朊蛋白在神经系统作用机制研究进展

细胞型朊蛋白(PrP~C)作为一种跨膜糖蛋白在哺乳动物中广泛存在。基因敲除的研究显示PrP~C在神经系统的活动中的关键作用包括周围神经髓鞘的形成以及对神经毒素刺激的保护。PrP~C在不同的细胞类型中也有不同的生物学作用。如PrP~C模块化结构、多种结合伴侣以及与脂质筏的密切关联的特性,使其具有组装多组分复合物的能力,从而触发不同的信号通路,调节细胞分化。PrP~C在大脑中参与的病理性作用仍然没有一致的定论,其错误折叠产生的异构体PrP~(SC)是朊病毒疾病的主要致病因素。但有证据指出PrP~C在朊病毒疾病中发挥的致病作用独立于羊瘙痒病朊蛋白亚型(PrP~(SC)),在朊病毒感染过程中,朊病毒疾病的临床和神经病理症状与大脑中PrP~C而不是PrP~(SC)的表达水平成正比。另外,PrP~C可能还是一种与神经退行性病变相关的蛋白,参与β淀粉样蛋白(Aβ)等聚集性蛋白的神经毒素信号转导,还充当α-突触核蛋白的细胞受体,促进其在细胞吸收以及大脑中传播。虽然朊病毒的研究已经取得很大的进展,但PrP~C在大脑中的作用仍然没有明确,因此探索PrP~C在细胞中作用具有十分重要的意义。     

Hypothalamic-pituitary-adrenal axis disregulation in PrPC-null mice

As manifestations of prion diseases include disturbances of hypothalamic and pituitary functions, we tested the hypothesis that the cellular prion protein (PrPC) has a role as modulator of the hypothalamic-pituitary-adrenal axis. The level of corticosterone and adrenocorticotropic hormone were compared in PrPC null (PrP 0/0) and wild-type (PrP+/+) mice. PrP 0/0 showed hypercorticism during the dark part of day. After acute stress, corticosterone and adrenocorticotropic hormone increased similarly in PrP+/+ and PrP 0/0 mice. Adrenocorticotropic hormone, however, remained elevated in PrP+/+ 0/0 mice at corticosterone levels that are inhibitory in PrP mice. Pretreatment with corticosterone or dexamethasone inhibited stress-induced elevation of adrenocorticotropic hormone in PrP+/+ but not in PrP 0/0 mice. Thus, PrPC may play a role in the negative feedback regulation of axis.     

Changes in the localization of brain prion proteins during scrapie infection

Prion proteins (PrP) were localized in the brains of normal and scrapie-infected hamsters by immunohistochemistry and Western blotting. PrP monoclonal antibodies and monospecific anti-PrP peptide sera, which react with both the cellular (PrPC) and scrapie (PrPSc) isoforms of the prion protein, were used to locate PrP in tissue sections. In normal hamsters, PrPC was located primarily in nerve cell bodies throughout the CNS; whereas, in the terminal stages of scrapie, PrP immunoreactivity was shifted to the neuropil and was absent from most nerve cell bodies. Prion proteins were not uniformly dispersed throughout the gray matter of scrapie-infected hamster brains; rather, they were concentrated in those regions that exhibited spongiform degeneration and reactive astrogliosis. Since earlier studies showed that the level of PrPC remains constant during scrapie infection as measured in whole brain homogenates and no antibodies are presently available that can distinguish PrPC from PrPSc, we analyzed individual brain regions by Western blotting. Analysis of proteinase K-digested homogenates of dissected brain regions showed that most of the regional changes in PrP immunoreactivity that are seen during scrapie infection are due to the accumulation of PrPSc. These observations indicate that the tissue pathology of scrapie can be directly correlated with the accumulation of PrPSc in the neuropil, and they suggest that the synthesis and distribution of the prion protein has a central role in the pathogenesis of this disorder.     

Presence of prion protein in peripheral tissues of Libyan Jews with Creutzfeldt-Jakob disease.

The prion protein (PrP) gene on chromosome 20 encodes a protein designated PrPC. An abnormal, protease-resistant isoform of PrPC, denoted PrPCJD or PrPSc, is present in the brains of patients with Creutzfeldt-Jakob disease (CJD). In Libyan Jews, CJD segregates with a point mutation at codon 200 of the PrP gene, resulting in the substitution of lysine for glutamate. In the present study, we examined the presence of PrP in fibroblasts and leukocytes derived from eight CJD patients with the codon 200 mutation. In cultured fibroblasts as well as in leukocytes, there was a significant increase in PrP as judged by immunocytochemistry in addition to immunoblotting. Most of the PrP in fibroblasts and leukocytes could be released from the external surface by phosphatidylinositol-specific phospholipase C, a property characteristic of PrPC. In leukocytes only, part of the protein was protease resistant, resembling PrPCJD. The concentration of PrP mRNA was similar in fibroblast lines derived from controls and CJD patients. These results suggest that in CJD patients carrying a mutation at codon 200 of the PrP gene, the metabolism of PrP, rather than PrP synthesis, is abnormal.     

