Diagnosis codes underestimate chronic kidney disease incidence compared with eGFR-based evidence: a retrospective observational study of patients with type 2 diabetes in UK primary care.

BACKGROUND: Type two diabetes (T2D) is a leading cause of both chronic kidney disease (CKD) and onward progression to end-stage renal disease. Timely diagnosis coding of CKD in patients with T2D could lead to improvements in quality of care and patient outcomes. AIM: To assess the consistency between estimated glomerular filtration rate (eGFR)-based evidence of CKD and CKD diagnosis coding in UK primary care. DESIGN & SETTING: A retrospective analysis of electronic health record data in a cohort of people with T2D from 60 primary care centres within England between 2012 and 2022. METHOD: We estimated the incidence rate of CKD per 100 person-years using eGFR-based CKD and diagnosis codes. Logistic regression was applied to establish which attributes were associated with diagnosis coding. Time from eGFR-based CKD to entry of a diagnosis code was summarised using the median and interquartile range. RESULTS: The overall incidence of CKD was 2.32 (95% confidence interval [CI] = 2.24 to 2.41) and significantly higher for eGFR-based criteria than diagnosis codes: 1.98 (95% CI = 1.90 to 2.05) versus 1.06 (95% CI = 1.00 to 1.11), respectively; P<0.001. Only 45.4% of CKD incidences identified using eGFR-based criteria had a corresponding diagnosis code. Patients who were younger, had a higher CKD stage (G4), had an observed urine albumin-to-creatinine ratio (A1), or no observed HbA1c in the past year were more likely to have a diagnosis code. CONCLUSION: Diagnosis coding of patients with eGFR-based evidence of CKD in UK primary care is poor within patients with T2D, despite CKD being a well-known complication of diabetes.

Loss of small GTPase Rab7 activation in prion infection negatively affects a feedback loop regulating neuronal cholesterol metabolism.

Prion diseases are fatal and infectious neurodegenerative diseases that occur in humans and animals. They are caused by the misfolding of the cellular prion protein PrPc into the infectious isoform PrPSc. PrPSc accumulates mostly in endolysosomal vesicles of prion-infected cells, eventually causing neurodegeneration. In response to prion infection, elevated cholesterol levels and a reduction in membrane-attached small GTPase Rab7 have been observed in neuronal cells. Here, we investigated the molecular events causing an impaired Rab7 membrane attachment and the potential mechanistic link with elevated cholesterol levels in prion infection. We demonstrate that prion infection is associated with reduced levels of active Rab7 (Rab7.GTP) in persistently prion-infected neuronal cell lines, primary cerebellar granular neurons, and neurons in the brain of mice with terminal prion disease. In primary cerebellar granular neurons, levels of active Rab7 were increased during the very early stages of the prion infection prior to a significant decrease concomitant with PrPSc accumulation. The reduced activation of Rab7 in prion-infected neuronal cell lines is also associated with its reduced ubiquitination status, decreased interaction with its effector RILP, and altered lysosomal positioning. Consequently, the Rab7-mediated trafficking of low-density lipoprotein to lysosomes is delayed. This results in an impaired feedback regulation of cholesterol synthesis leading to an increase in cholesterol levels. Notably, transient overexpression of the constitutively active mutant of Rab7 rescues the delay in the low-density lipoprotein trafficking, hence reducing cholesterol levels and attenuating PrPSc propagation, demonstrating a mechanistic link between the loss of Rab7.GTP and elevated cholesterol levels.

Gene-Edited Cell Models to Study Chronic Wasting Disease.

Prion diseases are fatal infectious neurodegenerative disorders affecting both humans and animals. They are caused by the misfolded isoform of the cellular prion protein (PrPC), PrPSc, and currently no options exist to prevent or cure prion diseases. Chronic wasting disease (CWD) in deer, elk and other cervids is considered the most contagious prion disease, with extensive shedding of infectivity into the environment. Cell culture models provide a versatile platform for convenient quantification of prions, for studying the molecular and cellular biology of prions, and for performing high-throughput screening of potential therapeutic compounds. Unfortunately, only a very limited number of cell lines are available that facilitate robust and persistent propagation of CWD prions. Gene-editing using programmable nucleases (e.g., CRISPR-Cas9 (CC9)) has proven to be a valuable tool for high precision site-specific gene modification, including gene deletion, insertion, and replacement. CC9-based gene editing was used recently for replacing the PrP gene in mouse and cell culture models, as efficient prion propagation usually requires matching sequence homology between infecting prions and prion protein in the recipient host. As expected, such gene-editing proved to be useful for developing CWD models. Several transgenic mouse models were available that propagate CWD prions effectively, however, mostly fail to reproduce CWD pathogenesis as found in the cervid host, including CWD prion shedding. This is different for the few currently available knock-in mouse models that seem to do so. In this review, we discuss the available in vitro and in vivo models of CWD, and the impact of gene-editing strategies.

