Ethanolamine Is a New Anti-Prion Compound

Prion diseases are a group of fatal neurodegenerative disorders caused by accumulation of proteinaceous infectious particles, or prions, which mainly consist of the abnormally folded, amyloidogenic prion protein, designated PrP^Sc. PrP^Sc is produced through conformational conversion of the cellular isoform of prion protein, PrP^C, in the brain. To date, no effective therapies for prion diseases have been developed. In this study, we incidentally noticed that mouse neuroblastoma N2a cells persistently infected with 22L scrapie prions, termed N2aC24L1-3 cells, reduced PrP^Sc levels when cultured in advanced Dulbecco’s modified eagle medium (DMEM) but not in classic DMEM. PrP^C levels remained unchanged in prion-uninfected parent N2aC24 cells cultured in advanced DMEM. These results suggest that advanced DMEM may contain an anti-prion compound(s). We then successfully identified ethanolamine in advanced DMEM has an anti-prion activity. Ethanolamine reduced PrP^Sc levels in N2aC24L1-3 cells, but not PrP^C levels in N2aC24 cells. Also, oral administration of ethanolamine through drinking water delayed prion disease in mice intracerebrally inoculated with RML scrapie prions. These results suggest that ethanolamine could be a new anti-prion compound.     

Cervid Prion Protein Polymorphisms: Role in Chronic Wasting Disease Pathogenesis

Chronic wasting disease (CWD) is a prion disease found in both free-ranging and farmed cervids. Susceptibility of these animals to CWD is governed by various exogenous and endogenous factors. Past studies have demonstrated that polymorphisms within the prion protein (PrP) sequence itself affect an animal’s susceptibility to CWD. PrP polymorphisms can modulate CWD pathogenesis in two ways: the ability of the endogenous prion protein (PrP^C) to convert into infectious prions (PrP^Sc) or it can give rise to novel prion strains. In vivo studies in susceptible cervids, complemented by studies in transgenic mice expressing the corresponding cervid PrP sequence, show that each polymorphism has distinct effects on both PrP^C and PrP^Sc. It is not entirely clear how these polymorphisms are responsible for these effects, but in vitro studies suggest they play a role in modifying PrP epitopes crucial for PrP^C to PrP^Sc conversion and determining PrP^C stability. PrP polymorphisms are unique to one or two cervid species and most confer a certain degree of reduced susceptibility to CWD. However, to date, there are no reports of polymorphic cervid PrP alleles providing absolute resistance to CWD. Studies on polymorphisms have focused on those found in CWD-endemic areas, with the hope that understanding the role of an animal’s genetics in CWD can help to predict, contain, or prevent transmission of CWD.     

Prion-Associated Neurodegeneration Causes Both Endoplasmic Reticulum Stress and Proteasome Impairment in a Murine Model of Spontaneous Disease

Prion diseases are a group of neurodegenerative disorders that can be spontaneous, familial or acquired by infection. The conversion of the prion protein PrP^C to its abnormal and misfolded isoform PrP^Sc is the main event in the pathogenesis of prion diseases of all origins. In spontaneous prion diseases, the mechanisms that trigger the formation of PrP^Sc in the central nervous system remain unknown. Several reports have demonstrated that the accumulation of PrP^Sc can induce endoplasmic reticulum (ER) stress and proteasome impairment from the early stages of the prion disease. Both mechanisms lead to an increment of PrP aggregates in the secretory pathway, which could explain the pathogenesis of spontaneous prion diseases. Here, we investigate the role of ER stress and proteasome impairment during prion disorders in a murine model of spontaneous prion disease (TgVole) co-expressing the Ub^G76V-GFP reporter, which allows measuring the proteasome activity in vivo. Spontaneously prion-affected mice showed a significantly higher accumulation of the PKR-like ER kinase (PERK), the ER chaperone binding immunoglobulin protein (BiP/Grp78), the ER protein disulfide isomerase (PDI) and the Ub^G76V-GFP reporter than age-matched controls in certain brain areas. The upregulation of PERK, BiP, PDI and ubiquitin was detected from the preclinical stage of the disease, indicating that ER stress and proteasome impairment begin at early stages of the spontaneous disease. Strong correlations were found between the deposition of these markers and neuropathological markers of prion disease in both preclinical and clinical mice. Our results suggest that both ER stress and proteasome impairment occur during the pathogenesis of spontaneous prion diseases.     