GFP-tagged prion protein is correctly localized and functionally active in the brains of transgenic mice

Prion diseases result from conversion of PrPC, a neuronal membrane glycoprotein of unknown function, into PrPSc, an abnormal conformer that is thought to be infectious. To facilitate analysis of PrP distribution in the brain, we have generated transgenic mice in which a PrP promoter drives expression of PrP-EGFP, a fusion protein consisting of enhanced green fluorescent protein inserted adjacent to the glycolipid attachment site of PrP. We find that PrP-EGFP in the brain is glycosylated and glycolipid-anchored and is localized to the surface membrane and the Golgi apparatus of neurons. Like endogenous PrP, PrP-EGFP is concentrated in synapse-rich regions and along axon tracts. PrP-EGFP is functional in vivo, since it ameliorates the cerebellar neurodegeneration induced by a truncated form of PrP. These observations clarify uncertainties in the cellular localization of PrPC in brain, and they establish PrP-EGFP transgenic mice as useful models for further studies of prion biology.     

Redefining primary headaches

To assess the role of tau protein, beta-amyloid_(1–42) and cystatin C in the diagnostics of Alzheimer dementia (AD) and other neurodegenerative diseases (ND) by comparing to the control groups (CG). The levels of tau protein, beta-amyloid_(1–42) and cystatin C were assessed in the set of 69 patients (AD + ND, 33 males, 36 females, aged 22–90, mean 60.5 + 16.1 years), and in a control group of 69 subjects without the affection of the central nervous system (CGAD + CGND, 33 males, 36 females, aged 20–91, mean 60.5 + 16.0 years). Statistically significant increased tau protein levels (P = 0.0001) and index tau/beta-amyloid_(1–42) levels (P = 0.0002) were shown in the group of AD patients, compared to the group of ND patients. One-way ANOVA analysis with Bonferonni post hoc test did not show any significant differences of the cystatin C values between any of the compared groups. ROC analysis showed at least one tie between the positive actual state group (AD) and the negative actual state group (ND) by CSF cystatin C and at least one tie between the positive actual state group and the negative actual state group by CSF tau protein. Our study confirmed previously reported results only in part. While tau protein seems to be quite a reliable marker of AD, the role of beta-amyloid_(1–42) and cystatin C in AD diagnosis remains at least questionable.     

Novel Compounds Identified by Structure-Based Prion Disease Drug Discovery Using In Silico Screening Delay the Progression of an Illness in Prion-Infected Mice

The accumulation of abnormal prion protein (PrP^Sc) produced by the structure conversion of PrP (PrP^C) in the brain induces prion disease. Although the conversion process of the protein is still not fully elucidated, it has been known that the intramolecular chemical bridging in the most fragile pocket of PrP, known as the “hot spot,” stabilizes the structure of PrP^C and inhibits the conversion process. Using our original structure-based drug discovery algorithm, we identified the low molecular weight compounds that predicted binding to the hot spot. NPR-130 and NPR-162 strongly bound to recombinant PrP in vitro, and fragment molecular orbital (FMO) analysis indicated that the high affinity of those candidates to the PrP is largely dependent on nonpolar interactions, such as van der Waals interactions. Those NPRs showed not only significant reduction of the PrP^Sc levels but also remarkable decrease of the number of aggresomes in persistently prion-infected cells. Intriguingly, treatment with those candidate compounds significantly prolonged the survival period of prion-infected mice and suppressed prion disease-specific pathological damage, such as vacuole degeneration, PrP^Sc accumulation, microgliosis, and astrogliosis in the brain, suggesting their possible clinical use. Our results indicate that in silico drug discovery using NUDE/DEGIMA may be widely useful to identify candidate compounds that effectively stabilize the protein.Electronic supplementary materialThe online version of this article (10.1007/s13311-020-00903-9) contains supplementary material, which is available to authorized users.     

Rationale for diagnosing human prion disease.