Astrocyte in prion disease: a double-edged sword.

Prion diseases are infectious protein misfolding disorders of the central nervous system that result from misfolding of the cellular prion protein (PrPC) into the pathologic isoform PrPSc. Pathologic hallmarks of prion disease are depositions of pathological prion protein PrPSc, neuronal loss, spongiform degeneration and astrogliosis in the brain. Prion diseases affect human and animals, there is no effective therapy, and they invariably remain fatal. For a long time, neuronal loss was considered the sole reason for neurodegeneration in prion pathogenesis, and the contribution of non-neuronal cells like microglia and astrocytes was considered less important. Recent evidence suggests that neurodegeneration during prion pathogenesis is a consequence of a complex interplay between neuronal and non-neuronal cells in the brain, but the exact role of these non-neuronal cells during prion pathology is still elusive. Astrocytes are non-neuronal cells that regulate brain homeostasis under physiological conditions. However, astrocytes can deposit PrPSc aggregates and propagate prions in prion-infected brains. Additionally, sub-populations of reactive astrocytes that include neurotrophic and neurotoxic species have been identified, differentially expressed in the brain during prion infection. Revealing the exact role of astrocytes in prion disease is hampered by the lack of in vitro models of prion-infected astrocytes. Recently, we established a murine astrocyte cell line persistently infected with mouse-adapted prions, and showed how such astrocytes differentially process various prion strains. Considering the complexity of the role of astrocytes in prion pathogenesis, we need more in vitro and in vivo models for exploring the contribution of sub-populations of reactive astrocytes, their differential regulation of signaling cascades, and the interaction with neurons and microglia during prion pathogenesis. This will help to establish novel in vivo models and define new therapeutic targets against prion diseases. In this review, we will discuss the complex role of astrocytes in prion disease, the existing experimental resources, the challenges to analyze the contribution of astrocytes in prion disease pathogenesis, and future strategies to improve the understanding of their role in prion disease.

Oral administration of repurposed drug targeting Cyp46A1 increases survival times of prion infected mice.

Prion diseases are fatal, infectious, and incurable neurodegenerative disorders caused by misfolding of the cellular prion protein (PrPC) into the infectious isoform (PrPSc). In humans, there are sporadic, genetic and infectious etiologies, with sporadic Creutzfeldt-Jakob disease (sCJD) being the most common form. Currently, no treatment is available for prion diseases. Cellular cholesterol is known to impact prion conversion, which in turn results in an accumulation of cholesterol in prion-infected neurons. The major elimination of brain cholesterol is achieved by the brain specific enzyme, cholesterol 24-hydroxylase (CYP46A1). Cyp46A1 converts cholesterol into 24(S)-hydroxycholesterol, a membrane-permeable molecule that exits the brain. We have demonstrated for the first time that Cyp46A1 levels are reduced in the brains of prion-infected mice at advanced disease stage, in prion-infected neuronal cells and in post-mortem brains of sCJD patients. We have employed the Cyp46A1 activator efavirenz (EFV) for treatment of prion-infected neuronal cells and mice. EFV is an FDA approved anti-HIV medication effectively crossing the blood brain barrier and has been used for decades to chronically treat HIV patients. EFV significantly mitigated PrPSc propagation in prion-infected cells while preserving physiological PrPC and lipid raft integrity. Notably, oral administration of EFV treatment chronically at very low dosage starting weeks to months after intracerebral prion inoculation of mice significantly prolonged the lifespan of animals. In summary, our results suggest that Cyp46A1 as a novel therapeutic target and that its activation through repurposing the anti-retroviral medication EFV might be valuable treatment approach for prion diseases.