Strain Typing of Classical Scrapie and Bovine Spongiform Encephalopathy (BSE) by Using Ovine PrP (ARQ/ARQ) Overexpressing Transgenic Mice

Transmissible spongiform encephalopathies (TSE), caused by abnormal prion protein (PrP^Sc), affect many species. The most classical scrapie isolates harbor mixtures of strains in different proportions. While the characterization of isolates has evolved from using wild-type mice to transgenic mice, no standardization is established yet. Here, we investigated the incubation period, lesion profile and PrP^Sc profile induced by well-defined sheep scrapie isolates, bovine spongiform encephalopathy (BSE) and ovine BSE after intracerebral inoculation into two lines of ovine PrP (both ARQ/ARQ) overexpressing transgenic mice (Tgshp IX and Tgshp XI). All isolates were transmitted to both mouse models with an attack rate of almost 100%, but genotype-dependent differences became obvious between the ARQ and VRQ isolates. Surprisingly, BSE induced a much longer incubation period in Tgshp XI compared to Tgshp IX. In contrast to the histopathological lesion profiles, the immunohistochemical PrP^Sc profiles revealed discriminating patterns in certain brain regions in both models with clear differentiation of both BSE isolates from scrapie. These data provide the basis for the use of Tgshp IX and XI mice in the characterization of TSE isolates. Furthermore, the results enable a deeper appreciation of TSE strain diversity using ovine PrP overexpressing transgenic mice as a biological prion strain typing approach.     

Discovery of a Class of Diketopiperazines as Antiprion Compounds

Prion diseases are fatal neurodegenerative and infectious disorders for which effective pharmacological tools are not yet available. This unmet challenge and the recently proposed interplay between prion diseases and Alzheimer's have led to a more urgent demand for new antiprion agents. Herein, we report the identification of a novel bifunctional diketopiperazine (DKP) derivative 1 d , which exhibits activity in the low micromolar range against prion replication in ScGT1 cells, while showing low cytotoxicity. Supported by properly addressed molecular modeling studies, we hypothesized that a planar conformation is the major determinant for activity in this class of compounds. Moreover, studies aimed at assessing the mechanism‐of‐action at the molecular level showed that 1 d might interact directly with recombinant prion protein (recPrP) to prevent its conversion to the pathogenic misfolded prion protein (PrP^Sc)‐like form. This investigation suggests that DKP based antiprion compounds can serve as a promising lead scaffold in developing new drugs to combat prion diseases.     

Prion protein gene expression in cultured cells

A single copy gene encodes both the scrapie (PrP^Sc) and cellular(PrP^C) isoforms of the prion protein (PrP). Cultured cell lineswere found to express the endogenous PrP mRNA at levels comparableto those observed in the brains of adult rodents; however, thesecells were invariably found to express greatly reduced levelsof PrP. In all the cell lines examined, PrP was undetectableby Western immunoblot analysis. These cells were also poor recipientsfor expression constructs linking the hamster PrP gene openreading frame to several strong eukaryotic promoters; stableclones derived by transfection of these expression vectors failedto show elevated expression of PrP. When extremely high levelsof PrP mRNA were produced using either an insect baculovirusor a mammalian SV40 based vector, significant quantities ofPrP were produced, although in both cases the proteins wereapparently processed differently from the PrP^C observed in brains.In an expression system using an SV40 late promoter vector inmonkey COS-7 cells, a significant fraction of PrP was transportedto the cell surface where PrP^C is found in vivo. PrP synthesizedby the baculovirus vector failed to induce scrapie in hamstersand did not possess the characteristics of the PrP^Sc isoformassociated with infectivity. The SV40 late promoter vector systemmay permit experiments designed to elucidate the role of PrP^Scduring scrapie infection as well as the function of PrP^C innormal metabolism.     

Structural Mechanism of Barriers to Interspecies Seeding Transmissibility of Full-Length Prion Protein Amyloid

A puzzling feature of prion diseases is the cross-species barriers. The detailed molecular mechanisms underlying these interspecies barriers remain poorly understood because of a lack of high-resolution structural information on the scrapie isoform of the prion protein (PrP^Sc). In this study we identified the critical role of the residues 165/167 in the barrier to seeding mouse PrP (mPrP) fibril seeds to human cellular prion protein (PrP^C). Solid-state NMR revealed a C-terminal β-sheet core spanning residues 165–230 and the packing arrangement of mPrP fibrils. Residues 165/167 are located on one end of the fibril core. Molecular dynamics simulations demonstrated that the stabilities of the seeding-induced β-strand structures are significantly impacted by hydrogen bonds involving the side chain of residue 167 and steric resistance involving residue 165. These findings suggest that the α2–β2 loop containing residues 165/167 could be the initial site of seed–template conformational conversion.     