Human prion diseases (PrD) like Creutzfeldt-Jakob disease (CJD) include sporadic, acquired and familial neurodegenerative disorders. The central events in the neuropathological process of PrDs are severe neuronal loss, spongiform change and accumulation of abnormal prion protein (PrPSc). The latter is a conformational variant of the host-encoded cellular PrP (PrPC), a copper-binding protein. The physiological role of PrPC is debated. Definitive diagnosis of PrD is based on post mortem demonstration of PrPSc by immunohistochemistry or Western blot. Mutations in the PrP gene (PRNP), the polymorphic site at codon 129, and the molecular characteristic of protease resistant PrP influence the phenotype. Clinical symptoms, cranial MRI scan, EEG and investigation of 14-3-3 protein in cerebrospinal fluid (CSF) suggest a diagnosis of probable CJD. Variant CJD, related to bovine spongiform encephalopathy, shows a different clinical course, symmetrical high intensity MRI signal in the pulvinar, presence of PrPSc in tonsil biopsy tissue, and a lower sensitivity of CSF 14-3-3 protein compared to sporadic CJD. Future possibilities in diagnosis of PrDs include either the demonstration of PrPSc in body fluids or disease associated changes in laboratory variables or gene expression.     

Mutant prion protein D202N associated with familial prion disease is retained in the endoplasmic reticulum and forms ‘curly’ intracellular aggregates

Transmissible Spongiform Encephalopathies are fatal neurodegenerative disorders of humans and animals that are familial, sporadic, and infectious in nature. Familial disorders of humans include Gerstmann–Straussler–Scheinker disease (GSS), familial Creutzfeldt–Jakob disease (CJD), and fatal familial insomnia, and result from point mutations in the prion protein gene. Although neurotoxicity in familial cases is believed to result from a spontaneous change in conformation of mutant prion protein (PrP) to the pathogenic PrP-scrapie (PrP^Sc) form, emerging evidence indicates otherwise. We have investigated the processing and metabolism of mutant PrP D202N (PrP^202N) in cell models to elucidate possible mechanisms of cytotoxicity. In this report, we demonstrate that PrP^202N expressed in human neuroblastoma cells fails to achieve a mature conformation following synthesis and accumulates in the endoplasmic reticulum as ‘curly’ aggregates. In addition, PrP^202N cells show increased sensitivity to free radicals, indicating that neuronal susceptibility to oxidative damage may account for the neurotoxicity observed in cases of GSS resulting from PrP D202N mutation. Yaping Gu and Susamma Verghese contributed equally.     

Expression of α<Subscript>2</Subscript>-macroglobulin,neutrophil elastase,and interleukin-1α differs in early-stage and late-stage atherosclerotic lesions in the arteries of the circle of Willis

Different types of atherosclerotic (AS) lesions can be distinguished histologically and represent different stages of AS plaque development. Late-stage lesions more frequently develop complications such as plaque rupture and thrombosis with vessel occlusion than early AS lesions. To clarify whether protective, destructive, and inflammatory proteins are differentially expressed in early-stage and late-stage AS plaques we examined the proteinase inhibitor α₂-macroglobulin (A2M), the neutrophil elastase (NE)—an enzyme degrading elastin and collagen fibers—and the proinflammatory protein interleukin-1α (IL-1α) in all types of AS plaques in the arteries of the circle of Willis from 78 human autopsy cases of both genders (61–91 years of age). Paraffin sections of AS plaques were immunostained with antibodies directed against A2M, NE and IL-1α. In initial AS lesions A2M was found, whereas NE and IL-1α were absent. NE and IL-1α became detectable as soon as a significant number of macrophages occurred within AS lesions. With increasing histopathological type of AS lesions, a marked increase of the area of the plaque exhibiting NE and IL-1α was observed. The area which exhibits A2M in AS plaques, on the other hand, did not vary significantly between the different stages. Thus, our results indicate a disproportionately high increase of the destructive enzyme NE and the proinflammatory protein IL-1α in relation to A2M with the progression of the grade of AS lesions pointing to the transgression of the protective capacity of A2M by NE and IL-1α in late-stage plaques. Therefore, our findings support the hypothesis that NE-induced tissue damage in late-stage AS plaques contributes to the development of plaque rupture and subsequent thrombosis.     

Variably Protease-Sensitive Prionopathy: a Novel Disease of the Prion Protein

Variably protease-sensitive prionopathy (VPSPr) is a novel disease involving the prion protein (PrP) that has clinical similarities with non-Alzheimer’s dementias especially frontotemporal dementia, diffuse Lewis body disease, and normal pressure hydrocephalus. VPSPr can be distinguished from sporadic Creutzfeldt–Jakob disease (sCJD) especially for the characteristics of the abnormal PrP. Furthermore, although VPSPr like sCJD affects patients with the three PrP genotypes as determined by the common methionine/valine polymorphism, the allelic prevalence is very different in the two diseases. These findings suggest that VPSPr is basically different from classical prion diseases such as sCJD being perhaps more akin to other neurodegenerative dementias.     