Early detection of prion protein aggregation with a fluorescent pentameric oligothiophene probe using spectral confocal microscopy.

Misfolding of the prion protein (PrP) and templating of its pathological conformation onto cognate proteins causes a number of lethal disorders of central nervous system in humans and animals, such as Creutzfeldt-Jacob disease, chronic wasting disease and bovine spongiform encephalopathy. Structural rearrangement of PrPC into PrPSc promotes aggregation of misfolded proteins into ß-sheet-rich fibrils, which can be visualized by conformationally sensitive fluorescent probes. Early detection of prion misfolding and deposition might provide useful insights into its pathophysiology. Pentameric formyl thiophene acetic acid (pFTAA) is a novel amyloid probe that was shown to sensitively detect various misfolded proteins, including PrP. Here, we compared sensitivity of pFTAA staining and spectral microscopy with conventional methods of prion detection in mouse brains infected with mouse-adapted 22L prions. pFTAA bound to prion deposits in mouse brain sections exhibited a red-shifted fluorescence emission spectrum, which quantitatively increased with disease progression. Small prion deposits were detected as early as 50 days post-inoculation, well before appearance of clinical signs. Moreover, we detected significant spectral shifts in the greater brain parenchyma as early as 25 days post-inoculation, rivaling the most sensitive conventional method (real-time quaking-induced conversion). These results showcase the potential of pFTAA staining combined with spectral imaging for screening of prion-infected tissue. Not only does this method have comparable sensitivity to established techniques, it is faster and technically simpler. Finally, this readout provides valuable information about the spatial distribution of prion aggregates across tissue in the earliest stages of infection, potentially providing valuable pathophysiological insight into prion transmission.

An astrocyte cell line that differentially propagates murine prions.

Prion diseases are fatal infectious neurodegenerative disorders in human and animals caused by misfolding of the cellular prion protein (PrPC) into the pathological isoform PrPSc Elucidating the molecular and cellular mechanisms underlying prion propagation may help to develop disease interventions. Cell culture systems for prion propagation have greatly advanced molecular insights into prion biology, but translation of in vitro to in vivo findings is often disappointing. A wider range of cell culture systems might help overcome these shortcomings. Here, we describe an immortalized mouse neuronal astrocyte cell line (C8D1A) that can be infected with murine prions. Both PrPC protein and mRNA levels in astrocytes were comparable with those in neuronal and non-neuronal cell lines permitting persistent prion infection. We challenged astrocytes with three mouse-adapted prion strains (22L, RML, and ME7) and cultured them for six passages. Immunoblotting results revealed that the astrocytes propagated 22L prions well over all six passages, whereas ME7 prions did not replicate, and RML prions replicated only very weakly after five passages. Immunofluorescence analysis indicated similar results for PrPSc Interestingly, when we used prion conversion activity as a readout in real-time quaking-induced conversion assays with RML-infected cell lysates, we observed a strong signal over all six passages, comparable with that for 22L-infected cells. These data indicate that the C8D1A cell line is permissive to prion infection. Moreover, the propagated prions differed in conversion and proteinase K-resistance levels in these astrocytes. We propose that the C8D1A cell line could be used to decipher prion strain biology.

The role of cellular prion protein in lipid metabolism in the liver.

Cellular prion protein (PrPC) is a plasma membrane glycophosphatidylinositol-anchored protein and it is involved in multiple functions, including neuroprotection and oxidative stress. So far, most of the PrPC functional research is done in neuronal tissue or cell lines; the role of PrPC in non-neuronal tissues such as liver is only poorly understood. To characterize the role of PrPC in the liver, a proteomics approach was applied in the liver tissue of PrPC knockout mice. The proteome analysis and biochemical validations showed an excessive fat accumulation in the liver of PrPC knockout mice with a change in mRNA expression of genes linked to lipid metabolism. In addition, the higher Bax to Bcl2 ratio, up-regulation of tgfb1 mRNA expression in PrPC knockout mice liver, further showed the evidences of metabolic disease. Over-expression of PrPC in fatty acid-treated AML12 hepatic cell line caused a reduction in excessive intracellular fat accumulation; shows association of PrPC levels and lipid metabolism. Therefore, based on observation of excessive fat globules in the liver of ageing PrPC knockout mice and the reduction of fat accumulation in AML12 cell line with PrPC over-expression, the role of PrPC in lipid metabolism is described.