High-level expression and characterization of a glycosylated covalently linked dimer of the prion protein

There is evidence that prion protein dimers may be involvedin the formation of the scrapie prion protein, PrP^Sc, from itsnormal (cellular) form, PrP^c. Recently, the crystal structureof the human prion protein in a dimeric form was reported. Herewe report for the first time the overexpression of a human PrPdimer covalently linked by a FLAG peptide (PrP::FLAG::PrP) inthe methylotrophic yeast Pichia pastoris. FLAG-tagged humanPrP (aa1-aa253) (huPrP::FLAG) was also expressed in the samesystem. Treatment with tunicamycin and endoglycosidase H showedthat both fusion proteins are expressed as various glycoforms.Both PrP proteins were completely digested by proteinase K (PK),suggesting that the proteins do not have a PrP^Sc structure andare not infectious. Plasma membrane fractionation revealed thatboth proteins are transported to the plasma membrane of thecell. The glycosylated proteins might act as powerful toolsfor crystallization trials, PrP^c/PrP^Sc conversion studies andother applications in the life cycle of prions.     

Circulation of Nor98 Atypical Scrapie in Portuguese Sheep Confirmed by Transmission of Isolates into Transgenic Ovine ARQ-PrP Mice

Portugal was among the first European countries to report cases of Atypical Scrapie (ASc), the dominant form of Transmissible Spongiform Encephalopathy (TSE) in Portuguese small ruminants. Although the diagnostic phenotypes observed in Portuguese ASc cases seem identical to those described for Nor98, unequivocal identification requires TSE strain-typing using murine bioassays. In this regard, we initiated characterization of ASc isolates from sheep either homozygous for the ARQ genotype or the classical scrapie-resistant ARR genotype. Isolates from such genotypes were transmitted to TgshpXI mice expressing ovine PrPARQ. Mean incubation periods were 414 ± 58 and 483 ± 107 days in mice inoculated with AL₁₄₁RQ/AF₁₄₁RQ and AL₁₄₁RR/AL₁₄₁RR sheep isolates, respectively. Both isolates produced lesion profiles similar to French ASc Nor98 ‘discordant cases’, where vacuolation was observed in the hippocampus (G6), cerebral cortex at the thalamus (G8) level, cerebellar white matter (W1) and cerebral peduncles (W3). Immunohistochemical PrP^Sc deposition was observed in the hippocampus, cerebellar cortex, cerebellar white matter and cerebral peduncles in the form of aggregates and fine granules. These findings were consistent with previously reported cases of ASc Nor98 transmitted to transgenic TgshpXI mice, confirming that the ASc strain present in Portuguese sheep corresponds to ASc Nor98.     

How an Infection of Sheep Revealed Prion Mechanisms in Alzheimer’s Disease and Other Neurodegenerative Disorders

Although it is not yet universally accepted that all neurodegenerative diseases (NDs) are prion disorders, there is little disagreement that Alzheimer’s disease (AD), Parkinson’s disease, frontotemporal dementia (FTD), and other NDs are a consequence of protein misfolding, aggregation, and spread. This widely accepted perspective arose from the prion hypothesis, which resulted from investigations on scrapie, a common transmissible disease of sheep and goats. The prion hypothesis argued that the causative infectious agent of scrapie was a novel proteinaceous pathogen devoid of functional nucleic acids and distinct from viruses, viroids, and bacteria. At the time, it seemed impossible that an infectious agent like the one causing scrapie could replicate and exist as diverse microbiological strains without nucleic acids. However, aggregates of a misfolded host-encoded protein, designated the prion protein (PrP), were shown to be the cause of scrapie as well as Creutzfeldt–Jakob disease (CJD) and Gerstmann–Sträussler–Scheinker syndrome (GSS), which are similar NDs in humans. This review discusses historical research on diseases caused by PrP misfolding, emphasizing principles of pathogenesis that were later found to be core features of other NDs. For example, the discovery that familial prion diseases can be caused by mutations in PrP was important for understanding prion replication and disease susceptibility not only for rare PrP diseases but also for far more common NDs involving other proteins. We compare diseases caused by misfolding and aggregation of APP-derived Aβ peptides, tau, and α-synuclein with PrP prion disorders and argue for the classification of NDs caused by misfolding of these proteins as prion diseases. Deciphering the molecular pathogenesis of NDs as prion-mediated has provided new approaches for finding therapies for these intractable, invariably fatal disorders and has revolutionized the field.     