Interaction of metals with prion protein: possible role of divalent cations in the pathogenesis of prion diseases

Prion diseases are fatal neurodegenerative disorders that affect both humans and animals. The rapid clinical progression, change in protein conformation, cross-species transmission and massive neuronal degeneration are some key features of this devastating degenerative condition. Although the etiology is unknown, aberrant processing of cellular prion proteins is well established in the pathogenesis of prion diseases. Normal cellular prion protein (PrP(c)) is highly conserved in mammals and expressed predominantly in the brain. Nevertheless, the exact function of the normal prion protein in the CNS has not been fully elucidated. Prion proteins may function as a metal binding protein because divalent cations such as copper, zinc and manganese can bind to octapeptide repeat sequences in the N-terminus of PrP(c). Since the binding of these metals to the octapeptide has been proposed to influence both structural and functional properties of prion proteins, alterations in transition metal levels can alter the course of the disease. Furthermore, cellular antioxidant capacity is significantly compromised due to conversion of the normal prion protein (PrP(c)) to an abnormal scrapie prion (PrP(sc)) protein, suggesting that oxidative stress may play a role in the neurodegenerative process of prion diseases. The combination of imbalances in cellular transition metals and increased oxidative stress could further exacerbate the neurotoxic effect of PrP(sc). This review includes an overview of the structure and function of prion proteins, followed by the role of metals such as copper, manganese and iron in the physiological function of the PrP(c), and the possible role of transition metals in the pathogenesis of the prion disease.     

Intracellular mechanisms mediating the neuronal death and astrogliosis induced by the prion protein fragment 106–126

Prion encephalopathies include fatal diseases of the central nervous system of men and animals characterized by nerve cell loss, glial proliferation and deposition of amyloid fibrils into the brain. During these diseases a cellular glycoprotein (the prion protein, PrP^C) is converted, through a not yet completely clear mechanism, in an altered isoform (the prion scrapie, PrP^Sc) that accumulates within the brain tissue by virtue of its resistance to the intracellular catabolism. PrP^Sc is believed to be responsible for the neuronal loss that is observed in the prion disease. The PrP 106–126, a synthetic peptide that has been obtained from the amyloidogenic portion of the prion protein, represents a suitable model for studying the pathogenic role of the PrP^Sc, retaining, in vitro, some characteristics of the entire protein, such as the capability to aggregate in fibrils, and the neurotoxicity. In this work we present the results we have recently obtained regarding the action of the PrP 106–126 in different cellular models. We report that the PrP 106–126 induces proliferation of cortical astrocytes, as well as degeneration of primary cultures of cortical neurons or of neuroectodermal stable cell lines (GH₃ cells). In particular, these two opposite effects are mediated by the same attitude of the peptide to interact with the L-type calcium channels: in the astrocytes, the activity of these channels seems to be activated by PrP 106–126, while, in the cortical neurons and in the GH₃ cells, the same treatment causes a blockade of these channels causing a toxic effect.     

Properties of the cellular prion protein expressed in Xenopus oocytes

The cellular prion protein (PrPC) from different species can be reproducibly expressed in Xenopus oocytes following injection of in vitro transcribed mRNAs. The level of PrPC accumulation increases with the amount of RNA injected and with the duration of incubation. PrPC expressed in oocytes is similar in size and abundance to PrPC protein in mouse brain and >100 ng of PrPC is expressed per oocyte allowing complete experiments to be carried out in single living cells. The protein is glycosylated, fully protease sensitive and expressed on the cell surface. Xenopus oocytes therefore provide a useful model system for the study of prion proteins and their associated disease processes.     

A neuronal cell line that does not express either prion or doppel proteins

Prions have been extensively studied since they represent a new class of infectious agents, the pathogenic prion protein (PrPSc). However, a central question on the physiological function of the normal prion protein (PrPC) remains unresolved. A cell model which was previously established from Rikn mice (PrP-/-) remains problematic because of its ectopic expression of the doppel (Dpl) which may have a neurotoxic effect. Here we established neuronal cell lines from Zürich I (PrP-/-) which do not express Dpl protein and ICR mice (PrP+/+) by transfecting with plasmid encoding for the large T antigen of SV40. The transformed cells have shown neuronal characteristics and, thus, these cell lines may provide a useful model to explore the function of neuronal PrPC.     