Combining autophagy stimulators and cellulose ethers for therapy against prion disease.

Prion diseases are fatal transmissible neurodegenerative disorders that affect animals and humans. Prions are proteinaceous infectious particles consisting of a misfolded isoform of the cellular prion protein PrPC, termed PrPSc. PrPSc accumulates in infected neurons due to partial resistance to proteolytic digestion. Using compounds that interfere with the production of PrPSc or enhance its degradation cure prion infection in vitro, but most drugs failed when used to treat prion-infected rodents. In order to synergize the effect of anti-prion drugs, we combined drugs interfering with the generation of PrPSc with compounds inducing PrPSc degradation. Here, we tested autophagy stimulators (rapamycin or AR12) and cellulose ether compounds (TC-5RW or 60SH-50) either as single or combination treatment of mice infected with RML prions. Single drug treatments significantly extended the survival compared to the untreated group. As anticipated, also all the combination therapy groups showed extended survival compared to the untreated group, but no combination treatment showed superior effects to 60SH-50 or TC-5RW treatment alone. Unexpectedly, we later found that combining autophagy stimulator and cellulose ether treatment in cultured neuronal cells mitigated the pro-autophagic activity of AR12 and rapamycin, which can in part explain the in vivo results. Overall, we show that it is critical to exclude antagonizing drug effects when attempting combination therapy. In addition, we identified AR-12 as a pro-autophagic drug that significantly extends survival of prion-infected mice, has no adverse side effects on the animals used in this study, and can be useful in future studies.

Cytoskeleton-Associated Risk Modifiers Involved in Early and Rapid Progression of Sporadic Creutzfeldt-Jakob Disease.

A high priority in the prion field is to identify pre-symptomatic events and associated profile of molecular changes. In this study, we demonstrate the pre-symptomatic dysregulation of cytoskeleton assembly and its associated cofilin-1 pathway in strain and brain region-specific manners in MM1 and VV2 subtype-specific Creutzfeldt-Jakob disease at clinical and pre-clinical stage. At physiological level, PrPC interaction with cofilin-1 and phosphorylated form of cofilin (p-cofilin(Ser3)) was investigated in primary cultures of mouse cortex neurons (PCNs) of PrPC wild-type and knockout mice (PrP-/-). Short-interfering RNA downregulation of active form of cofilin-1 resulted in the redistribution/downregulation of PrPC, increase of activated form of microglia, accumulation of dense form of F-actin, and upregulation of p-cofilin(Ser3). This upregulated p-cofilin(Ser3) showed redistribution of expression predominantly in the activated form of microglia in PCNs. At pathological level, cofilin-1 expression was significantly altered in cortex and cerebellum in both humans and mice at pre-clinical stage and at early symptomatic clinical stage of the disease. Further, to better understand the possible mechanism of dysregulation of cofilin-1, we also demonstrated alterations in upstream regulators; LIM kinase isoform 1 (LIMK1), slingshot phosphatase isoform 1 (SSH1), RhoA-associated kinase (Rock2), and amyloid precursor protein (APP) in sporadic Creutzfeldt-Jakob disease MM1 mice and in human MM1 and VV2 frontal cortex and cerebellum samples. In conclusion, our findings demonstrated for the first time a key pre-clinical response of cofilin-1 and the associated pathway in prion disease.

Molecular Alterations in the Cerebellum of Sporadic Creutzfeldt-Jakob Disease Subtypes with DJ-1 as a Key Regulator of Oxidative Stress.

