronal loss from CA1. There was a statistically significant reduction in synaptophysin staining in the stratum radiatum of the CA1 at 13 weeks indicating loss of presynaptic terminals. Damage to the dorsal hippocampus is known to disrupt burrowing and nesting behaviour. We have demonstrated a neuropathological correlate of an early behavioural deficit in prion disease and suggest that this should allow insights into the first steps of the neuropathogenesis of prion diseases.     

Atypical inflammation in the central nervous system in prion disease

The inflammatory response in prion diseases is dominated by microglial activation. Contrary to their profile in vitro none of the pro-inflammatory cytokines interleukin-1beta, interleukin-6, or tumour necrosis factor-alpha are significantly upregulated in the ME7 model of prion disease. However, two major inflammatory mediators are elevated: transforming growth factor-beta1 and prostaglandin E2. This cytokine profile is the same as that reported for macrophages during phagocytosis of apoptotic cells and indeed transforming growth factor-beta1 and prostaglandin E2 are responsible for the downregulated phenotype of these macrophages. Transforming growth factor-beta1 may also have roles in extracellular matrix deposition and in amyloidogenesis and may play a direct role in disease pathogenesis. There is also now evidence to suggest that a peripheral infection, and its consequent systemic cytokine expression, may drive central nervous system cytokine expression and perhaps exacerbate disease.     

Neuropathologically distinct prion strains give rise to similar temporal profiles of behavioral deficits

Mouse-adapted scrapie strains have been characterized by vacuolation profiles and incubation times, but the behavioral consequences have not been well studied. Here, we compared behavioral impairments produced by ME7, 79A, 22L, and 22A strains in C57BL/6J mice. We show that early impairments on burrowing, glucose consumption, nesting and open field activity, and late stage motor impairments show a very similar temporal sequence in ME7, 79A, and 22L. The long incubation time of the 22A strain produces much later impairments. However, the strains show clear late stage neuropathological differences. All strains showed clear microglial activation and synaptic loss in the hippocampus, but only ME7 and 79A showed significant CA1 neuronal death. Conversely, 22L and 22A showed significant cerebellar Purkinje neuron loss. All strains showed marked thalamic neuronal loss. These behavioral similarities coupled with clear pathological differences could serve to identify key circuits whose early dysfunction underlies the neurological effects of different prion strains.     

Oxidative stress in the brain at early preclinical stages of mouse scrapie

Oxidative stress has been shown to be involved in the pathogenesis of neurodegenerative diseases including prion diseases. Although a growing body of evidence suggests direct involvement of oxidative stress in the pathogenesis of prion diseases, it is still not clear whether oxidative stress is a causative early event in these conditions or a secondary phenomenon commonly found in the progression of neurodegenerative diseases. Using a mouse scrapie model, we assessed oxidative stress in the brain at various stages of the disease progression and observed significantly increased concentration of lipid peroxidation markers, malondialdehyde and 4-hydroxyalkenals, and mRNA level of an oxidative stress response enzyme, heme oxygenase-1, at early preclinical stages of scrapie. The changes preceded dramatic synaptic loss demonstrated by immunohistochemical staining of a synaptic protein, synaptophysin. These findings imply that the brain undergoes oxidative stress even from an early stage of prion invasion into the brain. Given the well-known deleterious effects of reactive-oxygen-species-mediated damage in the brain, it is considered that the oxidative stress at the preclinical stage of prion diseases may predispose the brain to neurodegenerative mechanisms that characterize the diseases.     

Aberrant Alterations of Mitochondrial Factors Drp1 and Opa1 in the Brains of Scrapie Experiment Rodents

The abnormal mitochondrial dynamics has been reported in the brains of some neurodegenerative diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), but limitedly described in prion disease. Dynamin-related protein 1 (Drpl) and optic atrophy protein 1 (Opa1) are two essential elements for mitochondria fission and fusion. To evaluate possible changes of mitochondria dynamics during prion infection, the situations of brain Drp1 and Opa1 of scrapie strains 139A, ME7, and S15 mice, as well as 263K-infected hamsters, were analyzed. Significant decreases of brain Drp1 were observed in scrapie-infected rodents at terminal stage by Western blots and immunohistochemical assays, while the levels of Opa1 also showed declined tendency in the brains of scrapie-infected rodents. Immunofluorescent assays illustrated well localization of Drp1 or Opa1 within NeuN-positive cells. Moreover, the S-nitrosylated forms of Drp1significantly increased in the brain tissues of 139A- and ME7-infected mice at terminal stage. Dynamic analysis of Drp1 and SNO-Dpr1 in the brains collected at different time points within the incubation period of 139A-infected mice demonstrated that the whole Drp1 decreased at all tested samples, whereas the SNO-Drp1 remarkably increased in the sample of 90-day post-infection (dpi), reached to the peak in that of 120 dpi and dropped down but still maintained at higher level at the end of disease. The levels of apoptotic factors cleaved caspase 9, caspase 3, and Bax were also markedly increased in the brain tissues of the mice infected with agents 139A and ME7. Our data indicate a disorder of mitochondria dynamics in the brains of prion infection, largely depending on the abnormal alteration of brain Drp1.     