Alpha-tocopherol at nanomolar concentrations increases the viability of PC12 cells under oxidative stress conditions. The effects of modulation of signaling systems

We found that long-term preincubation of neuronal-like PC12 cells with α-tocopherol at micro- as well as nanomolar concentrations significantly increased cell viability under oxidative stress conditions. We discovered that the protective effect of α-tocopherol increases with an increase in its concentration in the 1–100 nM range, while its effects at concentrations of 100 nM, 1, 10, and 100 μM were similar when α-tocopherol was applied to cells 12–18 h prior to H₂O₂. An important role in the protective effect of long-term pre-incubation of cells with α-tocopherol at various concentrations is probably related to its modulatory influence on the activities of protein kinase C, extracellular signal-activated protein kinase, and phosphatidylinositol 3-kinase. Short-term preincubation of PC12 cells with this antioxidant at nanomolar concentrations for 0.5 or 1.5 h practically did not influence cell viability, while the protective effect of α-tocopherol at micromolar concentrations was probably related to its ability to terminate free-radical reactions due to direct interactions with free radicals.     

Integrated signaling in heterodimers and receptor mosaics of different types of GPCRs of the forebrain: relevance for schizophrenia

Receptor–receptor interactions within receptor heterodimers and receptor mosaics formed by different types of GPCRs represent an important integrative mechanism for signaling in brain networks at the level of the plasma membrane. The malfunction of special heterodimers and receptor mosaics in the ventral striatum containing D₂ receptors and 5-HT_2A receptors in cortical networks may contribute to disturbances of key pathways involving ventral striato-pallidal GABA neurons and mediodorsal thalamic prefrontal glutamate neurons that may lead to the development of schizophrenia. The ventral striatum transmits emotional information to the cerebral cortex through a D₂ regulated accumbal–ventral pallidal–mediodorsal–prefrontal circuit which is of special interest to schizophrenia in view of the reduced number of glutamate mediodorsal–prefrontal projections associated with this disease. This circuit is especially vulnerable to D₂ receptor activity in the nucleus accumbens, since it produces a reduction in the prefrontal glutamate drive from the mediodorsal nucleus. The following D₂ receptor containing heterodimers/receptor mosaics are of special interest to schizophrenia: A_2A–D₂, mGluR5–D₂, CB₁–D₂, NTS₁–D₂ and D₂–D₃ and are discussed in this review. They may have a differential distribution pattern in the local circuits of the ventral striato-pallidal GABA pathway, predominantly located extrasynaptically. Specifically, trimeric receptor mosaics consisting of A_2A–D₂–mGluR5 and CB₁–D₂–A_2A may also exist in these local circuits and are discussed. The integration of receptor signaling within assembled heterodimers/receptor mosaics is brought about by agonists and allosteric modulators. These cause the intramembrane receptor–receptor interactions, via allosteric mechanisms, to produce conformational changes that pass over the receptor interfaces. Exogenous and endogenous cooperativity is discussed as well as the role of the cortical mGluR2–5-HT_2A heterodimer/receptor mosaic in schizophrenia (Gonzalez-Maeso et al. 2008). Receptor–receptor interactions within receptor heterodimer/receptor mosaics of different receptors in the ventral striatum and cerebral cortex give novel strategies for treatment of schizophrenia involving, e.g., monotherapy with either A_2A, mGluR5, CB₁ or NTS₁ agonists or combined therapies with some of these agonists combined with D₂-like antagonists that specifically target the ventral striatum. In addition, a combined targeting of receptor mosaics in the ventral striatum and in the cerebral cortex should also be considered. This review is dedicated to the special issue “Brain plasticity: aging and neuropsychiatric disorders”.     

Parkin Attenuates Wild-Type τ Modification in the Presence of β-Amyloid and α-Synuclein

Changes in tau (τ) metabolism comprise important pathological landmarks in the tauopathies with parkinsonism as well as Parkinson’s disease and Alzheimer’s disease. Mutations in the parkin gene are associated with Parkinson’s disease. Deposits of amyloid proteins, including Aβ and α-synuclein coexist in the brains of patients with dementia with Lewy bodies; however, it is not known how either of them interacts with τ to provoke neurofibrillary tangle formation across the tauopathies. Here, we show a role for parkin against τ pathology in the presence of intracellular Aβ or α-synuclein. Parkin attenuates four-repeat human τ, but not mutant P301L, hyperphosphorylation in the presence of intracellular Aβ_1–42, or α-synuclein and decreases GSK-3β activity in amyloid-stressed M17 human neuroblastoma cells. These data suggest that parkin may counteract the alteration of τ metabolism in certain neurodegenerative diseases with τ cytopathy and parkinsonism. An erratum to this article can be found at