Cerebellar damage and granular and Purkinje cell loss in sporadic Creutzfeldt-Jakob disease (sCJD) highlight a critical involvement of the cerebellum during symptomatic progression of the disease. In this project, global proteomic alterations in the cerebellum of brain from the two most prevalent subtypes (MM1 and VV2) of sCJD were studied. Two-dimensional gel electrophoresis (2DE) coupled mass spectrometric identification revealed 40 proteins in MM1 and 43 proteins in VV2 subtype to be differentially expressed. Of those, 12 proteins showed common differential expression in their expression between two subtypes. Differentially expressed proteins mainly belonged to (i) cell cycle, gene expression and cell death; (ii) cellular stress response/oxidative stress (OS) and (iii) signal transduction and synaptic functions, related molecular functions. We verified 10 differentially expressed proteins at transcriptional and translational level as well. Interestingly, protein deglycase DJ-1 (an antioxidative protein) showed an increase in its messenger RNA (mRNA) expression in both MM1 and VV2 subtypes but protein expression only in VV2 subtype in cerebellum of sCJD patients. Nuclear translocalization of DJ-1 confirmed its expressional alteration due to OS in sCJD. Downstream experiments showed the activation of nuclear factor erythroid-2 related factor 2 (Nrf2)/antioxidative response element (ARE) pathway. DJ-1 protein concentration was significantly increased during the clinical phase in cerebrospinal fluid of sCJD patients and also at presymptomatic and symptomatic stages in cerebellum of humanized PrP transgenic mice inoculated with sCJD (MM1 and VV2) brain. These results suggest the implication of oxidative stress during the pathophysiology of sCJD.

YKL-40 in the brain and cerebrospinal fluid of neurodegenerative dementias.

BACKGROUND: YKL-40 (also known as Chitinase 3-like 1) is a glycoprotein produced by inflammatory, cancer and stem cells. Its physiological role is not completely understood but YKL-40 is elevated in the brain and cerebrospinal fluid (CSF) in several neurological and neurodegenerative diseases associated with inflammatory processes. Yet the precise characterization of YKL-40 in dementia cases is missing. METHODS: In the present study, we comparatively analysed YKL-40 levels in the brain and CSF samples from neurodegenerative dementias of different aetiologies characterized by the presence of cortical pathology and disease-specific neuroinflammatory signatures. RESULTS: YKL-40 was normally expressed in fibrillar astrocytes in the white matter. Additionally YKL-40 was highly and widely expressed in reactive protoplasmic cortical and perivascular astrocytes, and fibrillar astrocytes in sporadic Creutzfeldt-Jakob disease (sCJD). Elevated YKL-40 levels were also detected in Alzheimer's disease (AD) but not in dementia with Lewy bodies (DLB). In AD, YKL-40-positive astrocytes were commonly found in clusters, often around ß-amyloid plaques, and surrounding vessels with ß-amyloid angiopathy; they were also distributed randomly in the cerebral cortex and white matter. YKL-40 overexpression appeared as a pre-clinical event as demonstrated in experimental models of prion diseases and AD pathology. CSF YKL-40 levels were measured in a cohort of 288 individuals, including neurological controls (NC) and patients diagnosed with different types of dementia. Compared to NC, increased YKL-40 levels were detected in sCJD (p < 0.001, AUC = 0.92) and AD (p < 0.001, AUC = 0.77) but not in vascular dementia (VaD) (p > 0.05, AUC = 0.71) or in DLB/Parkinson's disease dementia (PDD) (p > 0.05, AUC = 0.70). Further, two independent patient cohorts were used to validate the increased CSF YKL-40 levels in sCJD. Additionally, increased YKL-40 levels were found in genetic prion diseases associated with the PRNP-D178N (Fatal Familial Insomnia) and PRNP-E200K mutations. CONCLUSIONS: Our results unequivocally demonstrate that in neurodegenerative dementias, YKL-40 is a disease-specific marker of neuroinflammation showing its highest levels in prion diseases. Therefore, YKL-40 quantification might have a potential for application in the evaluation of therapeutic intervention in dementias with a neuroinflammatory component.

MicroRNA Expression in the Locus Coeruleus, Entorhinal Cortex, and Hippocampus at Early and Middle Stages of Braak Neurofibrillary Tangle Pathology.