Copper is toxic to PrP-ablated mice and exacerbates disease in a mouse model of E200K genetic prion disease

The pathogenesis of the diverse forms of prion disease was attributed solely to the accumulation of the misfolded PrP forms, and not to the potential loss of normal PrP(C) function during disease propagation. In this respect, it was also not established whether mutant PrPs linked to genetic prion diseases, as is the case for E200K PrP, preserve the function of PrP(C). We now show that fibroblasts generated from both PrP-ablated mice and TgMHu2ME199K, a transgenic mouse line mimicking E200KCJD, were significantly more sensitive to copper toxicity than wt fibroblasts. Long-term administration of copper significantly accelerated the onset and progression of spontaneous prion disease in TgMHu2ME199K mice and caused marked irritability and cerebellar associated tip-toe walking in PrP(0/0) mice, while wt mice were not affected. Our results are consistent with the hypothesis that a functional PrP(C) is required to protect cells from high levels of copper, and that its substitution for a nonfunctional mutant PrP may accelerate the onset of genetic prion disease during oxidative insults.     

Early changes in Abeta levels in the brain of APPswe transgenic mice--implication on synaptic density, alpha7 neuronal nicotinic acetylcholine- and N-methyl-D-aspartate receptor levels

Tg 2576 (APPswe) mice develop age-related amyloid deposition as well as behavioural- and electrophysiological changes in the brain. In this study, APPswe mice were investigated from 7 to 90 days of age. We observed high Abeta levels in the cortex of APPswe mice at 7 days of age, suggesting that these mice produce Abeta from birth. A positive correlation between Abeta and synaptophysin levels, followed by changes in ERK MAPK activity, indicated that Abeta causes altered synaptic function and an increase in the number of synaptic terminals. In addition, alterations in [(125)I]alphabungarotoxin- and [(3)H]MK-801 binding sites were also observed in APPswe mice compared to controls. In conclusion, over-expression of Abeta early in life causes changes in synaptophysin levels and number of [(125)I]alphabungarotoxin- and [(3)H]MK-801 binding sites. The results may provide important information about the onset and consequences of Abeta pathology in this transgenic mouse model.     

Alzheimer's disease Abeta assemblies mediating rapid disruption of synaptic plasticity and memory

ABSTRACT: Alzheimer's disease (AD) is characterized by episodic memory impairment that often precedes clinical diagnosis by many years. Probing the mechanisms of such impairment may provide much needed means of diagnosis and therapeutic intervention at an early, predementia, stage. Prior to the onset of significant neurodegeneration, the structural and functional integrity of synapses in mnemonic circuitry is severely compromised in the presence of amyloidosis. This review examines recent evidence evaluating the role of amyloid-beta protein (Abeta) in causing rapid disruption of synaptic plasticity and memory impairment. We evaluate the relative importance of different sizes and conformations of Abeta, including monomer, oligomer, protofibril and fibril. We pay particular attention to recent controversies over the relevance to the pathophysiology of AD of different water soluble Abeta aggregates and the importance of cellular prion protein in mediating their effects. Current data are consistent with the view that both low-n oligomers and larger soluble assemblies present in AD brain, some of them via a direct interaction with cellular prion protein, cause synaptic memory failure. At the two extremes of aggregation, monomers and fibrils appear to act in vivo both as sources and sinks of certain metastable conformations of soluble aggregates that powerfully disrupt synaptic plasticity. The same principle appears to apply to other synaptotoxicamyloidogenic proteins including tau, alpha-synuclein and prion protein.     

Mutation in the prion protein gene at codon 232 in Japanese patients with Creutzfeldt-Jakob disease: a clinicopathological,immunohistochemical and transmission study

We describe the clinical, neuropathological, immunohistochemical and transmission findings in three patients with Creutzfeldt-Jakob disease (CJD) with a substitution from methionine to arginine at codon 232 (M232R) in the prion protein (PrP) gene. The patients with M232R presented clinically with rapidly progressive dementia, myoclonus, and periodic synchronous discharges in the electroencephalogram. These findings were mostly consistent with those for sporadic CJD. All patients reached the stage of akinetic mutism between 2 and 6 months, and died between 4 and 24 months after the onset of the disease. Histopathological examination revealed spongiform changes, neuronal loss and severe astrocytosis. Immunohistochemical staining for PrP showed diffuse gray matter staining, including synaptic structures. However, no plaque-type PrP deposition was observed in the affected brain tissue sections. The brain homogenates from two patients were successfully transmitted to experimental animals. Since the same mutation was not found in 100 healthy control individuals, the mutation might be associated with the disease. The clinicopathological and experimental transmission studies of CJD patients with this PrP gene mutation may thus help us to determine both phenotypic variations and the potential infectivities in different forms of prion diseases. Received: 27 December 1995 / Revised, accepted: 25 March 1995     

Prion protein disease and neuropathology of prion disease

Human prion diseases, in common with other neurodegenerative diseases, may be sporadic or inherited and are characterized by the accumulation of cellular proteins accompanied by neuronal death and synaptic loss. Prion diseases are, however, unique in being transmissible. Central to the pathogenesis of all forms of prion disease is the prion protein. This article provides a brief overview of the biology of human prion diseases followed by a more in-depth discussion of the neuropathology of these diseases, including features of neuroradiologic relevance.     