The Cellular Prion Protein: A Promising Therapeutic Target for Cancer

Studies on the cellular prion protein (PrP^C) have been actively conducted because misfolded PrP^C is known to cause transmissible spongiform encephalopathies or prion disease. PrP^C is a glycophosphatidylinositol-anchored cell surface glycoprotein that has been reported to affect several cellular functions such as stress protection, cellular differentiation, mitochondrial homeostasis, circadian rhythm, myelin homeostasis, and immune modulation. Recently, it has also been reported that PrP^C mediates tumor progression by enhancing the proliferation, metastasis, and drug resistance of cancer cells. In addition, PrP^C regulates cancer stem cell properties by interacting with cancer stem cell marker proteins. In this review, we summarize how PrP^C promotes tumor progression in terms of proliferation, metastasis, drug resistance, and cancer stem cell properties. In addition, we discuss strategies to treat tumors by modulating the function and expression of PrP^C via the regulation of HSPA1L/HIF-1α expression and using an anti-prion antibody.     

Tau Exon 10 Inclusion by PrPC through Downregulating GSK3β Activity

Tau protein is largely responsible for tauopathies, including Alzheimer’s disease (AD), where it accumulates in the brain as insoluble aggregates. Tau mRNA is regulated by alternative splicing, and inclusion or exclusion of exon 10 gives rise to the 3R and 4R isoforms respectively, whose balance is physiologically regulated. In this sense, one of the several factors that regulate alternative splicing of tau is GSK3β, whose activity is inhibited by the cellular prion protein (PrP^C), which has different physiological functions in neuroprotection and neuronal differentiation. Moreover, a relationship between PrP^C and tau expression levels has been reported during AD evolution. For this reason, in this study we aimed to analyze the role of PrP^C and the implication of GSK3β in the regulation of tau exon 10 alternative splicing. We used AD human samples and mouse models of PrP^C ablation and tau overexpression. In addition, we used primary neuronal cultures to develop functional studies. Our results revealed a paralleled association between PrP^C expression and tau 4R isoforms in all models analyzed. In this sense, reduction or ablation of PrP^C levels induces an increase in tau 3R/4R balance. More relevantly, our data points to GSK3β activity downstream from PrP^C in this phenomenon. Our results indicate that PrP^C plays a role in tau exon 10 inclusion through the inhibitory capacity of GSK3β.     

TRAIL Mediates Neuronal Death in AUD: A Link between Neuroinflammation and Neurodegeneration

Although the cause of progressive neurodegeneration is often unclear, neuronal death can occur through several mechanisms. In conditions such as Alzheimer’s or alcohol use disorder (AUD), Toll-like receptor (TLR) induction is observed with neurodegeneration. However, links between TLR activation and neurodegeneration are lacking. We report a role of apoptotic neuronal death in AUD through TLR7-mediated induction of death receptor signaling. In postmortem human cortex, a two-fold increase in apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining in neurons was found in AUD versus controls. This occurred with the increased expression of TLR7 and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) death receptors. Binge ethanol treatment in C57BL/6 mice increased TLR7 and induced neuronal apoptosis in cortical regions that was blocked by TLR7 antagonism. Mechanistic studies in primary organotypic brain slice culture (OBSC) found that the inhibition of TLR7 and its endogenous ligand let-7b blocked ethanol-induced neuronal cell death. Both IMQ and ethanol induced the expression of TRAIL and its death receptor. In addition, TRAIL-neutralizing monoclonal antibodies blocked both imiquimod (IMQ) and ethanol induced neuronal death. These findings implicate TRAIL as a mediator of neuronal apoptosis downstream of TLR7 activation. TLR7 and neuronal apoptosis are implicated in other neurodegenerative diseases, including Alzheimer’s disease. Therefore, TRAIL may represent a therapeutic target to slow neurodegeneration in multiple diseases.     