The present study analyzes by RT-qPCR the expression of microRNA (miRNA)-27a-3p, miRNA-124-3p, miRNA-132-3p, and miRNA-143-3p in the locus coeruleus (LC), entorhinal cortex (EC), CA1 region of the hippocampus (CA1), and dentate gyrus (DG) of middle-aged (MA) individuals with no brain lesions and of cases at Braak and Braak stages I-II and II-IV of neurofibrillary tangle (NFT) pathology. The most affected region is the LC in which miRNA-27a-3p, miRNA-124-3p, and miRNA-143-3p show a trend to increase at stages I-II and are significantly up-regulated at stages III-IV when compared with MA. Only miRNA-143-3p is up-regulated in the EC at stages III-IV when compared with MA and with stages I-II. No modifications in the expression levels of miRNA-27a-3p, miRNA-124-3p, miRNA-132-3p, and miRNA-143-3p are found in CA1 at any stage, whereas miRNA-124-3p is significantly down-regulated in DG at stages I-II. Accompanying in situ hybridization reveals miRNA-27a-3p, miRNA-124-3p, and miRNA-143-3 localization in neurons, indicating that changes in miRNA expression are not a direct effect of changes in the numbers of neurons and glial cells. Present observations show for the first time important miRNA de-regulation in the LC at the first stages of NFT. Since the LC is the main noradrenergic input to the cerebral cortex, key regulator of mood and depression, and one of the first nuclei affected in aging and Alzheimer's disease (AD), these findings provide insights for additional study of the LC in aging and AD.

Altered Ca2+ homeostasis induces Calpain-Cathepsin axis activation in sporadic Creutzfeldt-Jakob disease.

Sporadic Creutzfeldt-Jakob disease (sCJD) is the most prevalent form of human prion disease and it is characterized by the presence of neuronal loss, spongiform degeneration, chronic inflammation and the accumulation of misfolded and pathogenic prion protein (PrPSc). The molecular mechanisms underlying these alterations are largely unknown, but the presence of intracellular neuronal calcium (Ca2+) overload, a general feature in models of prion diseases, is suggested to play a key role in prion pathogenesis.Here we describe the presence of massive regulation of Ca2+ responsive genes in sCJD brain tissue, accompanied by two Ca2+-dependent processes: endoplasmic reticulum stress and the activation of the cysteine proteases Calpains 1/2. Pathogenic Calpain proteins activation in sCJD is linked to the cleavage of their cellular substrates, impaired autophagy and lysosomal damage, which is partially reversed by Calpain inhibition in a cellular prion model. Additionally, Calpain 1 treatment enhances seeding activity of PrPSc in a prion conversion assay. Neuronal lysosomal impairment caused by Calpain over activation leads to the release of the lysosomal protease Cathepsin S that in sCJD mainly localises in axons, although massive Cathepsin S overexpression is detected in microglial cells. Alterations in Ca2+ homeostasis and activation of Calpain-Cathepsin axis already occur at pre-clinical stages of the disease as detected in a humanized sCJD mouse model.Altogether our work indicates that unbalanced Calpain-Cathepsin activation is a relevant contributor to the pathogenesis of sCJD at multiple molecular levels and a potential target for therapeutic intervention.

Strain-Specific Altered Regulatory Response of Rab7a and Tau in Creutzfeldt-Jakob Disease and Alzheimer's Disease.

There is an increasing demand for the understanding of pathophysiology on neurodegeneration diseases at early stages. Changes in endocytic machinery and the cytoskeleton-associated response are the first alterations observed in Creutzfeldt-Jakob disease (CJD) and Alzheimer's disease AD brain. In this study, we performed a targeted search for endocytic pathway proteins in the different regions of the brain. We found late endosome marker Rab7a which was significantly upregulated in the frontal cortex region in the rapid progressive CJD form (MM1) and rapid progressive AD (rpAD) forms. However, Rab9 expression was significantly downregulated only in CJD-MM1 brain frontal cortex region. In the cerebellum, Rab7a expression showed significant upregulation in both subtype MM1 and VV2 CJD forms, in contrast to Rab9 which showed significant downregulation in both subtype MM1 and VV2 CJD forms at terminal stage of the disease. To check regulatory response at pre-symptomatic stage of the disease, we checked the regulatory interactive response of Rab7a, Rab9, and known biomarkers PrPC and tau forms in frontal cortex at pre-symptomatic stage of the disease in tg340 mice expressing about fourfold of human PrP-M129 with PrP-null background that had been inoculated with human sCJD MM1 brain tissue homogenates (sCJD MM1 mice). In addition, we analyzed 5XFAD mice, exhibiting five mutations in the APP and presenilin genes related to familial Alzheimer's disease (FAD), to validate specific regulatory response of Rab7a, Rab9, tau, and phosphorylated form of tau by immunostaining 5XFAD mice in comparison with the wild-type age-matched mice brain. The cortical region of 5XFAD mice brain showed accumulated form of Rab7a in puncta that co-label for p-Tau, indicating colocalization by using confocal laser-scanning microscopy and was confirmed by using reverse co-immunoprecipitation. Furthermore, synthetic RNA (siRNA) against the Rab7a gene decreased expression of Rab7a protein, in cortical primary neuronal cultures of PrPC wild type. This depleted expression of Rab7a led to the increased accumulation of PrPC in Rab9-positive endosomal compartments and consequently an increased co-localization between PrPC/Rab9; however, total tau level decreased. Interestingly, siRNA against tau gene in cortical primary neuronal cultures of PrPC wild-type mice showed enhanced Rab7a and Rab9 expression and increase formation of dendritic spines. The work described highlighted the selective involvement of late endosomal compartment marker Rab7a in CJD, slow and rapid progressive forms of AD pathogenesis.