Lack of TAR-DNA binding protein-43 (TDP-43) pathology in human prion diseases

Aims: TAR-DNA binding protein-43 (TDP-43) is the major ubiquitinated protein in the aggregates in frontotemporal dementia with ubiquitin-positive, tau-negative inclusions and motor neurone disease. Abnormal TDP-43 immunoreactivity has also been described in Alzheimer's disease, Lewy body diseases and Guam parkinsonism–dementia complex. We therefore aimed to determine whether there is TDP-43 pathology in human prion diseases, which are characterised by variable deposition of prion protein (PrP) aggregates in the brain as amyloid plaques or more diffuse deposits. Material and methods: TDP-43, ubiquitin and PrP were analysed by immunohistochemistry and double-labelling immunofluorescence, in sporadic, acquired and inherited forms of human prion disease. Results: Most PrP plaques contained ubiquitin, while synaptic PrP deposits were not associated with ubiquitin. No abnormal TDP-43 inclusions were identified in any type of prion disease case, and TDP-43 did not co-localize with ubiquitin-positive PrP plaques or with diffuse PrP aggregates. Conclusions: These data do not support a role for TDP-43 in prion disease pathogenesis and argue that TDP-43 inclusions define a distinct group of neurodegenerative disorders.     

异常PrP沉积对人朊蛋白病的临床和病理影响

目的　探讨异常ＰｒＰ沉积形式与ＣｒｅｕｔｚｆｅｌｄｔＪａｋｏｂ 病（ＣＪＤ）、ＧｅｒｓｔｍａｎｎＳｔｒａｕｓｓｌｅｒ 综合征（ＧＳＳ）临床与病理关系。方法　经病理检查确诊的７ 例ＣＪＤ,１ 例ＧＳＳ脑切片进行朊蛋白ＰｒＰ免疫组化染色和实验动物传递。结果　（１） ７ 例ＣＪＤ均呈现突触型阳性,而ＧＳＳ则呈现斑块型阳性;（２） 突触型阳性者,病程短,平均１２ ．３ 个月,斑块型阳性者病程６０ 个月;（３） ７ 例突触型均有痴呆,６ 例有肌阵挛,小脑症状不明显,斑块型则与其相反;（４） 突触型中４ 例进行动物传递均获成功,斑块型则否;（５） 突触型ＰｒＰ沉积多位于大脑灰质,斑块型多在小脑分子层。结论　应用水解高压灭菌法,以ＰｒＰ抗血清为第一抗体的免疫组化方法,可以准确判定异常ＰｒＰ沉积的两种类型,二者神经症状、病程长短、脑电图改变以及动物传递结果等均不相同。     

Tubulovesicular structures are not labeled using antibodies to prion protein (PrP) with the immunogold electron microscopy techniques

Tubulovesicular structures (TVS) are disease-specific, intraneuronal particles found by thin-section electron microscopy in all of the transmissible spongiform encephalopathies. We used immunogold (both 10 nm immunogold and 1 nm immunogold silver enhanced) methods for ultrastructural localization of prion protein (PrP). In all scrapie models examined (263 K and 22CH in hamsters and 87V and ME7 in mice), TVS-containing processes were readily detected but neither these processes nor TVS themselves were decorated with gold particles. Even when amyloid plaques were observed in a close contact with TVS-containing neuronal processes, the processes remained unstained, while the plaques were decorated with gold particles. TVS located in areas adjacent to plaques in the 87V model and in areas of diffuse PrP immunolabelling in ME7 were also unlabelled with anti-PrP sera. Using immunogold techniques we were unable to label TVS with anti-PrP antibodies. As these technique proved to be sensitive enough to immunolabel not only amyloid plaques but also pre-amyloid accumulations of PrP, we strongly believe that the absence of staining reflects the structure of TVS and that they are not composed of PrP. That TVS are PrP negative may have several important implications for hypotheses about their nature. Principally, it does not support the suggestion that TVS are cross-sections of “thick tubules” visualized by touch-preparations of scrapie-affected mouse and hamster brains. If PrP is the infectious agent, as suggested by the prion hypothesis, the absence of stainable PrP in TVS would indicate that these are not the ultrastructural correlate of the agent. If, however, TVS turn out to be more than merely a useful ultrastructural marker for the whole group of transmissible spongiform encephalopathies, it may suggest that PrP and the agent are two separate entities. Received: 11 March 1996 / Revised: 9 May 1996 / Accepted: 26 September 1996