Altered mRNA and Protein Expression of Monocarboxylate Transporter MCT1 in the Cerebral Cortex and Cerebellum of Prion Protein Knockout Mice

The effect of a cellular prion protein (PrP^c) deficiency on neuroenergetics was primarily analyzed via surveying the expression of genes specifically involved in lactate/pyruvate metabolism, such as monocarboxylate transporters (MCT1, MCT2, MCT4). The aim of the present study was to elucidate a potential involvement of PrP^c in the regulation of energy metabolism in different brain regions. By using quantitative real-time polymerase chain reaction (qRT-PCR), we observed a marked reduction in MCT1 mRNA expression in the cortex of symptomatic Zürich I Prnp^−/− mice, as compared to their wild-type (WT) counterparts. MCT1 downregulation in the cortex was accompanied with significantly decreased expression of the MCT1 functional interplayer, the Na⁺/K⁺ ATPase α2 subunit. Conversely, the MCT1 mRNA level was significantly raised in the cerebellum of Prnp^−/− vs. WT control group, without a substantial change in the Na⁺/K⁺ ATPase α2 subunit expression. To validate the observed mRNA findings, we confirmed the observed change in MCT1 mRNA expression level in the cortex at the protein level. MCT4, highly expressed in tissues that rely on glycolysis as an energy source, exhibited a significant reduction in the hippocampus of Prnp^−/− vs. WT mice. The present study demonstrates that a lack of PrP^c leads to altered MCT1 and MCT4 mRNA/protein expression in different brain regions of Prnp^−/− vs. WT mice. Our findings provide evidence that PrP^c might affect the monocarboxylate intercellular transport, which needs to be confirmed in further studies.     

Single-Chain Fragment Variable Passive Immunotherapies for Neurodegenerative Diseases

Accumulation of misfolded proteins has been implicated in a variety of neurodegenerative diseases including prion diseases, Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD). In the past decade, single-chain fragment variable (scFv) -based immunotherapies have been developed to target abnormal proteins or various forms of protein aggregates including Aβ, SNCA, Htt, and PrP proteins. The scFvs are produced by fusing the variable regions of the antibody heavy and light chains, creating a much smaller protein with unaltered specificity. Because of its small size and relative ease of production, scFvs are promising diagnostic and therapeutic reagents for protein misfolded diseases. Studies have demonstrated the efficacy and safety of scFvs in preventing amyloid protein aggregation in preclinical models. Herein, we discuss recent developments of these immunotherapeutics. We review efforts of our group and others using scFv in neurodegenerative disease models. We illustrate the advantages of scFvs, including engineering to enhance misfolded conformer specificity and subcellular targeting to optimize therapeutic action.     

Predicted secondary structure and membrane topology of the scrapie prion protein

The integral membrane sialoglycoprotein PrP^Sc is the only identifiablecomponent of the scrapie prion. Scrapie in animals and Creutzfeldt-Jakobdisease in humans are transmissible, degenerative neurologicaldiseases caused by prions. Standard predictive strategies havebeen used to analyze the secondary structure of the prion proteinin conjunction with Fourier analysis of the primary sequencehydrophobicities to detect potential amphipathic regions. Severalhydrophobic segments, a proline- and glycine-rich repeat regionand putative glycosylation sites are incorporated into a modelfor the integral membrane topology of PrP. The complete aminoacid sequences of the hamster, human and mouse prion proteinsare compared and the effects of residue substitutions upon thepredicted conformation of the polypeptide chain are discussed.While PrP has a unique primary structure, its predicted secondarystructure shares some interesting features with the serum amyloidA proteins. These proteins undergo a post-translational modificationto yield amyloid A, molecules that share with PrP the abilityto polymerize into birefringent filaments. Our analyses mayexplain some experimental observations on PrP, and suggest furtherstudies on the properties of the scrapie and cellular PrP isoforms.     