Identification of new molecular alterations in fatal familial insomnia.

Fatal familial insomnia is a rare disease caused by a D178N mutation in combination with methionine (Met) at codon 129 in the mutated allele of PRNP (D178N-129M haplotype). FFI is manifested by sleep disturbances with insomnia, autonomic disorders and spontaneous and evoked myoclonus, among other symptoms. This study describes new neuropathological and biochemical observations in a series of eight patients with FFI. The mediodorsal and anterior nuclei of the thalamus have severe neuronal loss and marked astrocytic gliosis in every case, whereas the entorhinal cortex is variably affected. Spongiform degeneration only occurs in the entorhinal cortex. Synaptic and fine granular proteinase K digestion (PrPres) immunoreactivity is found in the entorhinal cortex but not in the thalamus. Interleukin 6, interleukin 10 receptor alpha subunit, colony stimulating factor 3 receptor and toll-like receptor 7 mRNA expression increases in the thalamus in FFI. PrPc levels are significantly decreased in the thalamus, entorhinal cortex and cerebellum in FFI. This is accompanied by a particular PrPc and PrPres band profile. Altered PrP solubility consistent with significantly reduced PrP levels in the cytoplasmic fraction and increased PrP levels in the insoluble fraction are identified in FFI cases. Amyloid-like deposits are only seen in the entorhinal cortex. The RT-QuIC assay reveals that all the FFI samples of the entorhinal cortex are positive, whereas the thalamus is positive only in three cases and the cerebellum in two cases. The present findings unveil particular neuropathological and neuroinflammatory profiles in FFI and novel characteristics of natural prion protein in FFI, altered PrPres and Scrapie PrP (abnormal and pathogenic PrP) patterns and region-dependent putative capacity of PrP seeding.

Application of capillary immunoelectrophoresis revealed an age- and gender-dependent regulated expression of PrPC in liver.

The cellular prion protein (PrPC) is a glycoprotein, anchored to the plasma membrane and abundantly expressed in the central nervous system. The expression of PrPC in the peripheral tissues is low and only little information is available on its functions in the nonneuronal tissues. The antioxidant function of PrPC during the activation of hepatic stellate cells has already been reported. Therefore, the aim of the study was to expand our knowledge on the functions of PrPC by detailed characterization of its expressional profile in the liver. In a combined strategy by using capillary immunoelectrophoresis and standard techniques, we have shown a sexually dimorphic expression of PrPC in mice and human liver tissues. Further, we showed a significant age-dependent upregulation of PrPC expression in the liver of 14- and 9-month-old mice as compared to 3 months of age. Therefore, this study may provide new insights into the gender-specific role of PrPC in the liver, which may further be linked to its protective role against oxidative stress during aging. In addition, the current study also shows an application of immunoelectrophoresis with a low coefficient of variation to analyze the miniscule amount of PrPC in the mouse liver tissue.

Creutzfeldt-Jakob Disease Subtype-Specific Regional and Temporal Regulation of ADP Ribosylation Factor-1-Dependent Rho/MLC Pathway at Pre-Clinical Stage.

Small GTPases of the Arf family mainly activate the formation of coated carrier vesicles. We showed that class-I Arf1 interacts specifically with full length GPI-anchored cellular prion protein (PrP(C)). Several recent reports have also demonstrated a missing link between the endoplasmic reticulum and the Golgi-complex role for proper folding, but the exact molecular mechanism is not yet fully understood. In the present study, we identified and characterized the interactive role of Arf1 during PrP(C) intracellular distribution under pathophysiological conditions. PrP(C) interaction with Arf1 was investigated in cortical primary neuronal cultures of PrP(C) wild type and knockout mice (PrP(-/-)). Arf1 and PrP(C) co-binding affinity was confirmed using reverse co-immunoprecipitation, co-localization affinity using confocal laser-scanning microscopy. Treatment with brefeldin-A modulated Arf1 expression and resulted in down-regulation and redistribution of PrP(C) into cytosolic region. In the pre-symptomatic stage of the disease, Arf1 expression was significantly downregulated in the frontal cortex in tg340 mice expressing about fourfold of human PrP-M129 with PrP null background that had been inoculated with human sCJD MM1 brain tissue homogenates (sCJD MM1 mice). In addition, the frontal cortex of CJD human brain demonstrated significant binding capacity of Arf1 protein using co-immunoprecipitation analysis. We also examined Arf1 expression in the brain of CJD patients with the subtypes MM1 and VV2 and found that it was regulated in a region-specific manner. In the frontal cortex, Arf1 expression was not significantly changed in either MM1 or VV2 subtype. Interestingly, Arf1 expression was significantly reduced in the cerebellum in both subtypes as compared to controls. Furthermore, we observed altered RhoA activity, which in turn affects myosin light-chain (MLC) phosphorylation and Arf1-dependent PI3K pathway. Together, our findings underscore a key early symptomatic role of Arf1 in neurodegeneration. Targeting the Arf/Rho/MLC signaling axis might be a promising strategy to uncover the missing link which probably influences disease progression and internal homeostasis of misfolded proteins.

Anchorless 23-230 PrPC interactomics for elucidation of PrPC protective role.

Accumulation of conformationally altered cellular proteins (i.e., prion protein) is the common feature of prions and other neurodegenerative diseases. Previous studies demonstrated that the lack of terminal sequence of cellular prion protein (PrPC), necessary for the addition of glycosylphosphatidylinositol lipid anchor, leads to a protease-resistant conformation that resembles scrapie-associated isoform of prion protein. Moreover, mice overexpressing the truncated form of PrPC showed late-onset, amyloid deposition, and the presence of a short protease-resistant PrP fragment in the brain similar to those found in Gerstmann-Sträussler-Scheinker disease patients. Therefore, the physiopathological function of truncated_/anchorless 23-230 PrPC (Δ23-230 PrPC) has come into focus of attention. The present study aims at revealing the physiopathological function of the anchorless PrPC form by identifying its interacting proteins. The truncated_/anchorless Δ23-230 PrPC along with its interacting proteins was affinity purified using STrEP-Tactin chromatography, in-gel digested, and identified by quadrupole time-of-flight tandem mass spectrometry analysis in prion protein-deficient murine hippocampus (HpL3-4) neuronal cell line. Twenty-three proteins appeared to interact with anchorless Δ23-230 PrPC in HpL3-4 cells. Out of the 23 proteins, one novel protein, pyruvate kinase isozymes M1/M2 (PKM2), exhibited a potential interaction with the anchorless Δ23-230 form of PrPC. Both reverse co-immunoprecipitation and confocal laser-scanning microscopic analysis confirmed an interaction of PKM2 with the anchorless Δ23-230 form of PrPC. Furthermore, we provide the first evidence for co-localization of PKM2 and PrPC as well as PrPC-dependent PKM2 expression regulation. In addition, given the involvement of PrPC in the regulation of apoptosis, we exposed HpL3-4 cells to staurosporine (STS)-mediated apoptotic stress. In response to STS-mediated apoptotic stress, HpL3-4 cells transiently expressing 23-230-truncated PrPC were markedly less viable, were more prone to apoptosis and exhibited significantly higher PKM2 expressional regulation as compared with HpL3-4 cells transiently expressing full-length PrPC (1-253 PrPC). The enhanced STS-induced apoptosis was shown by increased caspase-3 cleavage. Together, our data suggest that the misbalance or over expression of anchorless Δ23-230 form of PrPC in association with the expressional regulation of interacting proteins could render cells more prone to cellular insults-stress response, formation of aggregates and may ultimately be linked to the cell death.