Expression of Main Toll-Like Receptors in Patients with Different Types of Colorectal Polyps and Their Relationship with Gut Microbiota

Abnormal activation of Toll-like receptor (TLRs) signaling can result in colon cancer development. The aim of this study was to investigate the expression of important TLRs in different histological types of colorectal polyps and evaluate their relationship with intestinal microbiota. The expression levels of TLR2, 3, 4, and 5 were analyzed in intestinal biopsy specimens of 21 hyperplastic polyp (HP), 16 sessile serrated adenoma (SSA), 29 tubular adenoma (TA), 21 villous/tubulovillous (VP/TVP) cases, and 31 normal controls. In addition, selected gut bacteria including Streptococcus bovis, Enterococcus faecalis, Enterotoxigenic Bacteroides fragilis (ETBF), Fusobacterium nucleatum, Porphyromonas spp., Lactobacillus spp., Roseburia spp., and Bifidobacterium spp. were quantified in fecal samples using absolute qRT PCR, and, finally, the association between TLRs and these gut microbiota- was evaluated by Spearman’s correlation coefficient. Higher expression of TLR2 and TLR4 in VP/TVP and TA, and lower expression levels of TLR3 and TLR5 in all type of polyps were observed. The differences in TLR expression patterns was not only dependent on the histology, location, size, and dysplasia grade of polyps but also related to the intestinal microbiota patterns. TLR2 and TLR4 expression was directly associated with the F. nucleatum, E. faecalis, S. bovis, Porphyromonas, and inversely to Bifidobacterium, Lactobacillus, and Roseburia quantity. Furthermore, TLR3 and TLR5 expression was directly associated with Bifidobacterium, Roseburia, and Lactobacillus quantity. Our results suggest a possible critical role of TLRs during colorectal polyp progression. An abnormal regulation of TLRs in relation to gut microbial quantity may contribute to carcinogenesis.     

Association between Toll-Like Receptor 4 Expression and Neural Stem Cell Proliferation in the Hippocampus Following Traumatic Brain Injury in Mice

Whether or how neural stem cells (NSCs) respond to toll-like receptor 4 (TLR4) in an inflammatory environment caused by traumatic brain injury (TBI) has not been understood. In the present study, association between TLR4 expression and NSCs proliferation in the hippocampus was investigated in a mouse model of TBI using controlled cortical impact (CCI). Hippocampal proliferating cells were labeled with the thymidine analog 5-bromo-2-deoxyuridine (BrdU). In order to identify NSCs, the proliferating cells were further co-labeled with BrdU/sex determination region of Y chromosome related high mobility group box gene 2 (SOX2). Morphological observation on the expression of BrdU, SOX2, and TLR4 in the hippocampus was performed by inmmunofluorescence (IF). Relative quantification of TLR4 expression at the protein and mRNA level was performed using Western blotting and real-time polymerase chain reaction (PCR). It was observed that BrdU⁺/SOX2⁺ cells accounted for 95.80% ± 7.91% among BrdU⁺ cells; several BrdU⁺ cells and SOX2⁺ cells in the hippocampus were also TLR4-positive post injury, and that BrdU⁺ cell numbers, together with TLR4 expression at either protein or mRNA level, increased significantly in TBI mice over 1, 3, 7, 14, and 21 days survivals and changed in a similar temporal pattern with a peak at 3 day post-injury. These results indicate that hippocampal proliferating cells (suggestive of NSCs) expressed TLR4, and that there was a potential association between increased expression of TLR4 and the proliferation of NSCs post TBI. It is concluded that hippocampal TLR4 may play a potential role in endogenous neurogenesis after TBI.     

Regional Differences in Neuroinflammation-Associated Gene Expression in the Brain of Sporadic Creutzfeldt–Jakob Disease Patients

Neuroinflammation is an essential part of neurodegeneration. Yet, the current understanding of neuroinflammation-associated molecular events in distinct brain regions of prion disease patients is insufficient to lay the ground for effective treatment strategies targeting this complex neuropathological process. To address this problem, we analyzed the expression of 800 neuroinflammation-associated genes to create a profile of biological processes taking place in the frontal cortex and cerebellum of patients who suffered from sporadic Creutzfeldt–Jakob disease. The analysis was performed using NanoString nCounter technology with human neuroinflammation panel+. The observed gene expression patterns were regionally and sub-regionally distinct, suggesting a variable neuroinflammatory response. Interestingly, the observed differences could not be explained by the molecular subtypes of sporadic Creutzfeldt–Jakob disease. Furthermore, analyses of canonical pathways and upstream regulators based on differentially expressed genes indicated an overlap between biological processes taking place in different brain regions. This suggests that even smaller-scale spatial data reflecting subtle changes in brain cells’ functional heterogeneity and their immediate pathologic microenvironments are needed to explain the observed differential gene expression in a greater detail.